Immunology, Molecular Virology, Vaccines

Can miRNAs help further attenuate influenza A vaccines?

Contributed by Guest Blogger: Brittany Sider ’11

MicroRNA (miRNA) molecules, first characterized in the early 1990s, have been implicated in a variety of different biological mechanisms. It took almost a decade for researchers to detect and understand the role of miRNAs in regulation of translation. Since then, research has focused on how we can scientifically manipulate these regulating molecules to our advantage in order to further understand biological underpinnings of certain diseases, as well as potential miRNA-based therapies.

The ability of the influenza virus to undergo frequent and substantial genomic mutations forces us to continually monitor its prevalence, and modify yearly vaccines to target the prevailing viral strains. Recently, live attenuated influenza vaccines (LAIVs, e.g. FluMist) have been proven effective, and have been distributed to a large portion of the eligible population to combat the seasonal flu. These vaccines are manipulated to become much more temperature-sensitive, and therefore are only capable of replicating in temperatures found in the nose. The inability of these attenuated viruses to replicate in the respiratory tract (due to higher temperatures) allows the vaccinated individual to produce antibodies to the influenza strains in the vaccine from the infection in the nasal passage. Therefore, the individual can produce the correct immune response without the virus spreading to the respiratory tract and causing symptoms.

In 2009, a group of researchers from Mount Sinai School of Medicine found that using microRNA response elements (MREs) can supplement the effectiveness of LAIVs. In the study, the MREs for the miR-124 (neural tissue-specific) and miR-93 (a ubiquitous miRNA) were inserted into open reading frames of influenza A nucleoprotein coding regions. The investigators vaccinated mice with miR-93-seeded strains, and then inoculated them with a lethal dose of influenza A/PR/8/34 H1N1 21 days later. This resulted in 100% survival of the subjects, as well as a robust immune response. In an attempt to attribute these results to other influenza strains, the same experiment was done with H5N1 (MREs were inserted into the vaccine specific for H5N1, and methods were repeated). Subjects who had received mock vaccinations 21 days prior to being inoculated with H5N1 displayed rapid weight loss, as well as 100% mortality. On the other hand, mice that had received the MRE-containing H5N1 strain did not display any signs of disease. Furthermore, serum from these subjects exhibited neutralizing activity against the wild-type H5N1, and a wide array of antibody responses (high levels of IgM, IgG1, IgG2a and IgG2b).

The results from this study lead the researchers to believe that MRE-containing LAIVs can be used, and potentially be even more effective than currently available LAIVs in protecting against influenza A outbreaks. In addition, this technology provides the potential to control for the degree of attenuation of the vaccine by manipulating the number of MREs/miRNAs. Lastly, FluMist – although proven to be equally as effective as injected vaccines – has some age exclusions. Perhaps the addition of MREs/miRNAs could expand the target demographic of this method of vaccination.

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Antiviral Drugs, Guest Blogger, Molecular Virology

A different alphabet, a different treatment?

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Contributed by Guest Blogger: Sean Koerner ’11

It’s easy to think of viruses as alien or lifeless – after all, they can’t reproduce on their own, eat anything, or even move around without assistance. However, viruses have evolved to use the same toolbox that human cells use, right down to the way their genes and proteins are encoded. One of the most problematic viruses for humans, HIV, works by putting its own information into our cells’ genomes, turning host cells into viral factories. This information is formed from two types of alphabets: strung-together sequences of deoxyribonucleotides, which exist intracellularly as deoxyribonucleotide triphosphate (dNTP) monomers in our own cells and ribonucletides, which form the HIV genome as well as existing independently as ribonucleotide triphosphate (rNTP) monomers within our own cells. In order to infect our cells, HIV uses a protein known as reverse transcriptase to generate the DNA that our cells are used to reading from the viral RNA genome. This reverse transcription of RNA to DNA has long been a target of anti-HIV drugs, since without this step, HIV cannot successfully infect our cells.

Recently, a team at the University of Rochester discovered a previously unknown characteristic of this process. Two of the cells most commonly infected by HIV, CD4+ lymphocytes and macrophages, displayed different levels of dNTPs and rNTPs after being infected by HIV, with the lymphocytes containing much less rNTPs and more dNTPs than the macrophages. After a biochemical analysis of the cells, the research team discovered that HIV’s reverse transcriptase is capable of using cellular rNTPs to generate RNA based upon the HIV genome, which is then reverse transcribed into cellular DNA while in the macrophage environment. This allows HIV to use the higher concentrations of rNTPs in macrophages to continue replicating efficiently, despite the relative dearth of dNTPs as compared to lymphocytes. Since HIV uses one method (dNTPs) in lymphocytes and one method (rNTPs) in macrophages, it may be possible to target HIV replication in macrophages specifically. Why care about the difference between the two cell types? Well, macrophages travel the body much more rapidly than lymphocytes; if we can stop HIV infecting them, we may be able to slow the progression of HIV infection throughout the body.

How could we do that? In short, by targeting the synthesis of rNTP strands with new drugs. Although we would likely experience side effects, they could be negligible compared with the repression of HIV. The research team at Rochester have already demonstrated that rNTP string inhibitors slow HIV’s infection of macrophages, so specific drugs targeted for this process might be able to halt it altogether.

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Epidemics, Uncategorized

Hey, I wonder what’s in bat poop?

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I know you were thinking the same thing! Bats are suspected reservoirs for several zoonotic viruses that can cause significant disease in humans or other animals. These include the dreaded Ebola virus, Nipah virus (which causes outbreaks of encephalitis in South East Asia), Hendravirus (which causes disease in horses) and several others. So knowing what viruses are carried by bats will be important in understanding emerging zoonoses.

Several studies have identified a diverse array of viruses in bats, but using next-generation sequencing it is now possible to investigate the population of viruses carried in bats to a much deeper level. In a study published last summer, guano from bats in California and Texas was collected by placing plastic sheets below the bat roosts. The individual roosts were occupied by as many as four different bat species, so the guano collected was a mix from the different inhabitants. To isolate viral DNA and RNA, the samples were filtered to remove cells then treated with nucleases to destroy any free DNA or RNA, leaving only encapsidated viral genetic material.

In the sequenced “virome” or population of viruses in the samples, only 51-39% of the sequences(depending on collection site) had matches to genbank sequences. So once again, viromic sequencing shows us how little we know about the viral world. Of those sequences that matched known sequences, most were insect and plant viruses. The bats are insectivores and the insects are herbivores, so you can see the viral populations from each link in the food chain. Only a very small proportion of the virome was of bacteriophage origin, much less than other viromic studies in humans and horses, although its not clear why there would be such a difference. Among mammalian viruses, which made up less thatn 10% of the sequences, there were adenoviruses, coronaviruses, parvoviruses, circoviruses, astrovirsues, picornaviruses and even poxviruses. Most of these sequences only matched less than 60% to known mammalian viruses however, so its unlikely that they pose a zoonotic threat.

As researchers continue to sequence viral populations, we keep seeing mostly novel sequences, something that has decreased in bacterial and eukaryotic sequencing. That tells us we have a lot more sequencing to do if we want to understand global viral diversity. In bats however, the major question is not so much about the diversity but the threat of zoonoses. It will be interesting to see the guanome of bats in areas where zoonoses are a real problem, and I wonder if this will be a technique useful to monitor the threat of emergent diseases as the cost of high throughput sequencing continues to drop.

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Epidemics, Evolution, Immunology

Virus and Parasite Unite

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Contributed by Guest Blogger: Joseph Zaino ‘11

Recent research has found a unique relationship between the intracellular parasite, Leishmania, and it’s corresponding Leishmania RNA virus-1 (LRV1). Ives et. al. concluded that Leishmania parasites, in the presences of LRV1, suppressed the host immune response and strengthened the pathogen’s persistence. Leishmania infects the human immune system by attacking macrophages. The parasite causes the infection known as leishmaniasis, which is typically transmitted by sand files. This is a serious infection, affecting an estimated 12 million people in the Mediterranean basin, Africa, the Middle East, Asia, Central and South America. The strain of parasite investigated by this study was mucocutaneous leishmaniasis (MCL). MCL destroys the soft tissues of the face and nasopharyngeal regions, as well as damages host immune responses.

Leishmania parasites are dependent on proinflammatory protein mediators called Toll-like receptors (TLRs). TLRs are found in intracellular vesicles of the macrophage- presumably the same vesicles that host Leishmania. Ives et. al. confirmed that TLR3-TRIF dependent pathways are essential for macrophage infection by Leishmania. The unusual part is that TLRs usually help the mammalian immune system to eliminate pathogens. Specifically, TLR-3 recognizes the double stranded RNA of many viruses that are released from dead parasites, unable to survive within their host. Observations found that between virally infected and non-virally infected Leishmania, the virally infected ones were more likely to successfully infect a host. Similarly, metastasizing parasites had greater levels of the LRVI virus than non-metastasizing parasites. The authors verified this finding by treating macrophages with purified LRVI, and observing the same phenotypic infection as the viral-infected Leishmania. Further models concluded that when TLR3 is deleted from macrophages, parasitic persistence was diminished.

This apparent mutualism seems to benefit both Leishmania and the virus by allowing a more successful rate of host infection. Many Leishmania species have lost RNAi interference pathways, allowing viruses to inhibit and replicate within them. In this case, the virally infected parasite is more persistent against macrophages, and more damaging to the mammalian immune system. Thus, it is advantageous for the parasite to coexist with the LRV1 virus. If severe MCL infections are contingent on LRV1 for infection, then future research can perhaps focus on this relationship in order to better understand and cure leishmaniasis.

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Epidemics, Guest Blogger

Chicken Anemia Virus and its Similarities to Human Anelloviruses

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Contributed by Guest Blogger: Maggie Rasnake ’11

When a virus is not known to be associated with any disease, it is called an orphan virus. Human anelloviruses, like torque teno virus (TTV) and torque teno mini virus (TTMV), are orphan viruses because they do not have known symptoms. TTV was first discovered in a patient with liver disease. However, no definite link between liver disease and the virus has been shown. Anelloviruses are genetically similar to an avian virus called chicken anemia virus (or CAV). CAV has had a large, economic impact on the poultry industry. Unlike TTV, it is known to have symptoms, but it can have a long lag-time between infection and the development of disease.

Both CAV and TTV have similar, single-stranded, circular DNA and have highly variable sections of the genome. It is believed that they evolved from a plant virus. Researchers realized that much of what they learned about CAV could be applied to TTV and vice versa. For example, when they realized that TTV had more than just three proteins encoded by its three open reading frames, they found that the same was true for CAV. When CAV was found to replicate in the bone marrow, it was discovered that a great deal of TTV replication occurs in the bone marrow as well.

CAV is associated with developmental problems for fetuses and young chickens. The virus is less understood in adult chickens, but when chickens have CAV, they are much more likely to suffer from other diseases and have higher mortality rates. Similarly, in infected humans, the viral load of TTV is higher when the individual has other infections. In addition to liver disease, levels of TTV tend to be higher in those with respiratory infections, kidney disease, HPV, and certain cancers, among others. TTV may enhance the pathogenic effects of other pathogens. High levels of TTV are found in individuals with HIV, but it is not known if TTV simply reflects the immune system’s status or if it contributes to the damage. An effective medium for studying TTV has not yet been established. The authors suggest that the virus might be better studied in a novel primate cell line transformed by an oncogenic virus.

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Guest Blogger, Immunology, Vaccines

Eat Your Vaccines

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Contributed by guest blogger: Nicole Engelhardt ’11

Usually when you get a vaccine it means you get a needle and a bandage. Not only that, but you get an attenuated virus. These weakened virus particles are strikingly similar to viable ones; they even infect cells. Because of their weakened state, they infect slower than natural virus particles, giving the body time to react. However, people who have weakened immune systems can still exhibit symptoms as if they were infected by the natural virus.

But a new tool may make this issue obsolete. What really matters when it comes to a vaccine is the shape of the particle, not the contents. The shape is recognized by B-cells in the body which then reproduce creating antibodies that attack all of the virus particles. However, these B-cells are very specific and very picky. Normally, it makes sense to use a weakened virus because it has the exact same shape as a normal virus and your B-cells will react to the vaccine as if it were the real thing. Is there any way, then, to produce the exact shape of the virus and therefore the correct antibodies without having the harmful side effects?

This paper explores the rotavirus particle which is the leading cause of gastroenteritis in the world. In some parts of the world, gastroenteritis can be deadly for many children. As it happens, the shape of the rotavirus particle can be mimicked almost exactly in plants. The shape of this virus is a capsid made out of proteins. First, the authors take the genes that code for the capsid proteins and insert it into the genome of the plants. Then the plants express the viral genes, creating the virus capsid proteins inside the cells of the plants. More incredible than that, these proteins self-assemble into the exact shape of the rotavirus capsid. Now you have a plant containing just the shell of the virus!

The experiments are still in their early stages, but when mice were fed these plants, the authors found they were producing the same antibodies that are produced when mice are actually infected with rotavirus. This bodes well for future research in humans. Once the antibodies are created, the severity of future infections is greatly decreased. If these transgenic plants do work, it could mean a safer and perhaps more affordable form of the vaccine that could help people the world over fight rotavirus before it can infect.

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Epidemics, Guest Blogger

The Relationship Between Diabetes and Enteroviruses

Contributed by Guest Blogger: Charlie Gray ‘11

Enteroviruses are a genus of positive sense, single-stranded RNA viruses which include poliovirus, coxsackie A & B, echovirus, and enterovirus. These viruses can cause a variety of symptoms ranging from the common cold and conjunctivitis to paralytic poliomyelitis. Researchers have also found an association between enteroviruses and type 1 diabetes, a disease whose incidence has increased over the past 25 years at an annual rate of 3%, a rate that cannot be explained simply by genetics.

In a recently published paper, Wing-Chi G Yeung and his colleagues conduct a systematic review of controlled studies that use molecular virological methods in an effort to compile what is currently known about the association between enteroviruses and type 1 diabetes, and to aggregate their results. Their meta-analysis included 34 papers, 30 of which used reverse transcriptase PCR or in situ hybridization to detect the enterovirus RNA; the other four used immunostaining for the enterovirus capsid protein, vp1, on autopsy pancreas specimens. Although the studies varied in age distribution, most investigated children and adolescents (i.e. less than 16).

Yeung and his colleagues found a strong association between enterovirus infection and diabetes, with a 9 fold increase in the risk of infection in diabetic individuals. They conclude that their meta-analysis of these previous observational studies do provide support to the growing collection of findings that individuals with type 1 diabetes have increased odds of suffering from an enterovirus infection.

Despite Yeung et al.’s findings, there was quite a bit of variation in the designs and methods used in the various studies that the authors analyzed. Only 10 of the 34 studies matched for three or more potential confounding factors such as age, genetic risk, geographical location and sampling time. In addition, the studies varied greatly in the site selection from which they collected samples (e.g., serum, stool, throat swabs). Enteroviruses invade cells and replicate at mucosal surfaces; therefore, detection rates could be significantly higher in samples that were obtained from the gastrointestinal tract.

Although this paper does provide evidence for a diabetes-enterovirus link, it does leave several questions for future research. It is unclear how strong the association between enterovirus infection and diabetes is, and if the other factors such as geographic location and genetics may influence the observation of enterovirus infection and diabetes. For example, previous studies have examined varying HLA (a gene encoding an important immune system protein) genotypes and how certain genotypes can modify the association between enterovirus infection and diabetes; however, those results have provided conflicting evidence. Therefore, further study is needed to determine how these confounding factors affect one another and the enterovirus-diabetes link.

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Antiviral Drugs, Immunology

A cell model of HIV latency for finding novel small-molecule therapeutics

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Contributed by Guest Blogger: Jack Bulat, ’11

Highly active antiretroviral therapy (HAART) has extended the quality and expectancy of life for people infected with HIV-1, but has been unsuccessful in leading to a cure for AIDS. This is because it proves ineffective at targeting the latent HIV-1 reservoir – a pool of memory CD4+ T cells in the quiescent phase of the cell cycle that harbor inactive integrated virus. Should an HIV-infected patient ever come off HAART, activation of this latent pool would cause the virus to re-emerge. Because HAART has become both expensive and toxic in the long-run, significant efforts have been directed at targeting HIV-1 latency for more effective treatment.

A considerable obstacle to studying HIV-1 latency in memory CD4+ T cells has been the lack of a latency cell model. Because only a small portion of CD4+ T cells infected with HIV-1 survive to become latently-infected memory cells, a resilient cell line mimicking latency has practical value for therapeutic screening. In a study, Yang and colleagues transduced primary CD4+ T cells with a lentiviral vector for constitutive expression of Bcl-2, an antiapoptotic signaling factor implicated in the generation and maintenance of memory CD4+ T cells. Upon confirming that the physical and biochemical properties of these Bcl-2-expressing cells are highly similar to those of freshly-isolated primary resting CD4+ T cells, they activated and infected the cells with an HIV-1 strain mutated to mitigate cytopathic effects. After establishing latency in the infected cells, the researchers screened more than 4400 drugs and natural products for the ability to activate the latent HIV-1 mutant. 5-hydroxynaphthalene-1,4-dione (5HN), a compound found in the leaves, roots, and bark of the black walnut tree, was a promising hit because it did not cause global T cell activation, which would be too dangerous for clinical use.

Despite this, it looks like 5HN will not be hitting the pharmacy shelves any time soon, since it is chemically reactive, affects several cellular proteins, and leads to the stimulation of inflammatory genes. Nevertheless, the study is significant for presenting a methodology for generating potentially useful cell lines modeling HIV-1 latency. A noteworthy criticism has been that a mutated strain, rather than wild-type virus, was used to infect the model cells. The scientists contended that the strain is suitable to study latency specifically because the genes implicated in HIV-1 activation were not modified.

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Epidemics, Guest Blogger

The Role of Social Networks in H1N1 Transmission Within a School

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Contributed by Guest Blogger: Aaron Grober ’11

The H1N1 subtype of the Influenza type A virus, known colloquially as “swine flu,” was the most common cause of human influenza infection in 2009, and remained a major concern in sparking a pandemic throughout the 2009/2010 flu season.

This recent paper examines the role of grade, class, and social network in transmission of this virus in a school setting. Taking a closer look at the actual transmission pattern of this novel subtype of influenza is critical in developing models to better predict and combat pandemic spread. In the case of this school, closure due to outbreak did not significantly affect transmission among students, indicating that it may have occurred too late to be effective, stressing the importance of more exact models. The study encompassed 370 students from 295 households, surrounding an H1N1 pandemic that occurred in a Pennsylvania elementary school in April and May 2009.

The researchers found that the structuring of the school into grades and classes significantly affected the probability of transmission: 3.5% between students within a class, five times less than that between students of the same grade but different class, and five times less than that between students of different grades.

The researchers took an in-depth look at fourth-graders. They note that children are four times more likely to play with members of the same sex, and found that this behavior had a significant impact on disease transmission; the onset of epidemic transmission occurred among boys significantly before that of female classmates. In addition, they found no significant difference between recorded playmate transmission rates, and the expected proportion for if being a playmate was not a risk factor. The researchers used class seating charts to determine if proximity to an infected individual affects the risk of transmission; as it turns out, they found that sitting next to an infected individual did not significantly affect one’s risk.

In addition to school structure, the researchers looked at spread within households. The probability of a child to adult transmission within a household depended significantly on the household size, where probability of spreading the disease is much lower in larger households than smaller ones. The predominant means of adult infection was from outside the home.

These unique findings shed light on the extremely complex transmission pattern within structured populations. The biggest factors for transmission within school are grade and class, but not seating arrangement, sex, but not playmate transmission. A number of obvious questions remain: Why does sharing a class, but not a desk-space affect transmission? Why is one more likely to transmit the disease in a smaller household than a larger one? This study is an extremely insightful epidemiological tool to help explain transmission, but our knowledge of how this virus spreads remains incomplete; it seems that the flu is far more complex than we imagined.

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Epidemics, Guest Blogger

Feeling tired all the time? You might have a virus.

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Update (Janyary 2012): Two studies identifying XMRV in CFS patients have been retracted, including the original paper that proposed the association. The current, best supported evidence, in this area suggests that the association was actually due to contamination. There appears to be strong scientific agreement that CFS is not related to infection with this virus.

Contributed by Guest Blogger: Nicole Krenitsky ’11

Patients with Chronic Fatigue Syndrome (CFS) perked up when a paper published in Science in 2009 linked the symptom-defined illness to xenotropic murine leukemia virus-related virus (XMRV). XMRV, also connected to prostate cancer, is positive sense, single-stranded RNA retrovirus of the class murine leukemia viruses (MLV). Four subsequent studies failed to find any MLV-related viruses in CFS patients or controls. Then in 2010, a paper published in PNAS reinvigorated the debate. The study did not specifically find XMRV but did find MLV-related viruses in the blood cells of CFS patients tested. Yet one month later, the CDC published report, stating that they had not found any MLV-related viruses in their own study of CFS patients.

Reasons for the inconsistent results are presently unknown. One hypothesis is that the PCR could have picked up mouse DNA or mouse viruses, contaminating the tests and producing false positives. Another involves the samples of participants; CFS is diagnosed solely based on symptoms and clinical case definitions such as the one published by the CDC do not differentiate well between CFS and depression, resulting in overdiagnosis. A study conducted by Ian Lipkin is underway and seeks to standardize sampling and analysis methods and use a larger sample size to settle the controversy.

Relating CFS to a virus has far-reaching consequences for patients and for public health. Antiretrovirals used to treat HIV have been shown to inhibit XMRV replication in vitro and some CFS patients have already begun ART following the 2009 Science publication. The AABB and the American Red Cross, erring on the side of caution, have banned patients with CFS from donating blood erring on the side of caution. If CFS is caused by MLV-related viruses, the blood supply would be tainted the syndrome passed to transfusion recipients.

One million Americans are affected by CFS and experience sleep disorders, cognitive difficulties, chronic muscle pain and headaches. Many dismiss the disorder as psychosomatic and doubt its legitimacy as an illness. In addition to diagnostic testing and finding clinical treatment or a cure for CFS, a link to a virus would give CFS scientific credibility. Mary Schweitzer, historian and CFS sufferer explains, “Patients are hopeful that now the disease itself might be treated seriously, that they’ll be treated seriously, and that there might be some solution.”

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Teaching and Research on the Microbial World in the Liberal Arts

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