RxPG News : Virology

How West Nile virus evades immune defenses

Thu, 05 Oct 2006 01:04:37 PST

( from http://www.rxpgnews.com ) West Nile virus evades the body's immune defenses by blocking immune signaling by a protein receptor, a finding that could pave the way for a vaccine to protect against North American strains of the virus, UT Southwestern Medical Center researchers report.

Researchers discovered the receptor's key role in controlling West Nile infection by conducting a study, described in October's Journal of Virology, that compares the genetics of an illness-causing Texas strain of the virus to a harmless African strain.

The Texas strain can inflict illness because it blocks the signaling activity of a protein receptor called the interferon alpha/beta receptor, or IFNAR, disrupting a cell's ability to direct the immune system to fight off the virus.

The African strain does not block IFNAR activity, so the immune system renders it harmless. The strain is harmful, however, in mice with dysfunctional receptors.

"We now hope to harness the African strain as the basis for West Nile vaccine studies. The virus has spread across the country and infected more than 2,100 U.S. residents 180 in Texas this year alone, so we have to learn how to deal with it," said Dr. Michael Gale, associate professor of microbiology at UT Southwestern and director of the study. Brian Keller, a student in the Medical Scientist Training Program at UT Southwestern, is the first author of the study.

West Nile virus, which is transmitted by mosquito bite, arrived in the United States in 1999 and has become an epidemic that flares up in the summer and lasts into fall.

Infection causes mild flu-like symptoms in most people, but about one in every 150 develop serious illness, that can include high fever, coma, seizures and encephalitis and meningitis. Children, the elderly or people with weak immune systems are most at risk.

There is no vaccine. Doctors can only treat symptoms of the disease.

Searching for clues that might allow development of a vaccine, Dr. Gale and his research team compared one strain from each of West Nile's two basic categories: the harmful strains associated with outbreaks of encephalitis and meningitis in North America, and non-harmful strains from Madagascar and Cyprus.

They studied a harmful strain isolated from an infected grackle from Hall County, Texas, in 2002, and a harmless strain isolated from an infected parrot from Madagascar in 1978.

They mapped the genetic makeup of each strain, and then tested the viruses in mice.

West Nile infection triggers production of interferon, a group of proteins that are crucial in immune defense. Interferon, which binds to IFNAR, subsequently signals the JAK-STAT molecular pathway, a series of biochemical reactions essential for turning on immune-defense genes, allowing the body to clear out the virus. This process occurs normally in the African strain.

Infection by the Texas strain, however, blocked IFNAR signaling activity, allowing the virus to replicate and spread.

This highlights the integral role of interferon and IFNAR signaling in innate immunity.

Dr. Gale said the mechanisms at work in the African strain could be used as a basis for a vaccine, perhaps mutating North American strains so they no longer disrupt immune signaling. The remaining key is figuring out the exact mechanics of how the strains block signaling, a project Dr. Gale's team is already at work on.

Fortunately, North American strains are extremely similar in fact, the one that appeared in the United States in 1999 and the Texas strain used in this study are 99 percent identical. One vaccine could, in theory, prevent illness from many of the harmful strains, Dr. Gale said.

"We feel a vaccine could be highly effective in preventing infection," said Dr. Gale.

Innovative method for creating a human cytomegalovirus vaccine outlined

Wed, 02 Aug 2006 11:42:37 PST

( from http://www.rxpgnews.com ) Each year, about 40,000 children are born infected with human cytomegalovirus, or CMV, and about 8,000 of these children suffer permanent disabilities due to the virus almost one an hour. These disabilities can include hearing loss, vision loss, mental disability, a lack of coordination, and seizures. According to the Centers for Disease Control and Prevention, CMV is as common a cause of serious disability as Down syndrome, fetal alcohol syndrome, or neural tube defects.

Because of the dangers posed by the virus to infants, the Institute of Medicine has declared that development of a CMV vaccine should be one of the highest priorities for vaccine makers. Now, in a new study in the August 1 issue of The Journal of Virology, researchers at The Wistar Institute outline an innovative approach that could be used to create such a vaccine.

The Wistar scientists began with the observation that mice harbor a species-specific form of CMV that is unable to sustain an infection in humans and is completely harmless to them. They then asked whether, using recombinant technologies, there might not be a way to shift the mouse-specific virus closer to the human-specific virus to generate a version of the virus able to elicit a protective immune response but not a dangerous infection in humans.

With this goal, the researchers began to systematically introduce selected genes from human CMV into the genome of mouse CMV in the laboratory. The result was a novel form of CMV virus that infected human cells well enough that it might trigger an immune response but not well enough to sustain an infection.

"It should be possible to develop a safe and effective CMV vaccine using the method we've described in our study," says Gerd G. Maul, Ph.D., a professor in the Gene Expression and Regulation Program at Wistar and senior author on the new study. "Success will depend on achieving a certain balance between the immune-stimulating genes from the human virus and the basic safety of the mouse virus."

Over the years, a number of scientists have worked to create a vaccine against human CMV. Among these is former Wistar researcher Stanley A. Plotkin, M.D., who during his career at the Institute between 1960 and 1991 developed the rubella vaccine that eradicated the disease in the U.S. and co-developed a new rotavirus vaccine approved in the U.S. in 2006. Plotkin's approach to developing a CMV vaccine was to attenuate, or weaken, human CMV, as he had done to create the rubella vaccine. In contrast, the rotavirus vaccine was developed using co-infection techniques to incorporate selected elements from several human rotavirus strains into a bovine rotavirus backbone. The vaccine that resulted takes advantage of the immunological characteristics of both the human and bovine viruses. This approach is conceptually similar to Maul's strategy for developing a CMV vaccine, although the recombinant technologies available today should help to streamline the task.

CMV infection is widespread in the U.S., according to the Centers for Disease Control and Prevention. Between 50 and 80 percent of adults are infected with CMV by the time they reach 40 years of age, but the virus generally poses little threat to them. The exceptions are AIDS patients, transplant recipients, and others with compromised immune systems are at risk for complications. The virus is a member of the herpes virus family, which includes the herpes simplex viruses, the viruses that cause chicken pox (varicella-zoster virus), and infectious mononucleosis (Epstein-Barr virus.) Infectious transmission is via body fluids.

Cracking Virus Protection Shield

Mon, 19 Jun 2006 02:17:37 PST

( from http://www.rxpgnews.com ) Ebola, measles and rabies are serious threats to public health in developing countries. Despite different symptoms all of the diseases are caused by the same class of viruses that unlike most other living beings carry their genetic information on a single RNA molecule instead of a double strand of DNA. Now researchers from the Institut de Virologie Moléculaire et Structurale [IVMS] and the Outstation of the European Molecular Biology Laboratory [EMBL] in Grenoble have obtained a detailed structural picture of a protein that allows the rabies virus to withstand the human immune response and survive and replicate in our cells. The study that is published in this week's online edition of Science suggests new potential drug targets in rabies and sheds light on how similar approaches can help fighting other viral diseases.

When the rabies virus enters a human cell through the membrane, the RNA molecule that carries its genes is transported into the centre of the cell. Here it redirects the cellular machinery of the host to produce many new copies of the virus that go on to infect more cells. One molecule that is crucial in this process is a viral protein called nucleoprotein. The protein ensures that on its way through the cell the virus RNA is not destroyed by the immune response of the host.

"Nucleoprotein is vital for the rabies virus," says Rob Ruigrok, Head of the IVMS. "It is one of the few proteins that the virus brings into the host cell and it wraps around the RNA like a protection shield. Without this shield the RNA would be degraded by the enzymes of the human immune system that try to eliminate the invader."

To investigate how exactly this protection shield works, Aurélie Albertini from Ruigrok's team obtained crystals of nucleoprotein bound to RNA. Examining the crystals with high-intensity X-ray sources at the European Synchrotron Radiation Facility [ESRF], Amy Wernimont from Winfried Weissenhorn's group at EMBL Grenoble produced a high-resolution image of the protein.

"Nucleoprotein acts like a clamp," says Weissenhorn. "It consists of two domains that like two jaws clasp around the RNA strand. Many nucleoproteins bind side-by-side along the length of an RNA molecule and make it inaccessible for degrading enzymes but also for the machinery needed to replicate the virus. This means that the protection shield must be flexible and able to distinguish between different types of enzymes trying to gain access."

The detailed structural picture suggests that upon a signal a part of the protein located between the two main domains might act as a hinge that moves the upper jaw out of the way when time for replication has come.

"This dynamic mechanism makes nucleoproteins an excellent drug target," says Ruigrok, "Small agents that bind to the protein in such a way to block its flexibility and keep it in the closed state, would prevent replication of the virus and would stop it from spreading."

Rabies virus shares this protection strategy with other viruses of its class; in Ebola, measles and Borna virus similar complexes of RNA and nucleoproteins have been found.

"This means that our results do not only have implications for the design of new drugs against rabies, but they suggest new therapeutic approaches in a variety of diseases, some of which are much more threatening than rabies. On a different note, the conservation of the nucleoprotein system also leaves room for evolutionary speculations about common ancestors and primordial infectious units of RNA viruses," Weissenhorn concludes.

Viruses trade-off between survival and reproduction

Thu, 15 Jun 2006 12:08:37 PST

( from http://www.rxpgnews.com ) Living is an energy-intensive exercise that inevitably involves trade-offs. As many a mother may tell you, expending the energy necessary to raise a clutch of kids can shave years off one's life. Trade-offs between reproductive success and survival have been demonstrated for a wide variety of organisms and, in keeping with life history theory, should arise in any organism striving to maximize fitness under the constraints of finite resources.

In a new study, Marianne De Paepe and François Taddei asked whether these trade-offs extend to viruses. Though not universally considered alive because they can't replicate without the help of their host's molecular machinery, viruses pass through distinct life cycle stages, mutate, and evolve in response to selection pressures from their host. Viruses also have life history traits, such as multiplication rate in a host, survival outside the host, and mode of transmission.

Working with viruses that infect bacteria, called bacteriophages (or in this case, coliphages, which infect Escherichia coli), De Paepe and Taddei predicted that the phage, just like a full-fledged cellular organism, would display trade-offs between survival and reproduction. They discovered that, although coliphages don't wither and die like real organisms, they do experience life history trade-offs, with rapid reproducers suffering higher casualties outside the host. And, by investigating several physical properties of the coliphages, they found that two physical parameters account for most of the observed variation in survival.

During infection, phages rapidly reproduce until the bacterial cell ruptures (called lysis) and releases the virions (viral particles, which consist of little more than the viral genome encased in a capsid protein shell). A phage typically meets its end after encountering harsh conditions, such as heat or osmotic shock, that rupture the capsid, releasing its genetic contents. How well the phage can resist such stresses depends in part on the strength of the capsid, which must withstand the extreme pressure exerted by strong repulsive forces between charged DNA strands and bending of the tightly packed DNA.

To investigate the presence of life history trade-offs in phages, De Paepe and Taddei measured the decay rate (mortality) and multiplication rate of 16 coliphage strains. They determined the kinetics of mortality within and across the phage populations, by measuring the number of phage particles that completed an infectious cycle in E. coli cultures, at different times and temperatures. Although all the phages died at a constant rate over time, different strains showed considerable variation in these rates. The constant rate (meaning that the probability of death did not increase over time) suggests that the phages succumbed to a single eventindicating that they don't agerather than to a series of events, which would be consistent with aging. Decay rates increase exponentially with the inverse of the temperature, which is a characteristic of very simple chemical reactions.

To determine the factors underlying this variation, the researchers investigated the correlation between several physical parameters and decay rate. Among the parameters measured were two factors governing capsid stabilitydensity of the packaged genome and surfacic mass of the capsid, an indicator of capsid thicknessand two indicators of replication rateburst size (number of particles released during an infection cycle) and latency period (time between infection and host lysis). They found that decay rate increases significantly with the density of the packaged DNA, linking higher internal pressure with higher mortality. By contrast, decay rate decreases as surfacic mass increases, supporting the notion that a stronger capsid increases phage stability. The highest correlation was between decay rate and multiplication rate in the bacterial host. (Multiplication rate was determined by dividing burst size by latency period.) A model based on these three variablespackaged DNA density, surfacic mass, and multiplication rateaccounts for over 90% of the observed variability in phage mortality rates.

Even though they don't have their own metabolism, viruses experience the same sorts of trade-offs between survival and reproduction seen in a wide range of species. This finding suggests that models of virulence evolution, which assume that transmission rates increase along with virulence, may not be valid, since transmission depends not just on parasite multiplication rate but also on survivalwhich, they show, are negatively correlated. The fact that this trade-off is present in this very simple biological situation, the researchers write, suggests that it might be a fundamental property of evolving entities produced under constraints. If this is true, the nonliving phages that opened the door to some of the most important discoveries in molecular biology may well provide a similar service for a wide range of evolutionary phenomena.

New hybrid virus provides targeted molecular imaging of cancer

Sat, 22 Apr 2006 19:31:37 PST

( from http://www.rxpgnews.com ) Researchers at The University of Texas M. D. Anderson Cancer Center have created a new class of hybrid virus and demonstrated its ability to find, highlight, and deliver genes to tumors in mice.

Researchers say the advance, reported in the journal Cell, is potentially an important step in making human cancer both more visible and accessible to treatment; it may also allow prediction and monitoring of how specific anti-cancer agents are actually working.

"In tumor-bearing mice, we show that this hybrid virus can target tumors systemically to deliver an imaging or therapeutic gene," says the co-leader of the study, Renata Pasqualini, Ph.D., professor of Medicine and Cancer Biology. "The signal is specific only to tumors, so one can monitor drug effectiveness at the molecular level."

The team created and characterized the hybrid viruses by combining genetic elements and biological attributes of an animal virus (adeno-associated virus, or AAV) with those of a bacterial virus (phage). Unlike animal viruses that infect mammalian cells, bacterial viruses have evolved to infect only bacterial hosts. The paper shows how particles of the hybrid virus, called AAV phage or AAVP, can serve as a vehicle for targeted delivery of genes to experimental tumors in mice and to the tumors' blood vessel supply, providing a strategy for finding tumors and genetically marking them for imaging on a clinic-ready body scanner.

The AAVP hybrid combines the ability of the bacterial virus to target specific tissues with the capability of the mammalian virus to actually deliver genes to cells. The crucial vehicles, or vectors, in the AAVP hybrid retained the properties of their respective parental viruses, which the researchers called a surprising outcome.

"This is only a proof-of-concept, and although we have yet to translate these hybrid viruses for use in humans, we hope that this new system will have future clinical applications," says Wadih Arap, M.D., the co-leader of the study and professor of Medicine and Cancer Biology. "In addition to the obvious biological interest, when the vector is refined for patient use, it could perhaps help us diagnose, monitor and treat human tumors more accurately."

The finding is the latest in a series of studies by Pasqualini and Arap that are built around their discovery that the human vasculature system contains unique molecular addresses. Organs and specialized tissues also have specific "zip codes" on their blood vessels, as do tumor blood vessels. Knowing this, Pasqualini and Arap designed, constructed, evaluated, and validated the targeted AAVP system over the past several years. Amin Hajitou, Ph.D., a post-doctoral fellow in the Arap/Pasqualini laboratory and first author of the Cell study says, "we were pleased by the strong effects of gene transfer in mouse models of common diseases such as breast and prostate cancer."

Their next step was to work closely with the team of M. D. Anderson researcher Juri Gelovani, M.D., Ph.D., chair of the Department of Experimental Diagnostic Imaging, a pioneer in development of molecular-genetic imaging tools.

"We could see by using positron emission tomography that the reporter and therapeutic genes were being expressed throughout the tumors in the animals," Gelovani says. "This is an example of the so-called "theragnostic" approach, a combination of the words therapeutic and diagnostic."

Next, the international collaborative research team plans to evaluate the safety and efficacy of other hybrid vectors in animal models. The ultimate goal is to adapt and optimize the AAVP-based targeting prototype for use in patients.

Mass spectrometry to detect norovirus particles

Mon, 10 Apr 2006 14:07:37 PST

( from http://www.rxpgnews.com ) Scientists have used mass spectrometry for decades to determine the chemical composition of samples but rarely has it been used to identify viruses, and never in complex environmental samples. Researchers at the Johns Hopkins Bloomberg School of Public Health recently demonstrated that proteomic mass spectrometry has the potential to be applied for this purpose. Using a two-step process, researchers successfully separated, purified and concentrated a norovirus surrogate from a clinical sample within a few hours. Nanospray mass spectrometry was used to demonstrate the feasibility of detecting norovirus particles in the purified concentrates.

Human norovirus is responsible for an estimated 23 million cases of gastrointestinal illness in the United States each year. This pathogen is a particular problem aboard cruise ships. The researchers believe that their mass spectrometric method could potentially be used for biodefense and public health preparedness as a tool for rapidly detecting norovirus--a category B bioterrorism agent--and other viral public health threats. The study is published in the April 2006 edition of Applied and Environmental Microbiology.

In simplified terms, mass spectrometry is essentially a scale for weighing molecules. A laser turns a sample into ionized particles, which are then accelerated in a vacuum toward a detector. The time lapsed prior to registering on the detector helps researchers determine the mass--or weight--of the particles. By targeting characteristic particles, or peptides, belonging to the viral coat protein, the virus can be positively identified by matching the results to entries in genetic databases.

In the Hopkins study, the researchers analyzed a stool sample treated with virus-like particles, which closely resemble norovirus but are noninfectious. Using mass spectrometry, the researchers were able to detect the norovirus capsid protein down to levels typically found in clinical specimens from sick individuals.

"This is the first report of the use of mass spectrometry for the detection of norovirus," said David R. Colquhoun, lead author of the study and research fellow with the Johns Hopkins Center for a Livable Future. "This is a significant step towards using mass spectrometry as an environmental surveillance tool for the detection of pathogenic human viruses in complex environmental samples such as human and animal waste."

Typically, bacteria and viruses are identified by cultivation on selective media and cell lines. However, this process does not work for human norovirus, which cannot be cultured outside the human body.

Rolf Halden, PhD, assistant professor in the Department of Environmental Health Sciences and senior author of the study, pointed out that proteomic mass spectrometry is appealing because it has the potential to identify different types and strains of viruses regardless of whether their presence is suspected or not. "Unlike other processes, we do not need to know what we are looking for in advance. Any pathogen whose genetic information is contained in online genetic databases represents a suitable potential target. This makes the technique ideal for situations where you have an emerging infectious agent or pathogenic strain, such as in a potential terrorist attack," said Halden.

xCT molecule is a major gateway for KSHV to enter human cells

Fri, 07 Apr 2006 13:55:37 PST

( from http://www.rxpgnews.com ) Researchers at the National Institute of Allergy and Infectious Disease (NIAID), a component of the National Institutes of Health (NIH), have identified a critical human cell surface molecule involved in infection by Kaposi's sarcoma herpesvirus (KSHV), the virus that causes Kaposi's sarcoma and certain forms of lymphoma. Kaposi's sarcoma is a major cancer associated with HIV/AIDS, and it typically manifests as multiple purple-hued skin lesions.

In the March 31, 2006 issue of Science, NIAID research fellow Johnan Kaleeba, Ph.D. and senior investigator Edward A. Berger; Ph.D., describe how the molecule xCT is a major gateway that KSHV uses to enter human cells. The molecule may also play a role in the development of Kaposi's sarcoma and other syndromes associated with the virus.

The natural function of xCT in the body is to transport molecules necessary for protecting against stress into cells. When cells are stressed, they express more xCT on their surfaces. Of note, this sort of stress can be caused by KSHV itself. This suggests that the virus may facilitate its own infectivity and dissemination in the body by inducing a physiological state that results in increased numbers of its own receptor.

"The advancement of knowledge achieved in this study highlights the outstanding intramural research that takes place here on the NIH campus," says Elias A. Zerhouni, M.D., NIH director.

"Understanding the mechanisms of cell entry of Kaposi's sarcoma herpesvirus is a landmark achievement in and of itself," says NIAID director Anthony S. Fauci, M.D. "But the connection between the virus and expression of its own receptor on a cell is even more provocative because it might change the way we think about KSHV-associated diseases and their treatment."

Although less common in the United States now than early in the AIDS pandemic, Kaposi's sarcoma is still the most common cancer associated with HIV infection. Prior to the AIDS pandemic, it was an obscure disease. First identified as a multi-pigmented skin disease by a Hungarian doctor named Moritz Kaposi in 1872, it was considered to be quite rare--a medical curiosity usually found in particular populations such as older Italian men, transplant patients and young men in certain parts of sub-Saharan Africa. But then at the dawn of the AIDS pandemic in the early 1980s, the small purplish Kaposi's sarcoma skin lesions began appearing on the bodies of young American men, many of whom went on to develop opportunistic infections.

Dr. Berger became interested in KSHV because of his interest in how viruses enter cells. A decade ago, his research team was the first to identify CXCR4 as one of the coreceptors that allows HIV to gain entry into cells of the immune system. This discovery quickly led to the identification by Dr. Berger's group and several other research teams of CCR5 as the other HIV coreceptor.

By applying the same technology used to identify CXCR4, Drs. Kaleeba and Berger ultimately identified the protein xCT as the receptor that can make cells permissive for KSHV fusion.

The NIAID discovery may lead to new avenues for treating KSHV, says Dr. Berger. Moreover, their finding should enable scientists to determine whether levels of xCT determine disease severity. It also will allow researchers to study whether the expression of xCT on cells varies among different groups of people and whether these variations are genetic or environmental. This research may ultimately explain why certain groups are more at risk for Kaposi's sarcoma.

"Our finding provides a new perspective on the disease," says Dr. Kaleeba, who is originally from Uganda where Kaposi's sarcoma accounts for at least 10 percent of known tumors. "Hopefully this will be the beginning of exciting new directions in this field, as it is likely to provide a useful framework for integration of the cell biology and epidemiology of this clinically important virus."

Surprising discovery about the inner workings of vesicular stomatitis virus (VSV)

Fri, 07 Apr 2006 13:52:37 PST

( from http://www.rxpgnews.com ) Biochemists at Wake Forest University School of Medicine have made a surprising discovery about the inner workings of a powerful virus a discovery that they hope could one day lead to better vaccines or anti-virus medications.

Reporting in the April issue of the Journal of Virology, the researchers have identified a protein that plays an important role in the ability of the vesicular stomatitis virus (VSV) to invade healthy cells and reproduce itself. The finding could play a role in vaccine development and also help scientists develop anti-viral agents to stop similar viruses in their tracks.

Although VSV infects animals, it is not a human pathogen. Nevertheless, scientists study it because of its similarity to viruses such as Ebola and Marburg hemorrhagic fever viruses, as well as rabies virus. "VSV is a good model of a variety of other viruses," said John Connor, Ph.D., a research assistant professor of biochemistry. "Our research has given us a better understanding of how viruses like these are able to do the nasty things they do."

The scientists set out to study the role of a protein known as "matrix," which is produced by VSV. They suspected matrix was important in how VSV is assembled, but unexpectedly discovered the matrix protein is critical in how the virus reproduces and spreads. When they altered the matrix protein, they weakened the virus' ability to reproduce. The finding has several important implications, Connor said.

Normally, VSV is extremely powerful, with the ability to shut down a cell's system for making proteins. VSV then takes over the cell's protein-making machinery and makes its own proteins so it can replicate and spread. The scientists were able to weaken this power by altering the matrix protein, so that VSV cannot make as much protein and does not reproduce as well.

Weakened viruses such as this are often used to make vaccines because they are less likely to be harmful. Currently, another weakened form of VSV is being used for a HIV vaccine that is being tested in humans. To make the vaccine, scientists started with the weakened VSV virus and added a protein from the HIV virus so that VSV "expresses" or makes a fragment of the HIV virus. In theory, when people are inoculated with the vaccine, they will develop antibodies to the HIV protein, and if they are exposed to the actual HIV virus, their bodies will neutralize it and kill it before it infects them.

In all, several weakened forms of VSV have been developed and at least two are currently being tested in HIV vaccines. If they don't prove effective, vaccine developers can turn to one of the others, including the mutant VSV virus developed by Connor and colleagues.

"Right now, there's no way of knowing which way of weakening the virus will make the best vaccine," Connor said.

In addition to its potential for vaccine development, the new finding about VSV also provides basic information about how the virus shuts downs a cell's protein making-abilities and dominates the process.

"We always knew this happened, but the process was like a black box," said Connor. "Now, we know that the matrix protein is involved and is incredibly important in virus reproduction. This pushes forward our knowledge of how this virus is so effective at replicating."

Could the finding about matrix be used to weaken other types of viruses? The scientists aren't sure, yet. "It's a strong possibility that every virus will have an Achilles' heel like this, where they need the function of a viral protein to make lots of virus," said Connor.

New human retrovirus - Xenotropic MuLV-related virus (XMRV)

Sat, 01 Apr 2006 19:26:37 PST

( from http://www.rxpgnews.com ) Howard Hughes Medical Institute researchers and their colleagues have discovered a new retrovirus in humans that is closely related to a cancer-causing virus found in mice. Their findings describe the first documented cases of human infection with a retrovirus that is native to rodents.

The researchers discovered the virus in patients with a rare type of prostate cancer. The patients in the study have a genetic mutation that compromised some of their natural defenses against viral infection. Thus, the researchers said their discovery raises the possibility that increased susceptibility to viral infection may play a role in development of some cancers. However, they emphasized that their findings by no means implicate the virus, dubbed XMRV, in causing prostate cancer. The virus may well have flourished as a result of the failure of the defense mechanism; and other factors such as chronic inflammation may play a more direct role in the cancer.

The discovery of the new virus was made by an interdisciplinary research team led by Robert Silverman of Cleveland Clinic and HHMI investigators Joseph DeRisi and Don Ganem, both at the University of California at San Francisco. A paper describing the findings was published on March 31, 2006, in the journal, Public Library of Science Pathogens.

The search for the new virus began when Silverman and his colleagues provided samples of a rare familial prostate cancer in which the viral-defense gene, RNASEL, had been mutated in a specific way. This mutation compromised the function of the enzyme produced by RNASEL, which normally shreds viral genetic material. Infected cells carrying the shredded viral genetic material are usually targeted for destruction by the immune system. While some scientists believe that such vulnerability to viral infection is connected to prostate cancer in these rare cases, others have presented evidence contesting that theory.

To screen for viruses in the prostate tissue samples, DeRisi and Ganem used the Virochip, which was invented by DeRisi and his colleagues. The Virochip consists of a microarray of some 20,000 characteristic gene sequences -- called oligonucleotides -- representing a vast array of known viruses. The oligonucleotides are deposited as tiny spots on a small glass chip.

To detect viruses from tissue samples, the researchers isolated genetic material from each sample and tagged the genetic material with a fluorescent tracer. They then applied the fluorescently tagged genetic material to the microarray chip. Since genes tended to adhere to those with a complementary genetic sequence, any viral gene sequences in the sample would attach themselves to corresponding viral sequences on the chip. The telltale fluorescence on spots on the chip signaled the presence of viral genetic material in the sample.

Although the Virochip contains only sequences from known viruses, DeRisi said it can also detect new viruses because they invariably contain sequences that have been conserved in their evolution from related viruses.

The initial screen of the RNASEL-mutant prostate cancers revealed the presence of a genetic sequence that closely resembled that of a mouse virus called murine leukemia virus (MuLV). Murine leukemia virus is known as an endogenous virus because it normally exists as an integrated part of the mouse genome, rather than as independent, infective particle. MuLV is also a retrovirus, meaning its genetic material is in the form of RNA. The RNA is then reverse transcribed into DNA that is integrated into the DNA of the host cell the virus is infecting.

When the researchers isolated and sequenced the genome of the virus, they found that it was a xenotropic virus one that can only grow in foreign cells other than mouse cells. Thus, they named the virus, Xenotropic MuLV-related virus, or XMRV.

"This finding was a big surprise because most of these endogenous viral genomes have undergone such mutation and deletion that they are incapable of giving rise to viruses any more," said Ganem. "And while some of these viruses had been induced to grow in human cells in culture, the major question is whether such infection could ever happen in nature.

"So, one of the things that is important about our study from a virologic point of view, is that this is the first really solid example of an authentic xenotropic retroviral infection in a human being," said Ganem.

According to DeRisi, the Virochip made it possible to analyze these samples without preconceived biases about what viruses might be present. "Since the chip represents every known virus in one assay, it is agnostic as to what might be found," he said. "We would never have looked for this class of virus if it wasn't for the virus chip."

Importantly, the researchers found that prostate cancers in which both copies of the RNASEL gene were crippled by mutation showed much more frequent XMRV infection than did those cancers that still had one normal copy of the RNASEL gene.

"This link between the virus and RNASEL is the second finding that is important and is firmly established in this study," noted Ganem. "We don't see the infection in people who don't have the RNASEL mutation, which suggests strongly RNASEL is an important part of the defense against retroviral infection. This is the first evidence in humans of findings that were previously made only in vitro."

DeRisi pointed out that detailed comparison of samples of the virus between people found that although all were XMRV they showed tiny genetic variations. "So, while it is the same virus in each patient, the viruses are different enough to say that they are most likely independently acquired and are not the result of some contamination of the samples," he said.

Ganem cautioned that any link between XMRV and prostate cancer is tenuous at best. "First, the genetic variant we studied occurs in familial clusters that constitute only a very small sliver of prostate cancers," he said. "And secondly, there are many reasons to believe that the virus might not relate to prostate cancer."

For example, he pointed out, analysis of prostate tissue by Silverman and his colleagues indicated that the virus appears only in a small percentage of connective tissue cells, called stromal cells, rather than in the tumors themselves. "So, one interpretation could be that the infection is entirely incidental to prostate cancer," said Ganem. "The patients with RNASEL mutations may be more likely to get the infection or perhaps less likely to clear it. Clearly XMRV is not a classic oncogenic virus."

Nevertheless, said Ganem, an indirect link to cancer cannot be ruled out, since "in cancer research these days, there is a lot of interest in the stroma as the soil in which cancer arises." He added that the chronic inflammation from infection of stromal tissues may play a role in triggering such cancers.

DeRisi observed that "it may be that men who are so-called RNASEL-mutant are just more susceptible to viruses in general, and this susceptibility has little to do with their cancer. Nevertheless, the fact that this virus is found in tumor tissue and that it is a new virus and the first of its kind ever documented in humans is an intriguing finding that demands to be followed up. This initial finding raises many questions. For example, what is the route of transmission? How is the virus passed from person to person? And are people the natural reservoir of this virus, or is it some other organism?"

DeRisi and Ganem said they are planning studies to explore whether XMRV is restricted to prostate cancers or whether it is more widespread in the body and in other segments of the human population. To answer such questions, the researchers are developing a blood test that can be used in epidemiological studies.

Viruses can be forced to evolve as better delivery vehicles for gene therapy

Wed, 08 Feb 2006 11:33:37 PST

( from http://www.rxpgnews.com ) Viruses and humans have evolved together over millions of years in a game of one-upmanship that, often as not, left humans sick or worse.

Now, a University of California, Berkeley, researcher has shown that viruses - in this case, a benign one - can be forced to evolve in ways to benefit humans.

The adeno-associated virus, or AAV, is a common, though innocuous, resident of the body that has received a lot of attention in recent years as a possible carrier for genes in gene therapy. Because as many as 90 percent of people already have the virus, however, their immune systems are primed with antibodies to quickly tackle and neutralize it, thwarting any attempt at gene therapy.

UC Berkeley's David Schaffer, associate professor of chemical engineering and a member of the Helen Wills Neuroscience Institute, with colleagues Narendra Maheshri, James T. Koerber and Brian Kaspar, decided to speed up the process of viral evolution and direct the change in a way that would allow the virus to slip past the body's immune defenses, making it a more viable vehicle for gene therapy. In essentially two generations of accelerated evolution, requiring about two months of lab work, they succeeded.

"Directed evolution has mainly been done to change the activity of an enzyme - to make it more effective toward a new substrate or better able to catalyze a reaction, for example - or to make antibodies better binders against specific targets," said Schaffer, who also is an affiliate of the UC Berkeley wing of the California Institute for Quantitative Biomedical Research (QB3). "In the viral realm, this approach is essentially untapped."

This technique could be used to improve many other characteristics of AAV to make it a better delivery vector for genes.

"We think there are a huge variety of new problems we could address as well, such as targeting the virus to cells it is ordinarily not good at getting into, or speeding its transport through the body," he said.

Though Schaffer acknowledges that the technique could be used to help pathogenic viruses evade the human immune system, potentially making them more virulent, he said that other and easier techniques already allow this frightening possibility.

AAV consists of two genes enclosed within a ball, or capsid, of proteins. The capsid proteins are what antibodies recognize, and as a result were the target of directed evolution. To provide the raw material for evolution - the genetic variation from which nature selects the best-adapted organism - the researchers created mutant viruses by introducing small variations in the genes through an error-prone polymerase chain reaction (PCR) coupled with a test tube recombination technique. After reassembling the mutant viruses inside their capsids, they introduced them to blood serum pooled from rabbits immunized against AAV, and thus containing many types of antibodies to AAV. Only the mutant viruses good at evading antibodies to AAV survived the serum.

After passing the viruses three times through increasingly more potent serum, they isolated the survivors and subjected them to another round of PCR that introduced more mutations. After passing this second generation through serum three times, they isolated viruses that could survive AAV antibodies much better than the original strain of AAV. One strain of virus was 96 times more effective than the wild AAV, and two evolved strains survived injection into mice with nearly 1,000 times the level of antibodies required to neutralize the wild virus.

By sequencing the survivor strains, the researchers discovered that the capsid proteins of the survivors differed from those of the original strain by only seven amino acid building blocks, two of which were responsible for most of the altered interaction with antibodies.

"Starting from scratch, just trying to rationally decide which two amino acid changes to make on the virus, there is no way you would have guessed those two," Schaffer said. "Using the same algorithm as nature came up with - evolution - to solve the problem, is the best way to do it."

Since each generation takes about a month, Schaffer predicted that many types of new and improved strains could be created in a few months' time, and certainly in less than a year. He is pursuing experiments now using pooled human blood serum.

"This virus is kind of a gift from nature, a very safe and efficient virus, but nature never evolved it to be a human therapeutic. So, in a sense, we have to re-evolve it for that purpose," he said.

Epstein-Barr Virus Found in Breast Cancer Tissue May Impact Efficiency of Treatment

Fri, 20 Jan 2006 14:00:37 PST

( from http://www.rxpgnews.com ) Epstein-Barr virus has been detected in breast cancer tissue and tumor cells and may impact the efficiency of chemotherapeutic drug treatment say researchers from France and Japan. They report their findings in the January 2006 issue of the Journal of Virology.

A ubiquitous human herpesvirus, the Epstein-Barr virus (EBV), has been previously linked to skin and gastric cancer, as well as cancer of the salivary glands and thymus. New studies have detected EBV in breast cancer specimens and have prompted researchers to examine the effect of infection with EBV on anticancer drug treatment.

In the study biopsy specimens of breast cancer tissue and tumor cells were tested for the EBV genome. The genome was identified in about half of the specimens, however the viral load was highly variable from tumor to tumor. These findings indicate that although EBV isn't likely to cause breast cancer, it may contribute to tumor progression. In addition, researchers studied the EBV infected cells in vitro and found that the virus may contribute to the resistance of paclitaxel (taxol), chemotherapy commonly used in the treatment breast cancer, and cause overexpression of the multidrug resistance gene (MDRI).

"Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance," say the researchers.

Honeybees May Transmit Viruses to Their Offspring

Fri, 20 Jan 2006 13:55:37 PST

( from http://www.rxpgnews.com ) Researchers from the U.S. Department of Agriculture report what may be the first evidence of queen honeybees transmitting viruses to their offspring. They report their findings in the January 2006 issue of the journal Applied and Environmental Microbiology.

Honeybees contribute greatly to the annual 15 billion dollar agriculture market by assisting in the pollination of a wide variety of crops. The health of honeybee colonies is continuously threatened by various pathogens, with viruses posing the greatest risk due to lack of information concerning transmission and outbreaks.

In the study feces and tissue (including hemolymph, gut, ovaries, spermatheca, head, and eviscerated body) of individual queen bees were tested for viral presence. All tissue forms but one, as well as feces, were found to carry viral infections. Once the viruses in the queen bees were identified, their offspring (including eggs, larvae and adult workers) were tested and found to carry the same viruses.

"The present study, using the sensitive RT-PCR method, demonstrated the vertical transmission of multiple viruses from mother queens to their offspring by two findings: first, the presence of viruses in queen excretion and queen tissues, particularly in the tissue of ovaries; and second, detection of the same viruses in queens' eggs and young larvae that are not normally associated with V. destructor, which is an important vector of bee viruses," say the researchers.

Human Papillomavirus Could Spread Through Blood - Study

Thu, 17 Nov 2005 16:39:38 PST

( from http://www.rxpgnews.com ) Potentially transmissible human papillomavirus DNA has been identified in human blood cells suggesting that the virus, traditionally thought to be sexually transmitted, could also be spread through blood products. Researchers from the National Institutes of Health report their findings in the November 2005 issue of the Journal of Clinical Microbiology.

Human papillomavirus (HPV) is most commonly recognized as a sexually transmitted disease that can cause genital warts as well as cervical cancer. It had been widely accepted that HPVs could not be disseminated through blood. However, the successful experimental transmission of bovine papillomavirus through blood several years ago suggested that might not be the case.

In the study researchers examined HPV DNA in banked, frozen blood cells from pediatric HIV patients and fresh blood cells from healthy donors. Results showed that eight HIV patient samples (seven of which acquired HIV through blood transfusions) and three healthy donor samples were positive for two subgroups of the HPV type 16 genome and that the DNA could exist in a transmissible form.

"Peripheral blood mononuclear cells (PBMCs) might serve as a source of HPV in the infection of epithelial cells and contribute to their nonsexual spread," say the researchers. "However, additional work is needed to confirm this as a possible mode of HPV transmission."

Gene Identified in Epstein-Barr Virus that May Contribute to Cancer

Thu, 17 Nov 2005 16:36:38 PST

( from http://www.rxpgnews.com ) Researchers have identified a gene in the Epstein-Barr virus that may contribute to the development of lymphoproliferative disease (LPD) in humans. Their findings appear in the November 2005 issue of the Journal of Virology.

Epstein-Barr virus (EBV) is a form of human herpes virus that is the causative agent of mononucleosis. It is often associated with various types of human cancers, specifically lymphoproliferative disease (leukemia and hodgkins/non-hodgkins lymphoma), in immunosupressed patients. In the study immunodeficient mice infected with an EBV mutant missing a gene that controls cell lysis (the rupturing of the infected cell to release new viruses) did not develop LPD, however, when mice were challenged with EBV containing the lytic gene, development of LPD was enhanced. These results indicate that lytic gene expression contributes to EBV-associated LPD.

"Our results suggest that the decreased ability of immunosuppressed hosts to control the lytic form of EBV may promote the development of LPD not only by allowing enhanced horizontal transmission of the virus but also by increasing the number of lytically infected tumor cells," say the researchers.

New Gene Identified for Antiviral Activity

Thu, 17 Nov 2005 16:36:38 PST

( from http://www.rxpgnews.com ) Researchers have identified a gene in mice capable of producing an innate antiviral response to infection. Their findings appear in the November 2005 issue of the Journal of Virology.

The innate immune response, largely composed of the alpha/beta interferon system, is the first defense against controlling viral infections. These interferons are produced in response to viral infection and stimulate specific genes to produce antiviral compounds. These interferon-stimulated genes (ISGs) are responsible for many of the bodies' innate antiviral activities, but there are still some effects yet to be explained by the genes already identified.

In the study researchers used a modified Sindbis virus to express selected ISG responses in mice and looked for an attenuated infection. Through this approach they identified the interferon-stimulated gene 15 (ISG15) as having antiviral activity, protecting mice against mortality and decreasing viral replication in multiple organs.

"We show that expression of ISG15 in INF-á/âR mice attenuates Sindbis virus infection, providing in vivo evidence that ISG15 can function as an antiviral molecule," say the researchers.

Secrets to monoclonal antibody's success against West Nile Virus

Thu, 29 Sep 2005 20:53:38 PST

( from http://www.rxpgnews.com ) A monoclonal antibody that can effectively treat mice infected with West Nile virus has an intriguing secret: Contrary to scientists' expectations, it does not block the virus's ability to attach to host cells. Instead, the antibody somehow stops the infectious process at a later point.

"This was a complete surprise to us, but it gives us some very useful insights," says senior author Daved Fremont, Ph.D., associate professor of pathology & immunology and of biochemistry & molecular biophysics at Washington University School of Medicine in St. Louis. "Based on what we've learned, we are now developing therapeutic antibodies for related viruses that also are effective at stopping the process of infection after the virus attaches to host cells."

Detailed study of how the antibody physically binds to the virus has provided intriguing clues to how it may block infection. Scientists found evidence suggesting that the antibody prevents the virus from rearranging the protein envelope that surrounds its genetic material after it enters a host cell.

To reproduce, a virus must alter its envelope in order to inject its genetic material inside the cell. After that injection, the virus tricks the host cell into making more copies of the genetic material that can then be assembled into new viral particles or virions and sent out to infect other host cells and reproduce. But with the viral reproduction process blocked by the antibody, scientists suspect that the host cell eventually destroys the virion.

Fremont and colleagues, who publish their results in the Sept. 29 issue of Nature, hope to design a new diagnostic system that can determine whether vaccines for West Nile and related viruses undergoing clinical trials stimulate production of antibodies that stop infections at a similar point.

In 2004, West Nile virus, which is a mosquito-borne flavivirus, reportedly caused 2,470 infections and 88 deaths in the United States. First isolated in Africa in 1937, West Nile spread to the Middle East, Europe, and Asia before arriving in the United States in 1999. Most infections with the virus are mild or symptom-free, but infections in people with weakened immune systems and those over 50 sometimes lead to serious complications or death.

Like West Nile, dengue virus is a flavivirus spread by mosquito bites, but only in tropical regions of the world. The dengue virus is estimated by Centers for Disease Control and Prevention epidemiologists to cause100 million infections annually worldwide.

"Currently there are no effective and safe vaccines for pediatric dengue," says co-author Michael Diamond, M.D., Ph.D., assistant professor of molecular microbiology, of pathology & immunology and of medicine. "Thanks to our data from the West Nile virus antibody, we believe we now have a much better idea of how to evaluate vaccines for dengue."

Fremont and Diamond led a team of researchers at Washington University and Macrogenics Inc., a private company, that announced the identification of the effective West Nile antibody earlier this year. In a line of mice genetically altered to increase vulnerability to the virus, they found injection of the new antibodies could boost survival rates of mice infected with the virus to greater than 90 percent.

Scientists at Macrogenics are working on the preliminary studies required before the West Nile antibody can be tested in humans. Meanwhile, researchers at Washington University wanted to know why the new antibody was so effective.

Antibodies normally work by binding to invaders to flag them for consumption and destruction by immune system cells known as macrophages. In the prior study, which screened several potential West Nile antibodies, scientists found that all the most potent antibodies bound to a particular section of a protein that makes up the exterior of the viral envelope. The envelope of a single viral particle or virion is comprised of 180 copies of this protein.

For the new study, scientists determined the detailed structure of a single antibody bound to its envelope protein target region using the technique of protein crystallography. Scientists were able to affirm in greater detail earlier observations suggesting that the antibody will be therapeutic for all strains of West Nile Virus.

Based on this data, they predicted how multiple copies of the successful antibody would bind to a virion.

"We were startled to find that the antibody only seemed to be able to attach to 120 of the 180 copies of the target region in the complete viral envelope," says Grant Nybakken, a Washington University M.D./Ph.D. student who was lead author of the study.

Further tests showed that virions covered in infection-stopping antibodies could still bind to host cells, while antibodies that were less effective at stopping infection could more effectively prevent the virion from binding to host cells.

How does an antibody that's better at preventing the virus from binding to host cells actually turn out to be worse at treating infection? The key may lie in a theory known as antibody-dependent enhancement (ADE) of infection, which has been observed in test tube studies of dengue virus and may be important to the onset of dengue hemorrhagic fever.

This theory suggests that dengue and other viruses may have developed tricks that let them reproduce inside macrophages, the immune cells that normally consume and destroy any object that they find covered in antibodies. In effect, these tricks turn antibodies that should be death warrants into passes into cells where invaders can reproduce.

Fremont cautions that this phenomenon has not been seen in West Nile virus, but notes that when he and his colleagues tested the ability of several antibodies to prevent West Nile from reproducing inside macrophages, they found that only the therapeutic antibodies blocked the virus' reproduction. The therapeutic antibodies' ability to stop reproduction in macrophages even worked when the virions were simultaneously exposed to antibodies known to enhance infection.

"Do the therapeutic antibodies also prevent the virus from properly injecting its genetic material into macrophages? It's a tempting possibility, but we don't have the evidence to prove it yet," he says.

Flaviviruses use a novel mechanism to evade host defenses

Thu, 29 Sep 2005 06:23:38 PST

( from http://www.rxpgnews.com ) Researchers from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, have made the surprising discovery that flaviviruses, which cause such serious diseases as West Nile fever, yellow fever and forms of encephalitis, evade immune system defenses in different ways depending on whether they are transmitted by mosquitoes or ticks. This finding could lead to new approaches to developing vaccines and treatments against these illnesses.

"Flaviviruses exact an enormous toll in terms of illness and death worldwide," notes NIAID Director Anthony S. Fauci, M.D. "Because this is a relatively new field of study, everything we learn about how these viruses operate is significant. This elegant work opens an array of new questions and research opportunities to pursue as we strive to better understand this family of viruses and develop countermeasures against them."

Mosquito-borne flaviviruses include West Nile virus, yellow fever virus, dengue virus and Japanese encephalitis virus; the less-familiar tick-borne flaviviruses are just as serious, causing tick-borne encephalitis or hemorrhagic fevers. Currently, a Japanese encephalitis outbreak is raging in India and Nepal and has killed more than 1,000 people. In Europe and Southeast Asia, tick-borne encephalitis typically results in more than 10,000 patient visits to hospitals annually and has a fatality rate of up to 25 percent in some regions. Viruses that cause encephalitis lead to inflammation of the brain. Hemorrhagic fevers are viral infections that cause capillaries to burst, leading to unusual bleeding on or under the skin or in various organs.

The study released this week online in the Journal of Virology describes how a single virus protein--NS5--from the tick-borne Langat flavivirus counteracts the natural ability of interferon to combat the virus. Langat virus was originally isolated in the 1950s in Malaysia and Thailand. Langat virus can infect people following a tick bite, but there are no cases of natural disease recorded. In the 1970s Langat was briefly used as a live vaccine against more virulent tick-borne encephalitis viruses in Russia but caused encephalitis complications in about 1 of every 10,000 people.

Interferon, the body's first defense against many viruses, triggers a cascade of immune defenses. According to researchers at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, MT, NS5 blocks the body's attempt to signal for immune defenses, preventing the immune system from both stopping the spread of virus and helping the body recover from infection.

Interferon is so critical for recovery from these infections that it is being tested in clinical trials to treat infection with various flaviviruses. But the treatment appears to fail in about half of cases. Dengue virus, West Nile virus and yellow fever virus have a protein called NS4B that prevents interferon from functioning properly. It was thought that the tick-borne flaviviruses would use the same protein, so the NS5 finding was unexpected.

The RML group, directed by Marshall Bloom, M.D., chose Langat virus because it is spread by ticks--a trademark of RML expertise--and because it possesses the same survival mechanisms as the more serious tick-borne encephalitis, Omsk hemorrhagic fever (found in western Siberia) and the closely related Kyasanur forest disease (found in western India).

"These diseases are spread by the same tick that carries Lyme disease in the U.S.," says Dr. Bloom. "So, the fact that West Nile virus first appeared or emerged in the U.S. in 1999 should warn us about the potential for tick-borne flaviviruses to emerge on other continents." In preparation for such a development, Dr. Fauci notes that two other NIAID laboratories have similar flavivirus studies under way, and the three groups are building on the discoveries of each other.

Dr. Bloom says that all flaviviruses have a similar genomic structure, and many scientists thought they would use the same survival mechanism and respond to the same vaccines and therapies, but the RML work shows otherwise.

"NS5 prevents interferon from doing its sentry job and allows the virus to take over cells," says Dr. Bloom. "This is the first definitive study that dissects where the failure occurs in the signaling pathway, and then identifies some of the interacting partners in the cell and virus." Prior to this work, Dr. Bloom says, scientists knew only that NS5 helped tick-borne flaviviruses replicate.

RML's Sonja Best, Ph.D., who spearheaded the Langat virus work, says the group will continue to study tick-borne flaviviruses by examining the role and location of NS5 in Powassan virus. Powassan virus, found in North America, Russia, China and Southeast Asia, rarely infects people but is potentially fatal. If the research group can track the movement of NS5 in Powassan-infected cells and learn how it interacts with other proteins to block immune defenses, "that would provide a target for therapeutics to counteract tick-borne flaviviruses," says Dr. Best.

Adenovirus may deliver bird flu vaccine

Wed, 14 Sep 2005 01:45:38 PST

( from http://www.rxpgnews.com ) A harmless virus used as a delivery vehicle may help set a roadblock for a potentially catastrophic human outbreak of bird flu, according to researchers at Purdue University and the Centers for Disease Control and Prevention (CDC).

Purdue molecular virologist Suresh Mittal and his collaborators are investigating a new way to provide immunity against avian influenza viruses, or bird flu, the most lethal of which, H5N1, has a 50 percent fatality rate in humans. Under a $1.6 million grant from the National Institute of Allergy and Infectious Diseases (NIAID), the researchers are focusing on using a harmless virus, called adenovirus, as a transmitting agent for a vaccine to fight off highly virulent strains of the avian influenza viruses.

Current vaccines are designed for strains of flu found in local areas and are effective only as long as the virus doesn't change form. Existing vaccines will have limited success against new strains of avian influenza, he said. Every time a bird flu mutates, vaccines must be redesigned.

An additional important advantage to using an adenovirus as a vector, or transporter of vaccine into cells, is that is could be mass produced much more quickly than with current methods.

"Our approach is to use an adenovirus to deliver some components of the bird flu virus in a vaccine formulation," Mittal said. "We already know how to grow large amounts of adenovirus and how to purify it because adenoviruses already are used in clinical trials for gene therapy as vectors."

Mittal and collaborators Harm HogenEsch, co-principal investigator and head of Purdue's Department of Veterinary Pathobiology, and Jacqueline Katz and Suryaprakash Sambhara, both co-investigators from the CDC, are focused on creating better protection against the constantly mutating avian influenza viruses.

"The ultimate goal of our research is to develop an effective avian influenza virus vaccine that will provide long-lasting and broad immunity against multiple strains of this virus," Mittal said.

The proteins that form the basis for all of today's flu vaccines are grown in fertilized chicken eggs. It takes months to produce a new vaccine for a new virus strain using this method and limits vaccine supplies due to shortages in eggs for the purpose. The egg production method also creates difficulty in redesigning vaccine to keep pace with virus mutations.

"Do we still want to depend on the egg to make our flu vaccines?" Mittal said. "When these types of viruses strike humans, they also strike poultry. In that case, the availability of fertilized eggs to make enough vaccine will be compromised."

Even with a recently developed vaccine based on growing a single protein of H5N1, it would be difficult to rapidly produce enough protective medication to stem a pandemic, according to experts from the CDC, World Health Organization and NIAID. However, a large quantity of an adenovirus-based vaccine could easily be produced on short notice, Mittal said.

A "medium-level" bird flu pandemic in the United States would kill between 90,000 and 200,000 people with another 20 million to 47 million more sickened, CDC experts estimate. The economic impact on the United States alone would be between $71.3 billion and $166.5 billion.

Influenza viruses are respiratory pathogens responsible for widespread infections in humans, a variety of birds, marine mammals, pigs and horses. In Southeast Asia, millions of poultry have died or been euthanized because of H5N1 avian influenza.

Before the human cases of H5N1, there were no known cases of bird-to-human transmission that led to significant disease, Mittal said. However, this type of flu already has passed from poultry to people who had direct contact with ill birds or contact with contaminated bird feces, saliva or nasal mucous. A few cases have been documented of human-to-human infection, according to the World Health Organization.

In addition, migratory waterfowl at China's Qinghai Lake Nature Reserve have succumbed to H5N1. While migratory birds often can pass avian influenza to other animals, they generally don't become ill. Scientists from the U.S. Geological Survey National Wildlife Health Center believe this is further evidence that H5N1 could trigger a human flu pandemic. No cases of this type of bird flu have been found in the United States.

Since 2003 about 60 people have died in Southeast Asia from H5N1, about half of those afflicted. Outbreaks of H5N1 in birds have been confirmed in 11 countries, and the disease is spreading north into countries outside of Southeast Asia and already has been reported in Russia. The disease's spread, occurrences of other bird flu types and the already confirmed human cases, provide added impetus for finding more effective vaccine protection, Mittal said. The CDC reported two human cases of another bird flu in the United States in recent years - H7N2 in Virginia in 2002 and in New York in November 2003.

Viruses are classified according to the combination of two types of proteins found on the virus cell surface. The 15 types of hemagglutinin (H) protein and nine types of neuraminidase (N) protein form a large number of influenza viruses for which birds are the natural hosts.

New, often more dangerous, flu strains develop when the H and N combinations change. When the genes of a human or swine influenza mix with an avian variety, a highly pathogenic human flu likely will result, Mittal said.

These virus alterations work a bit like a jigsaw puzzle: As two viruses grow in the same animal, they replicate and reassemble their genome. If one virus takes a piece of genome from the other virus to fill an empty spot, then a new virus is born.

The potential for a pandemic exists when one of these new viruses is introduced into the human population. People with no previous exposure to this new flu strain have little or no immunity, making them highly susceptible to a virus that now can easily spread from person to person.

The last pandemic was 1968-69 when 34,000 Americans died of the Hong Kong flu (H3N2), a disease still circulating. In 1957-58 Asian flu (H2N2) killed 70,000 people in the United States. The worst flu pandemic was in 1918-19 when Spanish flu (H1N1) was fatal to 500,000 in the United States and as many as 50 million worldwide. Unlike other influenza outbreaks, the origin of that virus is still unknown.

Scientists believe that without the right vaccines and preparation, H5N1 has the potential to be one of the deadliest flu outbreaks if it mutates to easy human-to-human transmission. This flu strain was first identified in South Africa in 1961, with the first bird-to-human case recorded in 1997.

"Many people believe that this virus will mutate and become more widespread," Mittal said. "The question is when? Are we prepared? We hope that successful completion of this project will result in development of an adenovirus-based vaccine effective against pathogenic avian influenza viruses."

Coronavirus HCoV-NL63 associated strongly with croup

Tue, 23 Aug 2005 21:06:38 PST

( from http://www.rxpgnews.com ) A forthcoming paper in the international, open-access journal PLoS Medicine makes the strongest association yet between a newly identified virus and the pediatric respiratory disease commonly known as croup. Following their recent description of the coronavirus HCoV-NL63, Lia van der Hoek and colleagues suggest this is one of the most frequently detected viruses in children with lower respiratory tract infections (LRTIs). These infections are estimated by the World Health Organization to be responsible for one fifth of all deaths in children under five years old.

The team, including researchers from University Medical Centres in Amsterdam, Bochum and Freiberg, determined the incidence of this novel virus in a sample of children under three years old with such respiratory infections. Nine hundred and forty nine samples of nasopharyngeal secretions were collected from both hospitalized patients and outpatients in four different regions of Germany. The study found that forty-nine samples (5.2%) were positive for the virus HCoV-NL63 overall, with a greater incidence in outpatients (7.9%) than hospitalized patients (3.2%). Co-infection with two other viruses also known to be prominent in the cause of LRTIs, was also frequently observed.

The researchers also investigated the occurrence of HCoV-NL63 in cases of respiratory disease where no other virus could be detected. Here, a strong relationship with the clinical symptoms associated with croup was apparent: 43% of the HCoV-NL63 positive patients with high HCoV-NL63 load and absence of co-infection had croup, compared with 6% of HCoV-NL63 negative patients. Previous studies have reported trends in croup, such as the relative susceptibility of boys to the disease, its peak occurrence in the second year of life and its predominance in late autumn and earlier winter, that are matched by patterns of HCoV-NL63 occurrence.

This systematic association of croup with HCoV-NL63 is particularly timely as the newly identified virus has spread worldwide, reported in Australia, Japan, the US and Canada. The study will also contribute towards the clarification of the viral causes of lower respiratory tract infections generally; causes that have not always been apparent despite the frequency of such infections amongst children.

New Test May Simultaneously Identify Herpesviruses, Enteroviruses, and Flaviviruses

Thu, 18 Aug 2005 02:39:38 PST

( from http://www.rxpgnews.com ) Researchers from France may have developed a new method of simultaneously detecting viruses from three different families that cause diseases of the central nervous system in humans. Their findings appear in the August 2005 issue of the Journal of Clinical Microbiology.

Viruses afflicting the central nervous system are mostly caused by herpesviruses, enteroviruses, and flaviviruses. Human herpesvirus can lead to serious diseases such as encephalitis, myelitis, and meningitis. Eighty to ninety-two percent of aseptic meningitis cases are caused by human enteroviruses as well as several poliovirus serotypes. Tick-borne encephalitis and West Nile encephalitis are two of many viruses belonging to the flavivirus family.

In the study a new diagnostic tool that uses reactive primers to detect for each family of viruses followed by DNA probe technology to differentiate between virus species within each family was tested. Researchers were able to accurately identify herpesviruses, specifically herpes simplex virus type 1 and 2, all serotypes of human enteroviruses and five flaviviruses including West Nile, Dengue, and Langat virus.

"This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis," say the researchers.

Research team isolates receptor for deadly viruses

Sun, 31 Jul 2005 14:12:38 PST

( from http://www.rxpgnews.com ) A collaborative research team from the Uniformed Services University of the Health Sciences (USU), the Australian Animal Health Laboratory (AAHL) and the National Cancer Institute (NCI) have made a major breakthrough in efforts to combat two deadly viruses that could be engineered for use as bioweapons. The team isolated the functional receptor for the Nipah and Hendra viruses--naturally occurring and highly pathogenic paramyxoviruses for which no treatments or vaccines are currently available.

Christopher C. Broder, Ph.D., associate professor in USU's Department of Microbiology, and his NIH-funded team of researchers and investigators demonstrated that a cell surface protein called Ephrin-B2 is a functional receptor for both the Hendra and Nipah viruses. Many animal species are vulnerable to these viruses, making the potential for amplification in intermediate hosts and transmission greater. Ephrin-B2 is highly conserved in animals, and this finding sheds light on how these viruses can infest such a wide range of hosts.

"In addition to our concern about Nipah and Hendra viruses as emerging global health and economic threats, we worry about their potential use as bioterror agents," stated Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases, the arm of NIH that funded the research, in an NIH news release. "This work, funded through our biodefense research program, is a major step towards developing countermeasures to prevent and treat Nipah and Hendra viruses."

"Now that we've identified the cell receptor, we have a new target for activity, hopefully blocking the viruses from infecting cells," Dr. Broder explained. Team members Matthew Bonaparte, Ph.D., and Anthony Dimitrov, Ph.D., both at USU, identified the cell receptor by analyzing a human cell line that was resistant to virus infection against two susceptible cell lines. The results of the research were published in the July 26 edition of the Proceedings of the National Academy of Sciences.

"We identified genes that are coded for known and predicted cell surface proteins that were missing from the resistant cell line," Dr. Broder said. "The genes were put into cells that were then exposed to a live virus at AAHL."

Hendra virus was first isolated in 1994 when an outbreak of respiratory and neurologic disease emerged among horses and humans in Hendra, Australia, killing two people. Hendra recently reemerged in Queensland, Australia, and researchers there isolated the virus at the biosafety level 4 facility.

Nipah virus, which is similar to and in the same genus as Hendra, was initially isolated in 1999, when a large outbreak of encephalitis and respiratory illness occurred in Malaysia and Singapore, killing more than 100 people. Last year, two further Nipah virus outbreaks occurred in Bangladesh, killing roughly 75% of those infected. Scientists are disturbed by the fact that many of these recent cases involved human-to-human transmission of Nipah, which originates in bats.

Ephrin-B2 is found on cells in the central nervous system, as well as in cells lining blood vessels. It is essential for central nervous system development and blood vessel growth in the embryos of humans and other mammals.

Broder and his team are among only a handful of scientists focusing on the viruses, which have been under investigation by USU researchers since 2000. Broder is a principal investigator on one of six projects from the Mid-Atlantic Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases funded by NIH.

The research has led to two inventions on which USU and the Henry M. Jackson Foundation for the Advancement of Military Medicine have filed patent applications.

The first patent application, "Soluble forms of Hendra Virus and Nipah Virus G glycoprotein," covers the production and use of the soluble G glycoprotein. This protein has utility as a vaccine, in the development of pharmaceutical compositions and in diagnostic assays. The second patent application, "Compositions and Methods for the Inhibition of Membrane Fusion by Paramyxoviruses," covers the use of a novel peptide sequence of the soluble F glycoprotein, to block fusion of the virus with the host cell. This peptide can be used as a prophylactic, and/or to treat infections, and antibodies developed using this peptide can be utilized in diagnostic assays.

Mechanism that lets herpes simplex virus infect is discovered

Mon, 25 Jul 2005 16:11:38 PST

( from http://www.rxpgnews.com ) It's one of the most common viruses in America, and one that causes the most guilt and shame. It can get inside almost any kind of human cell, reproduce in vast numbers, and linger for years in the body, causing everything from recurrent genital blisters to sores around the mouth. Its complications can kill, and it may increase susceptibility to many nerve and brain disorders.

But until now, scientists haven't fully understood how the herpes simplex virus (HSV) manages to do all of this. And that has stood in the way of developing more targeted, effective treatments against it to help those infected.

New research from the University of Michigan Medical School may help change that.

An estimated 45 million Americans have genital herpes and millions more have the more visible oral variety. Once someone is infected, they're infected for a lifetime. New medicines for herpes infection are badly needed; currently, antiviral drugs can quell symptoms of an outbreak, but not eliminate the virus. And, there's increasing evidence that HSV may damage the nerve cells in which it hides between outbreaks, possibly contributing to neurological disease.

In a presentation Sunday at the International Congress of Virology and in two new papers in the Journal of Virology, U-M researchers are reporting the discovery of a receptor that appears to function as one "lock" that HSV opens to allow it to enter human cells. They've also found the gene that controls the production of that receptor, deciphered some aspects of the receptor's structure, and developed a pig-cell system that could be used to test new anti-herpes drugs.

The findings may help explain why the oral and genital herpes virus has such a successful track record: The receptor, dubbed B5, is made by most cells for another purpose not yet understood. HSV appears to have evolved a way to latch onto it, and fool the cell into letting the virus in. And since most cells express the gene for the B5 receptor, this may be a reason HSV can get into most kinds of cells.

"This may be one central part of the Achilles' heel in interactions of herpes virus with a cell to start infection. We can use the receptor molecule to try to understand the process and perhaps combat infection at this vulnerable site," says A. Oveta Fuller, Ph.D. the leader of the U-M team, senior author on the two papers and an associate professor in the U-M Medical School's Microbiology and Immunology Department. "While we're still a few years away from being able to use this new knowledge to find effective drug candidates, this is a very exciting confluence of discoveries."

The U-M holds a patent on the system and methods that the team used to make the discoveries.

Coincidentally, the U-M team's findings about the B5 receptor are being published at about the same time as an Italian team's reports about a possible 'key' on the herpes simplex virus surface that may match the 'lock' found by the U-M team. The Italian team has identified a region of a viral surface protein that matches the U-M team's predictions of what the virus likely would use to bind and engage the B5 receptor.

"It appears that B5 is a new class of viral receptor. Unlike other viruses so far, HSV seems to have evolved to take advantage of a broadly present cellular protein that has properties like that of known cellular fusion machinery," says Fuller. "No other virus has been shown to use a cellular fusion protein for entry into cells."

She explains that the search for the mechanisms by which HSV enters cells has been hindered by the fact that the virus is very good at entering so many kinds of cells. The many possibilities for virus binding to cells make deciphering the entry process a difficult problem to solve.

The gene that encodes B5 had in fact been sequenced, but not characterized, as part of the Human Genome Project. Discovering its role and studying the HSV entry mechanism was tricky and near impossible until Fuller's team discovered a type of pig kidney cell that isn't vulnerable to infection by human herpes virus. They searched the genome library to find genes essential to HSV infection, isolated the B5-coding sequence, and figured out how to get pig cells to express the human B5 protein to allow the pig cells to be infected with human herpes virus.

For these studies, Fuller credits the persistence of research team members in working with the genomic library and culture of human and pig cells, especially U-M doctorate graduate Aleida Perez and postdoctoral fellows Qingxue Li and Pilar Perez-Romero. Perez-Romero is first author of one of the two new papers, and a co-author on the other.

The two new papers show that the B5 receptor has important features that could explain why it is important to HSV's ability to fuse with the fluid membrane that encloses every human cell. The researchers were able to show that by placing only the DNA sequence that encodes B5 into HSV-resistant pig cells, they could make the pig cells susceptible to HSV. They were also able to block viral infection of both human cells and susceptible pig cells by adding to cell cultures a synthetic peptide made to mimic the structure of a smaller region of the B5 receptor. This peptide looks like a functional region of B5 and apparently interferes with virus engaging of the cell receptor.

The papers detail how the team isolated and characterized the gene that encodes B5, called hfl-B5, and used the DNA sequence to find out more about the protein structure of the B5 receptor. In the presentation at the International Congress for Virology, Fuller will describe recent findings that further confirm B5's importance in HSV infection.

The virology team reports that the B5 molecule appears to form a shape called a coiled coil. This intricately wound structure, they believe, may be similar to the structure of some fusion proteins of viruses and also to cellular proteins called SNAREs. Typically, SNARE proteins help cells to manage the fusion of membranes of vesicles inside the cell with other specific vesicles. Vesicles are tiny membrane-encased packets that encapsulate neurotransmitters, enzymes or other important substances and allow them to be transported within and between cells.

The researchers were able to show that B5 sits in the cell membrane with one end of the protein exposed outside of the cell ready to link up with viruses -- or to serve the receptor's "real" function, which still remains to be discovered. They also showed that HSV does not enter into pig cells that have an altered human B5 protein that is changed by mutations that affect a functional region important to forming a coiled coil.

"If B5 is a SNARE-like cell fusion receptor", Fuller says, "it may turn out to be useful for more than HSV drug treatment. It could act as a way to link vesicles containing drugs with cells, and deliver them inside". She is currently collaborating with U-M nanotechnology researchers on this concept.

The findings suggest that B5 or its viral ligand could be a target for antiviral treatment, much like cell receptors for the entry of human immunodeficiency virus (HIV) into cells have become targets for new AIDS drugs.

Why 2003 Monkeypox outbreak in the US wasn't deadly

Sat, 16 Jul 2005 00:10:38 PST

( from http://www.rxpgnews.com ) An outbreak of 72 cases of monkeypox in the United States during the summer of 2003 didn't produce a single fatality, even though the disease usually kills 10 percent of those infected.

Why did none of the patients die? New research from Saint Louis University Health Sciences Center and several partner institutions may provide an answer.

In this month's issue of Virology, researcher and senior author Mark Buller, Ph.D., from Saint Louis University Health Sciences Center and colleagues conclude that some strains of monkeypox are more virulent than others, depending on where in Africa the virus came from.

"We have at least two biological strains of monkeypox virus - one on the west coast of Africa, and the other in the Congo basin," Buller said. "The 2003 outbreak in the United States was from West Africa. If it had come from Congo, we might have had a bigger problem on our hands and very well might have seen patient deaths."

Researchers from the University of Victoria, Washington University School of Medicine, U.S. Army Research Institute of Infectious Diseases, the University of Alabama, East Carolina University and the Centers for Disease Control and Prevention are co-authors of the research.

Buller said that recent studies suggest the incidence of monkeypox is increasing due to encroachment of human into habitats of animal reservoirs. Monkeypox is classified as a "zoonosis," which means that it is a disease of animals that can be transmitted to humans under natural conditions. The first cases of monkeypox reported in humans involved contact between humans and animals in Africa.

The first outbreak of monkeypox in the Western hemisphere occurred in the U.S. Midwest from April to June of 2003. The virus entered the U.S. in a shipment of African rodents from Ghana in West Africa destined for the pet trade. At a pet distribution center, prairie dogs became infected and were responsible for 72 confirmed or suspected cases of human monkeypox.

"Unlike African outbreaks, the U.S. outbreak resulted in no fatalities and there was no documented human-to-human transmission," Buller said.

Monkeypox is part of a family of viruses that cause human smallpox, cowpox, and camelpox as well as monkeypox. Monkeypox usually produces a less severe illness with fewer fatalities than smallpox. But its symptoms are similar: fever, pus-filled blisters all over the body, and respiratory problems.

"Our finding may explain the lack of case-fatalities in the 2003 monkeypox outbreak in the United States, which was caused by a West African virus," Buller said.

Biologists see combined structure of Coxsackievirus A21 and ICAM-1

Wed, 13 Jul 2005 12:29:38 PST

( from http://www.rxpgnews.com ) Biologists at Purdue University have determined the combined structure of a common-cold virus attached to a molecule that enables the virus to infect its host, information that ultimately may help researchers develop methods for treating certain viral infections.

Coxsackievirus A21 infects host cells first by recognizing a "receptor molecule" called ICAM-1, which is located on the cell's surface, and then by anchoring itself to the molecule. ICAM-1 stands for intracellular adhesion molecule 1.

"ICAM-1 is the same receptor molecule used by the vast majority of viruses that cause the common cold," said Chuan Xiao, a doctoral student who is leading the research in the laboratory of Michael Rossmann, the Hanley Distinguished Professor of Biological Sciences in Purdue's College of Science.

Findings will appear in the July issue of the journal Structure.

"The real objective of this work is to study the whole complex of ICAM-1 and the virus as a single entity," Rossmann said. "Being able to characterize the combined structure of the virus and ICAM-1 will teach us how the virus recognizes a particular kind of molecule and how it then anchors to the cell, which represents the initial stages of infection."

Ultimately, researchers are trying to learn more about the binding mechanisms because such knowledge might eventually lead to new treatments.

"One of the many different ways of inhibiting viral infection is to stop the virus from binding to cells," Rossmann said. "That has not been our objective in this case. We just want to learn how this virus infects its host cell. In other words, how does the virus get into the host?"

Coxsackievirus A21 is one of several viruses that cause the common cold.

The researchers used two methods to learn the structure of the virus-molecule complex. One method, a technique called X-ray crystallography, yielded images of the virus with a resolution of 2.5 angstroms, which is nearly fine enough to see individual atoms. Using this technique, researchers create crystals of a substance, in this case the virus. Then, X-rays are passed through the crystals, creating a "diffraction pattern" that can be interpreted with various computational procedures to produce an image.

The other method, a powerful imaging tool called cryo-electron microscopy, was used to determine the entire three-dimensional structure of the virus-molecule complex. With this technique, specimens are first frozen before they are studied with an electron microscope. Cryo-electron microscopy enables scientists to study details as small as 8 angstroms, resolution high enough to see groups of atoms. An angstrom is one ten-billionth of a meter, or roughly a millionth as wide as a human hair.

"The electron microscopy is necessary for studying the entire complex because you can't crystallize the complex of ICAM-1 and the virus," Rossmann said. "That's because crystallization often takes days, weeks or months, but the complex is only stable for hours, which means it doesn't stay together long enough to crystallize."

The researchers pieced together the overall structure of the virus and ICAM-1 by combining the high-resolution X-ray crystallography images of the virus with the lower resolution electron microscope view.

The findings represent the first time researchers have seen fine details of the complex's structure.

"It's important to see the shape of the complex because that could tell us how the virus recognizes the host cell," Rossmann said. "Knowing the structure might also reveal the initial stages of what happens after attachment, and indeed there probably are different steps in the attachment process.

"The receptor apparently binds into what we call a canyon, which is a surface depression on the virus, and it might do that in at least two different steps. Perhaps it binds once loosely on the surface, and then it might bind again deeply into the canyon to strengthen its attachment."

The research paper was written by Xiao; Carol M. Bator-Kelly, a technical assistant in Rossmann's lab; Elizabeth Rieder, a technical assistant for Eckard Wimmer, a virologist at the State University of New York at Stony Brook; Paul R. Chipman, an electron microscopist at Purdue; Alister Craig, a researcher from the Liverpool School of Tropical Medicine in the United Kingdom; Richard J. Kuhn, a professor of biological sciences at Purdue; Wimmer; and Rossmann.

The images are revealing new details about how amino acids, which are the building blocks of proteins, interact during the binding of Coxsackievirus A21 and ICAM-1.

"In general, some amino acids have a positive charge, and some have a negative charge, and these opposite charges can play a critical role in attracting a virus to a host cell," Rossmann said. "It turns out the attraction between negative and positive charges on the virus and on the host cell seem to be a dominating feature but not the entire story of the recognition process.

"There is also shape involved, and the canyon on the virus and features on the ICAM-1 molecule have to match each other, like a key going into a keyhole."

Xiao said the research is ongoing, and future work may delve into how Coxsackievirus A21 binds to another cell-receptor molecule called DAF, or decay acceleration factor. The virus binds to both ICAM-1 and DAF at the same time, so future work may result in finding the structure of a complex that includes both molecules attached to the virus.

How Nipah and Hendra viruses gain entry into cells

Thu, 07 Jul 2005 18:04:38 PST

( from http://www.rxpgnews.com ) Working independently, two research teams funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), have identified how Nipah and Hendra viruses, closely related viruses first identified in the mid-1990s, gain entry into human and animal cells.

Nipah and Hendra are emerging viruses that cause serious respiratory and neurological disease. People can acquire these deadly viruses from animals. Beginning in 1994, public health officials have recognized disease outbreaks in Malaysia, Singapore, Bangladesh and Australia.

Both viruses use a protein essential to embryonic development to enter cells and begin their often-fatal attack, report researchers at the University of California, Los Angeles (UCLA) and the Uniformed Services University of the Health Sciences (USUHS) in Bethesda, MD.

The UCLA team, headed by Benhur Lee, M.D., describes its findings in a Nature paper posted online on July 6. The report by the USUHS researchers, led by Christopher Broder, Ph.D., is appearing online the week of July 4 in the Proceedings of the National Academy of Sciences.

The first reported outbreak of Nipah virus occurred in 1998-1999 in Malaysia, sickening 265 people and killing 105, according to the World Health Organization. This outbreak, which in this case spread from pigs to humans, was contained by culling more than a million pigs. Hendra virus, so far less of a threat to human health, was first identified in 1994 in Australia when it spread from horses to humans.

"In addition to our concern about Nipah and Hendra viruses as emerging global health and economic threats, we worry about their potential use as bioterror agents," says Anthony S. Fauci, M.D., director of NIAID. "This work, funded through our biodefense research program, is a major step towards developing countermeasures to prevent and treat Nipah and Hendra infection."

Using different methods, both teams identified a specific cell surface receptor, Ephrin-B2, as the doorway used by Nipah and Hendra viruses to get inside cells. This receptor is found on cells in the central nervous system and those lining blood vessels. It is crucial for the normal development of the nervous system and the growth of blood vessels in human and other animal embryos. Ephrin-B2 is found in humans, horses, pigs, bats and other mammals, which explains the unusually broad range of species susceptible to Nipah and Hendra virus infection.

Dr. Broder and his colleagues collaborated with researchers at the National Cancer Institute, also part of the NIH, and the Australian Animal Health Laboratory. The team narrowed the search for the Nipah/Hendra receptor by first sifting through the genetic sequences of 55,000 possible receptors using microarray technology as a molecular sieve.

The scientists compared microarray signals from the 55,000 genetic sequences in one set of Nipah virus-resistant human cells with microarray signals from three sets of human cells that the virus can infect. This enabled the research team to narrow the possible number of receptor proteins to 120 by identifying those present in the virus-susceptible cells but absent in the virus-resistant cells. They winnowed the possibilities further--to just 21--by selecting only those candidate receptors within the molecular weight range they expected. They selected 10 expressed at high levels in the susceptible cell lines and inserted them, one by one, into the cells that resisted Nipah virus to identify the receptor. When they inserted the gene for Ephrin-B2, the previously Nipah-resistant cells admitted the virus.

The UCLA team, with collaborators at the University of Pennsylvania, Philadelphia, took a different approach, using tools of advanced molecular biology as well as old-fashioned detective work to identify the Ephrin-B2 receptor. They knew the receptor would be abundant among the type of cells Nipah virus attacks, specifically, nerve cells and cells lining blood vessels.

To identify the human cell receptor, they created a bait: the Nipah protein that docks to the unknown receptor was attached to part of a human antibody, like a worm on a fishing hook. When they placed this bait onto cells susceptible to Nipah virus infection, it attached to a protein on the cell surface. When placed on Nipah-resistant cells, however, the antibody did not attach to the cells. The scientists used an instrument that sorts molecules by weight to identify that Ephrin-B2 was the cell receptor protein that bound to the antibody/Nipah protein "fishing pole" they had made.

They wanted to confirm their findings, but since they did not have access to a high-level biosafety laboratory as Dr. Broder's team did, the UCLA researchers engineered a harmless virus with Nipah virus proteins embedded in its coat. The UCLA team found that this artificial construct could infect cells vulnerable to Nipah virus but was unable to infect Nipah virus-resistant cells. They also showed that this engineered virus could infect nerve cells and cells lining blood vessels using Ephrin-B2 as the receptor, in the same way as actual Nipah virus would infect these cells.

Knowing the identity of the Nipah and Hendra receptor will not only help in developing vaccines and treatments, but also promises to lead to better understanding of how the viruses cause disease in people and a variety of animals, the researchers say.

The Role of the Rab7 Protein

Wed, 22 Jun 2005 13:04:38 PST

( from http://www.rxpgnews.com ) When Robert Hooke first looked at cork bark with a light microscope in 1655, he saw small empty chambers, reminiscent of monastery cells. We now know that living cells are full of organellesspecialized subcompartments surrounded by membranes in which different cellular life functions occur. This complex organization raises major transport and sorting problems similar to those encountered in a large city in which trains and trucks carrying different cargos arrive at peripheral distribution centers.

The cargos must be sorted and transported to individual factories where goods are made for delivery to other city destinations or for export. At the same time, the different areas of the city produce waste products that also need to be sorted and transported correctly. Somehow, thousands of cargos must end up in exactly the right place in both the city and the cell.

One cellular system that sorts and transports cargos is the clathrin-mediated endocytic pathway. Endocytosisthe ingestion of materials into the cellis important for the interaction of cells with the environment because it allows the uptake of nutrients (the equivalent of the raw materials brought into the city) and signaling molecules (the letters brought in by the mail service). In clathrin-mediated endocytosis, materials arriving at the outside surface of the cell are engulfed in special areas of membrane known as coated pits, which pinch off to form intracellular vesicles. These lose their clathrin coat and other molecules involved in their formation to become early endosomes, a specific sort of intracellular vesicle. The cargos are then transferred to late endosomes, which have different proteins and functions than early endosomes. From endosomes, cargo can go either to lysosomes, where they are degraded, or to the Golgi apparatus, which sends cargo back to the cell surface.

Although many details of clathrin-mediated endocytosis have been uncovered, cell biologists still hotly debate whether early endosomes mature into late endosomes or whether transport vesicles take cargos from early to late endosomes. Unraveling such details will improve our understanding of normal cellular processes and should help in the design of intracellularly targeted drugs. Andreas Vonderheit and Ari Helenius now provide new insights into this controversy by examining how Semliki forest virus (SFV) is sorted and transported to late endosomes.

Like many animal viruses, SFV enters its host cells using clathrin-mediated endocytosis. One well-established way to study this process is to attach a fluorescent tag to individual virus particles and observe their travels through the cell. Vonderheit and Helenius now track this journey in greater detail than ever before by attaching different colored fluorescent tags to SFV and to protein markers of early and late endosomes. They then use video-enhanced triple-color microscopy to follow all the markers as they move through living cells. This analysis reveals that the virus is initially present in endosomes containing only proteins associated with early endosomes. Then, Rab7, a late endosome marker that is involved in transport of cargo from early to late endosomes, appears in distinct domains of these early endosomes. Finally, the viral cargo is transferred to a detached organelle that contains Rab7 but no early endosome markers. The researchers show that SFV transport to late endosomes requires Rab7 and the presence of intact microtubules, which often serve as a highway network along which vesicles travel.

The researchers conclude that, at least for SFV, the mechanism underlying sorting and transport from early to late endosomes falls somewhere in between the two existing models for clathrin-mediated endocytosis. Early endosomes, they postulate, have to acquire some characteristics of late endosomes before SFV can be transported to late endosomes in Rab7-positive vesicles. But other cargos, the authors point out, may follow different pathways through the cell.

Virus Uses Tiny RNA to Evade the Immune System

Fri, 03 Jun 2005 16:52:38 PST

( from http://www.rxpgnews.com ) In the latest version of the hide-and-seek game between pathogens and the hosts they infect, researchers have found that a virus appears to cloak itself with a recently discovered gene silencing device to evade detection and destruction by immune cells.

The report by Howard Hughes Medical Institute (HHMI) researchers in an article published in the June 2, 2005, issue of Nature may be the first to show how a virus uses the gene silencing machinery for its own infectious purposes.

In people, plants, and worms, hundreds of tiny RNA molecules can silence specific genes by interfering with larger messenger RNAs (mRNAs). That interference prevents mRNAs from making proteins. Scientists do not know which genes are hushed by the microRNAs in people, but the new study bolsters growing evidence that the little molecules can play important roles not only in normal human cells but in infected cells as well.

A popular notion is that the whole system of generating small RNAs was designed to be a defense by cells against viruses. Our study shows that a virus can also adapt it to evade the immune response, said HHMI investigator Don Ganem, who is at University of California, San Francisco.

Ganem studies how viruses infect people and cause disease. When scientists found that RNA interference appeared to be a basic and widespread gene regulatory mechanism, it became clear that such a fundamental pathway could of course be pirated by a virus, said postdoctoral fellow Adam Grundhoff, co-first author of the paper.

Thomas Tuschl, a newly selected HHMI investigator at The Rockefeller University, had already reported the existence of several microRNAs encoded by Epstein-Barr virus, although their functions were unknown. Grundhoff and co-first author Christopher Sullivan, a postdoctoral fellow in Ganem's lab, started their search for viral microRNAs with a small virus, known as SV40, in the belief that its diminutive size would make it easier to understand the functions of any microRNAs they found.

SV40 is a relatively harmless monkey virus that can cause kidney infections in its natural simian host. In rodents, however, it can cause cancer. Although the SV40 genome has been found in some human tumors, its role in human cancer has been debated. The virus is better known as a model system that has greatly contributed to major scientific advances about how genes work.

To launch their study, Grundhoff wrote a computer program to screen the SV40 genome for possible microRNA precursors. MicroRNAs are made from messenger RNA molecules with distinctive hairpin folds. The hairpin structure is diced into a microRNA segment that works with another complex to disable other messenger RNAs with complementary sequences.

Among several dozen predicted microRNAs, the top candidate turned out to be abundantly expressed in human cells infected with SV40.

Sullivan soon found the target of the plentiful SV40 microRNA. It effectively targeted the messenger RNA for a protein known as T antigen, leading to its cleavage. SV40 may be the world's most studied virus, Sullivan said, and T antigen is its most studied part.

When SV40 enters a cell, it produces T antigen, which functions to trigger viral DNA replication. Unfortunately for the virus, T antigen also serves as a target for immune (T) cells, which can destroy infected cells and prevent the virus from spreading.

Conveniently, the microRNA that targets T antigen is made late in the infectious cycle, just when T antigen is no longer essential for virus replication. Further experiments showed that cytotoxic immune cells were more likely to kill cells infected with a mutant virus that cannot make the microRNA than the normal virus. Thus, microRNA-induced reductions in T antigen expression promote escape from antiviral T cells without affecting virus growth.

Viruses can use the host RNA inference machinery, which is often speculated to have evolved as an antiviral mechanism, to generate small RNAs that serve their own purposes the latest chapter in the long cat-and-mouse game known to virologists as host-virus coevolution, the researchers conclude in their Nature article.

Virus Subverts Cellular Defense for Reproduction and Escape

Wed, 27 Apr 2005 02:26:38 PST

( from http://www.rxpgnews.com ) Against the constant threat of infection by bacteria or viruses, one line of defense for the eukaryotic cell is the autophagosome. This double-membrane structure, which buds off from the endoplasmic reticulum, traps cytoplasmic intruders and, upon maturation, merges with a lysosome to destroy them. In this issue, however, Karla Kirkegaard and colleagues show that for one class of viruses, the autophagosome is not a holding cell but a breeding ground, and may even provide a novel escape route.

The viruses in question are picornaviruses, which include polioviruses and rhinoviruses. Infection of human cells with poliovirus is known to induce proliferation of double-membrane cytoplasmic vesicles that are morphologically similar to autophagosomes, but the origin and ultimate identity of these vesicles has not been resolved. To test whether these viral-laden vesicles are truly autophagosomes, the authors visualized two proteins: LC3, a specific marker for autophagosomes, and 3A, a part of the poliovirus RNA replication complex. After infection, these proteins colocalized, indicating the poliovirus was indeed within the autophagosome-like vesicles. LC3 also colocalized with LAMP1, a marker for lysosomes, indicating these vesicles mature in a manner similar to that of autophagosomes. This same effect could be induced simply by expressing two viral proteins.

All these results indicate that the virus stimulates production of vesicles that bear the traits of autophagosomes and contain the virus, but they dont indicate what the consequence is for viral replication. To determine that, the authors increased autophagosome production with two known stimulators of autophagy, tamoxifen and rapamycin. But rather than protecting the cell, this stimulation increased viral yield either 4-fold, in the case of tamoxifen, or 3-fold, for rapamycin. Conversely, inhibiting autophagosome production reduced viral yield. From these results, it seems the virus has subverted the components of the autophagy pathway for its own uses.

Inhibiting autophagosome production reduced viral yield inside the cell, but even more so outside. While they were not able to exclude other mechanisms, the authors argue that one possible explanation is that these vesicles are used by the virus to exit from the cell. Supporting this view, they produced electron micrographic images consistent with the fusion of the autophagosome with the plasma membrane and the extracellular release of its contents. This suggests that the virus, which is known to lyse cells to release new viral particles, has another, less lethal means of escape. This may increase the viruss chance of avoiding immune system detection as it infects new cells.

Monoclonal antibody cures West Nile Virus

Mon, 25 Apr 2005 19:58:38 PST

( from http://www.rxpgnews.com ) A newly developed monoclonal antibody can cure mice infected with the West Nile virus, scientists at Washington University School of Medicine in St. Louis report. If further studies confirm the effectiveness and safety of the antibody, it could become one of the first monoclonal antibodies used as a treatment for an infectious disease.

In a strain of mice that normally only has about a 10 percent survival rate after West Nile infection, scientists found that single doses of the antibodies given soon after infection could boost survival rates to 90 percent or higher.

"To our knowledge, these experiments are the first successful demonstration of the use of a humanized antibody as a post-exposure therapy against a viral disease," says senior investigator Michael Diamond, M.D., Ph.D., assistant professor of molecular microbiology, pathology and immunology and of medicine. "They also suggest antibody-based therapeutics may have a broader utility against other infectious diseases."

Diamond points out that Macrogenics Inc., of Rockville, Md., a company that contributed to the study and licensed the antibody from Washington University, must complete other preliminary studies before the antibody can be tested in humans. But he and his colleagues are excited both by the apparent potency of the antibody and its potential to help them explore new possibilities for treating related viruses that are more prolific causes of human disease and death.

"We could give a single dose of this antibody to mice as long as five days after infection, when West Nile virus had entered the brain, and it could still cure them," says Diamond. "It also completely protected against death from the disease."

Diamond and his colleagues will report their results in the May issue of Nature Medicine.

In 2004, West Nile virus reportedly caused 2,470 infections and 88 deaths in the United States. The mosquito-borne virus, first isolated in Africa in 1937, spread to the Middle East, Europe, and Asia before arriving in the United States in 1999. Most infections with the virus are mild or symptom-free, but infections in people with weakened immune systems and those over 50 sometimes lead to serious complications or death.

Scientists initially produced a panel of many West Nile virus antibodies from mouse cells. The human immune system would clear out these foreign antibodies quickly, so when they had identified a potent antibody, scientists at Macrogenics clipped out the genetic material that controls the antibody's targeting and cloned it into a human antibody. The "humanized" antibody should be less likely to induce an adverse human immune system response. A second round of tests in mice confirmed that the new antibodies retained their ability to stop West Nile virus.

Other monoclonal antibodies are currently in development or use as anti-cancer and anti-inflammatory treatments. An antibody against respiratory syncytial virus (RSV) is approved for use as a prophylactic treatment in children at risk of the disease in hospitals. Unlike the West Nile virus antibody, though, the RSV antibody has to be given prior to infection.

West Nile virus belongs to a family of viruses known as flaviviruses, several of which are spread by mosquito bites. Other flaviviruses include the virus that causes dengue fever, a potentially life-threatening infection prevalent in tropical cities. Centers for Disease Control and Prevention epidemiologists estimate that there are100 million cases of dengue worldwide every year.

"A lot of what we're learning from the West Nile virus antibody will be of consequence for the development of a pediatric dengue vaccine," says co-author Daved Fremont, Ph.D., associate professor of biochemistry and molecular biophysics and of pathology and immunology. "Currently there are no safe vaccines for dengue infections."

Important insights from the production and selection of the new antibody include a close fix on where the antibody binds to West Nile virus. Antibodies typically work by attaching to a piece of a foreign cell or substance, which causes immune system cells known as macrophages to pick up the substance and clear it from the body.

Binding to the invader is just the beginning of the battle, though. Some antibodies can bind to an invader but do so in a way that fails to slow the invader down or trigger a response from macrophages. In one rare case that involves the dengue fever virus, antibodies can adhere to the virus in a way that accelerates the infection.

From their initial pool of West Nile virus antibodies, researchers identified 46 that could bind to the West Nile virus' envelope (E) protein. Further testing showed that 12 could bind to the virus in a way that consistently neutralized it, shutting down infections in cell cultures and in mice.

To determine where these potently neutralizing antibodies were binding to the envelope protein, a task known as epitope mapping, researchers modified a yeast-based screening system. The system let them test individual antibodies for their ability to bind to many versions of the E protein, each with slight alterations. By analyzing the changes in the versions of the protein that antibodies had difficulty binding to, they isolated first a region of the E protein, known as domain III, and then a group of amino acids in that domain.

"The big surprise for us was that all of the potently neutralizing antibodies appear to recognize the same general region of this domain," says Fremont. "It was very consistentall the neutralizing antibodies that bind this domain adhere to that area; all the non-neutralizing antibodies that bind this domain adhere to different areas."

Fremont notes that while the E proteins of various flaviviruses are generally very similar, domain III can vary significantly. He and others are working to detail the precise mechanisms that allow the new West Nile antibody to block viral infection.

Diamond and Fremont are looking for other areas of the West Nile virus E protein that antibodies can bind to and neutralize the virus. Diamond is also using the yeast screening system to epitope map the sites on the dengue fever virus where antibodies can bind and inadvertently enhance infection instead of fighting it.

"We don't really understand on a molecular level what's happening in these cases, which are called enhancing antibodies," Diamond explains. "Epitope mapping may help us better understand this potentially dangerous interaction."

Genomes Offer Ecological Clues to Viruses

Tue, 19 Apr 2005 17:11:38 PST

( from http://www.rxpgnews.com ) Cyanobacteria have a long and checkered past. When their ancestors first appeared some 3 billion years ago, earths atmosphere likely contained mostly carbon dioxide, along with hydrogen sulfide, ammonia, nitrogen, and water vapor. Thought to be the first photosynthesizers, cyanobacteria forebears used water from their ocean habitat, carbon dioxide, and sunlight to make sugar, and produced oxygen as wastethe kiss of death for most ancient microorganisms, which eventually died from oxygen poisoning.

Modern cyanobacteria continue to exert disproportionate influence for their size. The Prochlorococcus group of cyanobacteriawhich measure in at less than a micron in diameter, allowing 500-plus individuals to fit comfortably on the head of a pinaccount for a significant fraction of global photosynthesis by virtue of their ubiquitous presence in nutrient-depleted ocean waters. Even tinier agentsthe viruses that infect these bacteria, called cyanophagesappear capable of wielding equally surprising influence on global cycles by affecting the population dynamics and evolutionary path of Prochlorococcus.

To better understand the nature of virushost interactions at sea, Sallie Chisholm and colleagues investigated the genetic makeup of three cyanophages. The marine phages resemble two terrestrial phagescalled T4 and T7that infect Escherichia coli but carry genes that appear specially adapted to infecting photosynthetic bacteria in nutrient-poor oceans.

Of over 430 completed phage genomes, only one (P60) infects cyanobacteria. Since marine phages likely face different selection pressures than their terrestrial equivalents, the authors explain, genome analysis can shed light on the agents of selection, besides providing a survey of marine phage types. Chisholm and colleagues chose to sequence three marine phagesone podovirus (P-SSP7) and two myoviruses (P-SSM2 and P-SSM4)based on their morphology and host range, and characterized their genomes. The P-SSP7 virus has genes that closely match many of T7s so-called core genessignature genes required for that viruss mode of infection, which involves killing its host. P-SSP7 also has the same genome structure as other T7-like phages, though it appears capable of coexisting with its host (based on the presence of an integrating enzyme) while T7 kills as it infects. Chisholm and colleagues go on to characterize the two myoviruses and find that both viruses share most of the core genes found in T4-like phages. And like T4 phages, both myoviruses lack the integrating enzymes, suggesting they share T4 phages homicidal approach to infection.

Beyond the core phage genes, Chisholm and colleagues also present a survey of genes likely derived from cyanobacteria that could play defining functional roles in marine phagehost interactions. All three cyanophages contain photosynthesis-related genes, some of which, the authors propose, may mean the virus helps the host maintain photosynthesis during infection. The podovirus also has a candidate gene involved in DNA synthesis, which the authors speculate might help the virus reproduce in nutrient-poor environments, and all three cyanophages carry genes involved in metabolizing carbon. The absence of such genes in terrestrial phages, the authors argue, lends support to the notion that marine phages have evolved different adaptive mechanisms in response to the ocean environment.

Given the intimate relation between virus and host, the effects of gene swapping between virus and host is likely to be a two-way street. Just as cyanophages may help shape the fate of their hosts, its likely that cyanobacterial genes influence phage ecology and perhaps even its range. The cyanophages characterized here take after two phages that were central to many fundamental breakthroughs in molecular biology, including the discovery that genes are made of DNA. It remains to be seen how the marine versions of these legendary laboratory viruses contribute to our understanding of phage infections in one of the most abundant, ecologically diverse primary producers in the open seas. See also the related Primer The Third Age of Phage (DOI: 10.1371/journal.pbio.0030182).

Tegument Proteins Help Route Herpesvirus

Tue, 29 Mar 2005 17:49:38 PST

( from http://www.rxpgnews.com ) Tegument proteins, which lie between the viral capsid and membrane envelope, route herpesviruses to either the cell bodies or axon terminals of neurons, according to Gant Luxton et al.

The α-herpesviruses, which cause cold sores and shingles, enter sensory neurons, where they take up lifelong residence. When the viruses become reactivated, the progeny virus particles travel down axons to the periphery, resulting in physical symptoms.

The viral proteins associated with the microtubule motors that allow this transport remain unknown. To determine which viral proteins were involved in trafficking, Luxton et al. used correlative motion analysis to simultaneously track fluorescently labeled capsid and tegument proteins in living neurons.

The researchers found that the tegument proteins are the key components of the capsid transport complex; when tegument proteins were associated with the capsid, the viral particles moved toward the axon (anterograde motion). Conversely, when tegument proteins were removed, viral particles moved toward the cell body (retrograde motion). Identifying tegument proteins as an important component of the capsid transport complex reveals a key mechanistic step in the infectious cycle of human herpesvirus.

How a Latent Virus Eludes Immune Defenses

Tue, 22 Mar 2005 20:50:38 PST

( from http://www.rxpgnews.com ) For a virus to survive, it must elude the ever vigilant immune sentinels of its host. A latent virus can escape immune detection if it resides in nondividing cells and doesnt produce any proteins. No viral proteins means no red flags for immune cells. If the virus targets one of the many cell types that rarely divide, its relatively safe while latent. But some viruses, like the gamma-herpesvirus, infect B cells of the immune system, which occasionally divide. The gamma-herpesvirus genome persists as circular pieces of DNA called episomes. When an infected B cell divides, the latent gamma-herpes virus episome must replicate and segregate into daughter cells along with the cells genome. Viral replication and segregation requires the services of a protein called the episome maintenance proteina potentially recognizable target for immune cells.

Gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposis sarcomaassociated herpesvirus (KSHV), can induce uncontrolled lymphocyte (immune cell) proliferation and result in lymphoma, Hodgkins disease, and Kaposis sarcoma. These diseases arise from the persistent latent infections that take hold after initial infections are controlled by immune defenses. The episome maintenance protein produced by EBV, called EBNA-1, harbors an amino acid element in its epitopethe region that binds to a T cell and triggers an immune responsethat helps the viral protein evade the killer T cells that could destroy it. Lab studies show that the amino acid element limits EBNA-1s interaction with T cells by inhibiting synthesis and, to a lesser degree, degradation of the protein. How this evasive action works or helps the virus in a living organism is not entirely clear. But if T cells arent presented with bits of viral protein, they have no way of knowing the virus is present.

In a new study, Neil Bennett, Janet May, and Philip Stevenson explore this question by studying virushost interactions in mice infected with the murine gamma-herpesvirus-68 (MHV-68). Though MHV-68 infects mice, it behaves similarly to EBV and KSHV infections in humans, producing an acute mononucleosis-like illness and a pervasive pool of latently infected B cells. The episome maintenance protein in MHV-68 and KSHV is called ORF73. None of the viruses can maintain latent infections with deficient episome maintenance proteins.

Stevenson and colleagues first demonstrated that ORF73 limits T cell recognition and then identified a key region responsible for immune evasion by modifying different regions of the viral protein. In the next round of experiments, the authors asked how the viral protein manages this feat. They discovered that ORF73 limits T cell recognition much like EBNA-1 does, by reducing synthesis and degradation of the protein. One region strongly associated with inhibiting epitope presentation to killer T cells corresponded to reduced protein synthesis. When the authors modified the ORF73 transcript to circumvent T cell evasion, the T cells wiped out latent virus. These results indicate that avoiding epitope presentation during episome maintenance is key to the viruss survival.

Interestingly, the MHV-68 episome maintenance protein mediates immune evasion even though it lacks the amino acid element that does the job for EBV. Future studies will have to determine the responsible MHV-68 epitope and the mechanisms that engineer immune avoidance. Since a majority of epitopes that killer T cells recognize come from aborted translation events, it may be that evasive action is taken at the RNA transcript stage, before RNA is translated into protein. Evading killer T cells, the authors argue, is key to the survival of the gamma-herpesvirus. By figuring out just how evasion occurs, scientists can identify a promising target for controlling infection.

Norovirus Prevalent in Those Suffering from Traveler's Diarrhea

Sat, 19 Mar 2005 15:06:38 PST

( from http://www.rxpgnews.com ) Norovirus may be the most common cause of travelers' diarrhea for United States citizens returning from Mexico and Guatamala say researchers from the U.S., Guatemala, Mexico and Sweden.

It is estimated that 20 to 50 percent of the people traveling to tropical areas of the world will experience traveler's diarrhea (TD). TD, defined as three or more loose stool movements in a 24-hour period, frequently results from exposure to bacterial or viral pathogens. Noroviruses (NoV's) are considered to be one of the leading causes of nonbacterial gastro-related illnesses resulting in over 23 million cases annually.

In the study stool samples were collected from 34 patients with traveler's diarrhea and tested for the presence of norovirus. The virus was detected in 65 percent of the samples tested, with time spent at travel destinations playing an important role in determining infection frequency.

"This study is the first of its kind to indicate that NoVs may be a major cause of illness among United States travelers who experience TD during extended stays in developing countries," say the researchers. The high frequency of NoV infection among TD cases examined in this study suggests that further investigations concerning the role of these viruses in TD are warranted."

New Test May Differentiate Between Poultry Vaccinated Against or Infected with Avian Flu

Fri, 18 Feb 2005 16:34:38 PST

( from http://www.rxpgnews.com ) Avian influenza (AI), a viral disease of poultry, causes a wide range of diseases affecting multiple organs and often resulting in death. In recent years, new strains of the virus have continued to emerge and cross over from the wild bird reservoir to domestic poultry. A new diagnostic test monitoring antibody response to the NS1 virus protein may allow for differentiation between poultry vaccinated or infected with avian influenza say researchers from Georgia.

"Over the last decade, AI viruses circulating in live-bird markets have provided a secondary reservoir from which influenza viruses have crossed over to infect commercial chicken and turkey operations," say the researchers.

A vaccine incorporating the inactivated whole-virus has been used in an attempt to prevent AI infection. While these traditional vaccines can protect against clinical infection and death, they can interfere with surveillance programs. Vaccination produces the same antibodies as an actual AI infection, and can commonly cause false positives with traditional diagnostic tests.

In the study, the researchers designated the NS1 virus protein, typically found in large amounts in virus-infected cells but not the virus itself, as a potential differential marker. They monitored both infected and vaccinated poultry for antibodies reactive to NS1. While live virus infection induced high levels of the NS1 antibody, commercial vaccines induced little or no antibody response.

"These results demonstrate the potential benefit of a simple, specific enzyme-linked immunosorbent assay (ELISA) for anti-NS1 antibodies that may have diagnostic value for the poultry industries," say the researchers.

Bat-CoV - New Coronavirus in Bats

Fri, 18 Feb 2005 16:30:38 PST

( from http://www.rxpgnews.com ) Transmission of animal viruses to humans poses a growing threat worldwide. The recent emergence of SARS, a coronavirus transmitted to humans from wild animals in live animal markets, reinforces the need for virus surveillance in exotic wildlife. Chinese researchers have identified a novel coronavirus found in bats.

Most coronaviruses are disease-causing agents associated with respiratory and gastrointestinal illness in humans and respiratory and neurological symptoms in animals. In the study respiratory and fecal samples were collected from twelve bat species of which three tested positive for the novel virus (Bat-CoV). All bats testing positive for the virus were healthy when physically examined so it remains unclear as to whether or not the virus is pathogenic in bats.

"Here, we report the identification of a novel bat coronavirus," say the researchers. "It is not known whether this virus would cause zoonotic disease in humans or other animals, but given the catastrophic consequences of SARS, further surveillance work on viruses in wildlife should be encouraged."

New Coronavirus Identified in Pneumonia Patients

Sun, 16 Jan 2005 13:26:38 PST

( from http://www.rxpgnews.com ) Researchers from Hong Kong have identified a novel coronavirus in patients suffering from pneumonia. Their findings appear in the January 2005 issue of the Journal of Virology. Coronaviruses are responsible for 5 to 30 percent of human respiratory tract infections largely due to their unique ability to replicate. Because so many cases of respiratory tract infections are reported each year, researchers are actively trying to identify new causative agents.

The new virus, labeled CoV-HKU1, was first identified in a 71-year old pneumonia patient that had just returned from China. Following the discovery, nasal samples were taken from patients suffering from respiratory illness, but negative for SARS and screened for the presence of CoV-HKU1. Samples taken from a 35-year old woman suffering from pneumonia were positive for the virus, supporting the identification of a new group 2 coronavirus.

"Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans," say the researchers. "Further clinical, seroepidemiological and phylogenetic studies would be required to determine the relative importance of CoV-HKU1 compared to other respiratory tract viruses in causing upper and lower respiratory tract infections, its seroprevalence, and the origin of the virus."

New Herpes Vaccine May be Ready for Human Trials

Thu, 23 Dec 2004 12:50:38 PST

( from http://www.rxpgnews.com ) New research suggests that a promising herpes vaccine may be ready for testing in humans say researchers from the National Institutes of Health and Harvard Medical School. Their findings appear in the January 2005 issue of the Journal of Virology.

Herpes simplex virus type 2 (HSV-2), or genital herpes, is a virus that infects approximately 22% of adult Americans. Bearing physical, psychological, and social effects on those who acquire it, it can pose an even more severe risk for immuno-compromised patients further emphasizing the need for an effective vaccine.

"In the aggregate, the burden of genital herpes has made development of more effective prevention strategies a health priority," say the researchers.

The study compared three different vaccines, a DNA vaccine, an antigenic vaccine and a live mutant strain of the type 2 virus, d15-29, in mice and guinea pigs. The live mutant strain, d15-29, showed minimal risk of causing disease as it is missing two of the genes necessary for replication and it stimulated a stronger immune response in both animals.

"Given its efficacy, its defectiveness for latency, and its ability to induce rapid, virus-specific CD8+-T-cell responses, the dl5-29 vaccine may be a good candidate for early-phase human trials," say the researchers.

(Y. Hoshino, S.K. Dalai, K. Wang, L. Pesnicak, T.Y. Lau, D.M. Knipe, J.I. Cohen, S.E. Straus. 2004. Comparative efficacy and immunogenicity of replication-defective, recombinant glycoprotein, and DNA vaccines for herpes simplex virus 2 infections in mice and guinea pigs.)