Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (K_A = 3.40 × 10⁸ m⁻¹) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (K_A = 3.40 × 10⁵ m⁻¹), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely ³²SFMPK³⁶ (denoted as F1 binding) and ²⁰LDKDLFTEEQ²⁹ (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.     

Assessing the Viral Fitness of Oseltamivir-Resistant Influenza Viruses in Ferrets,Using a Competitive-Mixtures Model

To determine the relative fitness of oseltamivir-resistant strains compared to susceptible wild-type viruses, we combined mathematical modeling and statistical techniques with a novel in vivo “competitive-mixtures” experimental model. Ferrets were coinfected with either pure populations (100% susceptible wild-type or 100% oseltamivir-resistant mutant virus) or mixed populations of wild-type and oseltamivir-resistant influenza viruses (80%:20%, 50%:50%, and 20%:80%) at equivalent infectivity titers, and the changes in the relative proportions of those two viruses were monitored over the course of the infection during within-host and over host-to-host transmission events in a ferret contact model. Coinfection of ferrets with mixtures of an oseltamivir-resistant R292K mutant A(H3N2) virus and a R292 oseltamivir-susceptible wild-type virus demonstrated that the R292K mutant virus was rapidly outgrown by the R292 wild-type virus in artificially infected donor ferrets and did not transmit to any of the recipient ferrets. The competitive-mixtures model was also used to investigate the fitness of the seasonal A(H1N1) oseltamivir-resistant H274Y mutant and showed that within infected ferrets the H274Y mutant virus was marginally outgrown by the wild-type strain but demonstrated equivalent transmissibility between ferrets. This novel in vivo experimental method and accompanying mathematical analysis provide greater insight into the relative fitness, both within the host and between hosts, of two different influenza virus strains compared to more traditional methods that infect ferrets with only pure populations of viruses. Our statistical inferences are essential for the development of the next generation of mathematical models of the emergence and spread of oseltamivir-resistant influenza in human populations.The neuraminidase (NA) inhibitors are a class of influenza antiviral drugs that are specifically designed to inhibit the enzymatic function of the NA, thereby preventing normal viral replication. Since 1999, two NA inhibitors (NAIs), oseltamivir (Tamiflu) and zanamivir (Relenza), have been shown to be effective for the treatment and prophylaxis of patients infected with not only seasonal influenza, but also highly pathogenic A(H5N1) and the newly emerged A(H1N1) pandemic virus. Prior to 2007, resistance to this class of drugs was considered relatively uncommon, particularly in comparison with the other class of influenza antivirals, the adamantanes, which readily select for viral resistance in treated patients. During early clinical trials, oseltamivir resistance was detected in only 1 to 2% of adults (14) and 5 to 6% of children (33) under treatment, although later studies detected resistance in up to 18% of oseltamivir-treated children (16). In contrast, resistance following zanamivir treatment is rare, with only one reported case observed in an immunocompromised patient (6). Influenza viruses that develop resistance to these drugs typically contain mutations in the NA which, either directly or indirectly, alter the shape of the NA enzymatic site, thereby reducing the ability of the drugs to bind to this specific pocket. One of the most commonly observed mutations in oseltamivir-resistant A(H3N2) viruses is an arginine-to-lysine mutation at residue 292 (R292K) of the NA, while the predominant NA mutation in oseltamivir-resistant A(H1N1) viruses is a histidine-to-tyrosine mutation at residue 274 (H274Y) (N2 NA amino acid numbering, equivalent to residue 275 based on N1 numbering). Both of these mutations have an indirect impact on drug binding, as they affect the ability of the glutamic acid residue at position 276 to reorientate, as required for slow binding by oseltamivir (3). Many mutations that cause NAI resistance also cause reduced NA enzyme activity and, consequently, can compromise viral fitness.Previous studies have demonstrated that viruses with an R292K NA mutation demonstrated compromised growth in vitro (36) and in ferrets were significantly less infectious and did not transmit (9). The replication and transmission fitness of the H274Y mutation has also been studied previously. An H274Y mutant A(H1N1) strain isolated from a patient under oseltamivir treatment demonstrated compromised growth in cell culture compared to a wild-type (WT) virus (13), although a strain carrying the same mutation selected in vitro was found to replicate as well as the wild type (32). The infectivity and transmissibility of an H274Y mutant were found to be restricted in ferrets (13), although a second study demonstrated that transmission of the mutant virus between ferrets was possible, but required a greater viral dose of the mutant compared to the wild type (10). These results suggest that resistant virus variants with the same NA mutation may differ in replication or transmission fitness depending on other viral components. Nevertheless, based on these data and the viral fitness of other resistant mutants, it was believed that NAI-resistant viruses were unlikely to spread throughout the community due to their compromised viral fitness in the absence of drug selective pressure. This was proven incorrect during the Northern Hemisphere 2007-2008 influenza season, when large numbers of oseltamivir-resistant seasonal A(H1N1) viruses with an H274Y mutation were detected in patients who had not been treated with oseltamivir (4, 24). The mutant strain continued to spread to the Southern Hemisphere, such that by late 2008 virtually all circulating seasonal A(H1N1) viruses were oseltamivir resistant (11). The rapid global spread of this strain clearly suggested that the oseltamivir-resistant seasonal A(H1N1) virus had fitness equivalent to or greater than that of the previous oseltamivir-sensitive A(H1N1) strain. The reasons for enhanced viral fitness in this strain, when previous studies demonstrated that the acquisition of an H274Y mutation led to reduced viral fitness, remain unclear but probably involve compensatory mutations or reassortment events which may have improved the hemagglutinin (HA)/NA balance, allowing efficient transmission (5, 26).Experimental methods have been developed to assess the relative fitness of NAI-resistant strains compared with respective wild-type viruses, both in vitro and in vivo. Ferrets have been considered the most appropriate model animal for influenza research, and fitness studies have assessed variables such as minimum dose required to achieve infection, duration of viral shedding, and levels of viral load to allow comparisons between viruses. The guinea pig model has also been previously used to assess the viral fitness of influenza viruses, particularly in comparing the transmissibility of strains via either the contact or aerosol route (2). As an alternative to these traditional approaches, we have investigated a methodology that involves coinfection of ferrets with a mixture of two influenza viruses. Daily monitoring of changes in the relative proportion of those viruses over the course of the infection allows determination of the relative replication fitness of the viruses. Monitoring of recipient ferrets exposed to the infected ferrets enables the relative transmissibility of the viruses (henceforth, the relative transmission fitness) to be determined. In this study, the “competitive-mixtures” methodology was used to assess the relative replication and transmission fitness of an oseltamivir-resistant R292K mutant A(H3N2) virus compared with an oseltamivir-sensitive A(H3N2) wild-type strain and also to asses the relative replication and transmission fitness of an oseltamivir-resistant H274Y seasonal A(H1N1) mutant compared with an oseltamivir-sensitive A(H1N1) wild-type strain. Quantitative estimates for the replication fitness of mutant viruses were determined using a simple mathematical model of within-host viral replication and mixed-effects statistical tests. Transmission fitness was evaluated by application of a graphical technique that demonstrated the relationship between the proportion of mutant virus in the infectee ferrets as a function of the proportion of mutant virus in the infector ferrets.Inferences drawn from the statistical analyses presented here are essential for the refinement of existing mathematical models that simulate the spread of influenza in the human population and model the deployment of antiviral agents. These models are designed to assess the likely impact of different antiviral agent deployment strategies to control pandemic influenza (18, 21, 35). At present, data on the probability of emergence of NAI-resistant strains, the relative transmission fitness of these strains, and the probability of an individual''s infection reverting to an NAI-sensitive strain in the absence of ongoing selective pressure are severely limited. In consequence, human population-level models of influenza spread must make gross assumptions on the likely characteristics of NAI-resistant strains. Data such as those presented here will be used to inform new models of drug deployment and result in improved pandemic policy advice (20, 23).     

Viral Production, Decay Rates, and Life Strategies along a Trophic Gradient in the North Adriatic Sea

Although the relationships between trophic conditions and viral dynamics have been widely explored in different pelagic environments, there have been few attempts at independent estimates of both viral production and decay. In this study, we investigated factors controlling the balance between viral production and decay along a trophic gradient in the north Adriatic basin, providing independent estimates of these variables and determining the relative importance of nanoflagellate grazing and viral life strategies. Increasing trophic conditions induced an increase of bacterioplankton growth rates and of the burst sizes. As a result, eutrophic waters displayed highest rates of viral production, which considerably exceeded observed rates of viral decay (up to 2.9 × 10⁹ VLP liter⁻¹ h⁻¹). Viral decay was also higher in eutrophic waters, where it accounted for ca. 40% of viral production, and dropped significantly to 1.3 to 10.7% in oligotrophic waters. These results suggest that viral production and decay rates may not necessarily be balanced in the short term, resulting in a net increase of viruses in the system. In eutrophic waters nanoflagellate grazing, dissolved-colloidal substances, and lysogenic infection were responsible together for the removal of ca. 66% of viral production versus 17% in oligotrophic waters. Our results suggest that different causative agents are primarily responsible for the removal of viruses from the water column in different trophic conditions. Factors other than those considered in the past might shed light on processes responsible for the removal and/or decay of viral particles from the water column.     

Viral production, decay rates, and life strategies along a trophic gradient in the North Adriatic Sea

Although the relationships between trophic conditions and viral dynamics have been widely explored in different pelagic environments, there have been few attempts at independent estimates of both viral production and decay. In this study, we investigated factors controlling the balance between viral production and decay along a trophic gradient in the north Adriatic basin, providing independent estimates of these variables and determining the relative importance of nanoflagellate grazing and viral life strategies. Increasing trophic conditions induced an increase of bacterioplankton growth rates and of the burst sizes. As a result, eutrophic waters displayed highest rates of viral production, which considerably exceeded observed rates of viral decay (up to 2.9 x 10(9) VLP liter(-1) h(-1)). Viral decay was also higher in eutrophic waters, where it accounted for ca. 40% of viral production, and dropped significantly to 1.3 to 10.7% in oligotrophic waters. These results suggest that viral production and decay rates may not necessarily be balanced in the short term, resulting in a net increase of viruses in the system. In eutrophic waters nanoflagellate grazing, dissolved-colloidal substances, and lysogenic infection were responsible together for the removal of ca. 66% of viral production versus 17% in oligotrophic waters. Our results suggest that different causative agents are primarily responsible for the removal of viruses from the water column in different trophic conditions. Factors other than those considered in the past might shed light on processes responsible for the removal and/or decay of viral particles from the water column.     

Viral contamination of a mosquito cell line,Aedes albopictus,associated with syncytium formation

Summary Viral contamination associated with syncytium formation in two sublines of Singh’sAedes albopictus cell cultures was investigated. Electron microscopy of the syncytia revealed the presence of five different types of virus-like particles, which morphologically resembled the parvo-, picorna-, toga-, and orbi-, and bacterial viruses. When a virusfree subline of theA. albopictus cells (SL3) was inoculated with extracts of the syncytium-formingA. albopictus cells, the parvo-, toga-, and orbi-type viral agents were consistently observed. Among these three agents, the togavirus-type agent is most likely responsible for the syncytium induction. Serological examination of the infected cell extract indicated that at least one of three virus-like agents, presumably the togavirus-type agent, was related to Chikungunya, O’nyong-nyong, and Western equine encephalomyelitis viruses (alphaviruses of the Togaviridae), but separable from these. Supported, in part, by USPHS-NIH General Research Grant No. PH S 5 SO1 RRO5679-06 from U.S. Pubic Health Service, Washington, D.C., to Boyce Thompson Institute.     

Avian Viral Pathogens in Swallows,Zimbabwe

We sampled 417 swallows in a wetland ecosystem of Zimbabwe in February 2010 and October 2011. RT-PCR tests revealed circulation of avian paramyxovirus type I, avian influenza and West Nile disease viruses in these populations. We discuss the relevance of these findings in relation to what is known on the epidemiology of these viruses in these hosts and in relation to the host ecology. We conclude with recommendations to focus more research on Passeriformes in disease ecology and in particular on the hirundinidae family.     

Viral Detection: Past,Present, and Future

Viruses are essentially composed of a nucleic acid (segmented or not, DNA, or RNA) and a protein coat. Despite their simplicity, these small pathogens are responsible for significant economic and humanitarian losses that have had dramatic consequences in the course of human history. Since their discovery, scientists have developed different strategies to efficiently detect viruses, using all possible viral features. Viruses shape, proteins, and nucleic acid are used in viral detection. In this review, the development of these techniques, especially for plant and mammalian viruses, their strengths and weaknesses as well as the latest cutting‐edge technologies that may be playing important roles in the years to come are described.     

Identification of a Plant Viral RNA Genome in the Nucleus

Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus (non-retroviral) is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf (Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.     

Liposomes in the Treatment of Parasitic,Viral, Fungal and Bacterial Infections

Abstract

The applicability of liposomes as carriers of immunomodulatory agents or antibiotics for improvement of treatment of severe infections is under investigation. The use of “classical'’ liposomes for targeting of macrophage modulators to enhance non-specific host resistance to infections caused by a variety of micro-organisms shows good results. The therapeutic prospects of “classical'’ liposomes as carriers of antibiotics are good, however are limited to the treatment of intracellular infections in mononuclear phagocyte system (MPS) tissues. The recent development of liposome formulations with reduced affinity to the MPS and long circulation half-lives creates new possibilities for obtaining improved delivery of antibiotics to infected tissues in general including infections in non-MPS tissues.     

Mechanochemistry of a Viral DNA Packaging Motor

The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage ?29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P_i release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage ?12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA decreases towards the end of the power stroke and the DNA rolls to the lever on the next subunit. Each power stroke facilitates ATP hydrolysis in the next catalytic site by inserting an Arg -finger into the site, as captured in ?12-P4. At the end of every four power strokes, ADP release happens slowly, so the cycle pauses constituting a dwell phase during which four ATPs are loaded into the catalytic sites. The next burst phase of four power strokes starts once spontaneous ATP hydrolysis takes place in the fifth site without insertion of an Arg finger. The push and roll model provides a new perspective on how a multimeric ATPase transports DNA, and it might apply to other ring motors as well.     

Viral contamination of a mosquito cell line, Aedes albopictus, associated with syncytium formation.

Viral contamination associated with syncytium formation in two sbulines of Singh's Aedes albopictus cell cultures was investigated. Electron microscopy of the syncytia revealed the presence of five different types of virus-like particles, which morphologically resembled the parvo-, picorna-, toga-, and orbi-, and bacterial viruses. When a virus-free subline of the A. albopictus cells (SL3) was inoculated with extracts of the syncytium-forming A. albopictus cells, the parvo-, toga-, and orbi-type viral agents were consistently observed. Among these three agents, the togavirus-type agent is most likely responsible for the syncytium induction. Serological examination of the infected cell extract indicated that at least one of three virus-like agents, presumably the togavirus-type agent, was related to Chikungunya. O'nyong-nyong, and Western equine encephalomyelitis viruses (alphaviruses of the Togaviridae), but separable from these.     

Viral sex, levels of selection, and the origin of life.

Several issues in Chao's related paper J. theor. Biol. (1991, 153, 229-246) are revisited. It is argued that mixes of segments from different viral coinfection groups cannot be regarded as sex, unless one is willing to accept that these groups are replicators and individuals. But, because selection in coinfection groups is dynamically analogous to that in trait groups in structured demes, one should also regard these latter groups as replicators. This approach is unacceptable since the groups in question have irregular ploidies, an unfixed number of parents, and no rules analogous to those of meiosis. It is emphasized, however, that the effective presence of neighbour-modulated fitness can ensure dynamical coexistence of covirus segments, even if the equal net reproduction rate within groups is not warranted. It seems that during the origin of coviruses from complete viruses, a higher-level evolutionary unit has become disintegrated, whereas during the origin of life a higher-level unit, the protocell, has emerged from lower-level ones, i.e. unlinked, replicating genes. These two gene-level systems are not homologous, but analogous. Although it is true that the resistance to parasites and the need to avoid a mutational collapse of the genome are likely to have called for some compartmentation in precellular stages of evolution, no clear demonstration, that the proposed mechanisms (the compartmentalized hypercycle and the stochastic corrector model) do in fact solve the error threshold problem, exists. Neither has a plausible mode of protocellular sex been suggested.     

The Role of Viral Mutation in the Pathogenesis of Chronic Viral Hepatitis

The quasispecies nature of hepatitis B and C virus (HBV, HCV) plays an important role in the pathogenesis, immune escape and drug resistance during chronic infection. Although there is still a lack of effective treatment for hepatitis C, a series of nucleoside analogs (NA) have been developed for the treatment of hepatitis B. NA resistant HBV mutants can accumulate during prolonged therapy and lead to the failure of anti-HBV therapy. Switching to other sensitive NAs can inhibit the emerged resistant mutants. Therefore, understanding the evolution of viral quasispecies under drug pressure is crucial for the establishment of antiviral strategy and the monitoring of antiviral process. Immune response and escape are complicated process, during which both host and virus factors may play their roles. Further understanding of the interaction and interrelationship between host and these viruses may lead to optimized prevention, diagnosis and treatment for chronic hepatitis.     

Phylogeography,Transmission, and Viral Proteins of Nipah Virus

Nipah virus (NiV), a zoonotic paramyxovirus belonging to the genus Henipavirus, is classified as a Biosafety Level-4 pathogen based on its high pathogenicity in humans and the lack of available vaccines or therapeutics. Since its initial emergence in 1998 in Malaysia, this virus has become a great threat to domestic animals and humans. Sporadic outbreaks and person-to-person transmission over the past two decades have resulted in hundreds of human fatalities. Epidemiological surveys have shown that NiV is distributed in Asia, Africa, and the South Pacific Ocean, and is transmitted by its natural reservoir, Pteropid bats. Numerous efforts have been made to analyze viral protein function and structure to develop feasible strategies for drug design. Increasing surveillance and preventative measures for the viral infectious disease are urgently needed.