Insertion of a Multibasic Cleavage Motif into the Hemagglutinin of a Low-Pathogenic Avian Influenza H6N1 Virus Induces a Highly Pathogenic Phenotype

The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA₀ (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.Wild birds represent the natural reservoir of avian influenza A viruses in nature (43). Influenza A viruses are classified on the basis of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. In wild birds throughout the world, influenza A viruses representing 16 HA and 9 NA antigenic subtypes have been found in numerous combinations (also called subtypes, e.g., H1N1, H6N1) (12). Besides classification based on the antigenic properties of HA and NA, avian influenza A viruses can also be classified based on their pathogenic phenotype in chickens. Highly pathogenic avian influenza (HPAI) virus, an acute generalized disease of poultry in which mortality may be as high as 100%, is restricted to subtypes H5 and H7. Other avian influenza A virus subtypes are generally low-pathogenic avian influenza (LPAI) viruses that cause much milder, primarily respiratory disease in poultry, sometimes with loss of egg production (6).The HA protein of influenza A virus is initially synthesized as a single polypeptide precursor (HA₀), which is cleaved into HA₁ and HA₂ subunits by host cell proteases. The mature HA protein mediates binding of the virus to host cells, followed by endocytosis and HA-mediated fusion with endosomal membranes (43). Influenza viruses of subtypes H5 and H7 may become highly pathogenic after introduction into poultry and cause outbreaks of HPAI. The switch from an LPAI phenotype to the HPAI phenotype of these H5 and H7 influenza A viruses is achieved by the introduction of basic amino acid residues into the HA₀ cleavage site by substitution or insertion, resulting in the so-called multibasic cleavage site (MBCS), which facilitates systemic virus replication (4, 5, 14, 44). The cleavage of the HA₀ of LPAI viruses is restricted to trypsin-like proteases which recognize the XXX(R/K)↓G cleavage motif, where the downward arrow indicates the site of cleavage. Replication of these LPAI viruses is therefore restricted to sites in the host where these enzymes are expressed, i.e., the respiratory and intestinal tract (32, 38). The introduction of an RX(R/K)R↓G or R(R/K)XR↓G minimal MBCS motif into the H5 and H7 subtype viruses facilitates the recognition and cleavage of the HA₀ by ubiquitous proprotein convertases, such as furin (20, 32, 41, 45). H5 influenza A viruses with a minimal MBCS motif only have the highly pathogenic phenotype if the masking glycosylation site at position 11 in the HA is replaced by a nonglycosylation site. Otherwise, at least one additional basic amino acid has to be inserted to allow the shift from an LPAI virus phenotype to an HPAI virus phenotype to occur (15, 18, 21, 22, 28). No information is available on the minimal prerequisites of H7 influenza A viruses to become highly pathogenic, but all HPAI H7 viruses have at least 2 basic amino acid insertions in the HA₀ cleavage site (22). HA₀ with the MBCS is activated in a broad range of different host cells and therefore enables HPAI viruses to replicate systemically in poultry (46). To date, little is known about the apparent subtype-specific nature of the introduction of the MBCS into LPAI viruses and the evolutionary processes involved in the emergence of HPAI viruses. When an MBCS was introduced in a laboratory-adapted strain of influenza virus, A/Duck/Ukraine/1/1963 (H3N8), it did not result in a dramatic change in pathogenic phenotype (35). Here, the effect of the introduction of an MBCS into a primary LPAI H6N1 virus, A/Mallard/Sweden/81/2002, is described. The introduction of an MBCS resulted in trypsin-independent replication in vitro and enhanced pathogenesis in a chicken model. Understanding the basis of the HA subtype specificity of the introduction of an MBCS into avian influenza viruses will lead to a better understanding of potential molecular restrictions involved in emergence of HPAI outbreaks.     

Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.Two classes of antiviral drugs are currently available for the treatment and prophylaxis of influenza, the adamantanes and the neuraminidase (NA) inhibitors (NAIs). The adamantanes were the first agents to be recognized to have anti-influenza virus activities as early as 1964 (2) although the rapid emergence of drug-resistant influenza virus strains has limited their clinical effectiveness (12). The NAIs, zanamivir (Relenza) and oseltamivir (Tamiflu), were the first drugs to be specifically designed as anti-influenza virus agents and have been available on the market in many countries since 1999. During oseltamivir clinical trials, 1 to 4% of treated adults (6) and 5 to 6% of treated children were found to shed resistant viruses (30) although more recent studies have reported resistance in 16 to 18% of viruses from oseltamivir-treated children (20, 29). In contrast to the frequency of resistance seen following oseltamivir treatment, only one occurrence of significant zanamivir resistance has been observed following zanamivir treatment. The zanamivir-resistant strain, an influenza B virus with an R152K NA mutation, was isolated from an immunocompromised patient undergoing prolonged zanamivir treatment (7).In addition to the analysis of influenza viruses isolated from patients undergoing either oseltamivir or zanamivir treatment, surveillance studies that analyze the NAI susceptibility of circulating viruses, predominantly from patients not undergoing NAI treatment, have also been conducted. Studies that have tested viruses isolated prior to the release of the NAIs (1996 to 1999) (23) and after the initiation of clinical use of these drugs (2000 to 2006) (16, 24) have found either no resistance or a very low frequency of resistance. In contrast, analysis of circulating seasonal influenza viruses from Europe during the 2007 to 2008 season revealed that 14% (59/437) of influenza A (H1N1) viruses had significantly decreased susceptibility to oseltamivir (21). Since this initial report, oseltamivir-resistant influenza A (H1N1) strains have spread throughout Europe (11) and have been detected at high frequencies in other countries including the United States (4), Japan (28), South Africa (1) and Oceania and Southeast Asia (17). These influenza A (H1N1) viruses have a mutation of histidine to tyrosine at residue 274 of the NA (N2 NA numbering; residue 275 by N1 NA numbering), which confers a high level of resistance to oseltamivir (10) but has no effect on susceptibility to zanamivir or to the adamantanes.Prior to May 2008, when the oseltamivir-resistant variants became the dominant influenza A (H1N1) strain in Oceania and Southeast Asia (17), NAI sensitivity monitoring conducted at the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, identified a number of influenza A (H1N1) viruses with reduced zanamivir susceptibility. These viruses contained a previously undescribed mutation at residue 136 of the NA. Here, we report on the detection of these mutant viruses from geographically distinct locations, the in vitro and in vivo fitness of the strains, and the finding that the mutant viruses appear to have been preferentially propagated during viral culture in Madin-Darby canine kidney (MDCK) cells.     

Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay

Background

Immunosuppressive viruses are frequently found as co-infections in the chicken industry, potentially causing serious economic losses. Because traditional molecular biology methods have limited detection ability, a rapid, high-throughput method for the differential diagnosis of these viruses is needed. The objective of this study is to develop a GenomeLab Gene Expression Profiler Analyser-based multiplex PCR method (GeXP-multiplex PCR) for simultaneous detection of eight immunosuppressive chicken viruses.

Results

Using chimeric primers, eight such viruses, including Marek's disease virus (MDV), three subgroups of avian leucosis virus (ALV-A/B/J), reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), chicken infectious anaemia virus (CIAV) and avian reovirus (ARV), were amplified and identified by their respective amplicon sizes. The specificity and sensitivity of the optimised GeXP-multiplex PCR assay were evaluated, and the data demonstrated that this technique could selectively amplify these eight viruses at a sensitivity of 100 copies/20 μl when all eight viruses were present. Among 300 examined clinical specimens, 190 were found to be positive for immunosuppressive viruses according to this novel assay.

Conclusion

The GeXP-multiplex PCR assay is a high-throughput, sensitive and specific method for the detection of eight immunosuppressive viruses and can be used for differential diagnosis and molecular epidemiological surveys.

     

High-level expression of a foreign gene by a recombinant baculovirus with an expanded host range

The usefulness of host range expanded viruses as an expressionvector system was investigated by following the expression ofthe E. coli lacZ gene. The host range expanded recombinantviruses were obtained from Sf-21 or BmN-4 cells coinfected withAutographa californica and Bombyx mori nuclearpolyhedrosis viruses. Among the host range expanded viruses,RecB-8 and RecS-B6 have similar enzyme digestion profiles butdifferent infection characteristics in cells. Therefore, tostudy the foreign gene expression efficiency of these twoviruses, we constructed recombinant viruses RecB8-LacZ andRecSB6-LacZ containing the lacZ gene instead of the polyhedringene. Also, the host range expanded recombinant AcNPV, Bac-BH,containing lacZ gene in the polyhedrin gene locus was constructedby substitution of the 0.6 kb region within the helicase gene ofBacPAK6 with that of BmNPV. -Galactosidase expressionefficiency by these viruses were determined and compared in Sf-21and BmN-4 cells. The result showed that Bac-BH has highexpression efficiency only in Sf-21 cells, whereas RecB8-LacZhas high expression efficiency both in Sf-21 and BmN-4 cells.Also, in BmN-4 cells, -galactosidase expressionefficiency of RecB8-LacZ was higher than that of recombinantBmNPV (BmK1-LacZ containing lacZ gene in polyhedrin gene locus).In addition, the expression efficiency was not correlated withvirus titer.     

Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused devastating outbreaks in avian species during the past decade. After emerging in the Guangdong province of China in 1996, H5N1 viruses have extended their geographic distribution from Asia into Europe and Africa (45, 51). Sporadic transmission of H5N1 viruses from infected birds to humans has resulted in over 380 laboratory-confirmed infections and a case fatality rate of ∼60% since 2003 (48). Currently circulating H5N1 viruses lack the ability to undergo efficient and sustained transmission among humans although instances of limited human-to-human transmission have been reported (13, 41). If H5N1 viruses were to acquire genetic changes that confer efficient transmissibility among humans, then another pandemic would likely occur.The pandemics of 1957 and 1968 highlight the importance of genetic reassortment between avian and human influenza viruses as a mechanism for the generation of human pandemic strains (15, 46, 47). The structural separation of the influenza virus genome into eight independent genes allows formation of hybrid progeny viruses during coinfections. The 1957 H2N2 and 1968 H3N2 pandemic viruses acquired the hemagglutinin (HA) and PB1 genes, with or without the neuraminidase (NA) gene, respectively, from an avian virus progenitor (14, 33). The remaining genes of these pandemic reassortants were derived from a contemporary human virus (14, 33). The host species in which such human pandemic strains were generated by reassortment between human and avian viruses is not known. However, coinfection of the same cell with both human and avian viruses must have occurred, even though human and avian influenza viruses have preferences for different sialic acid receptor structures present on cell surface glycoproteins and glycolipids (20, 30). The HA of human viruses preferentially binds α(2,6)-linked sialic acids while that of avian viruses preferentially bind α(2,3)-linked sialic acids (3, 12). Cells possessing both of these receptors could support coinfection of avian and human viruses, leading to reassortment.Human respiratory tract epithelial cells can possess surface glycans with α(2,3)- and α(2,6)-linked sialic acids and as such represent a potential host for the generation of avian-human reassortant viruses (24, 35). The general distribution of surface α(2,3)- and α(2,6)-linked sialic acids varies among cells of the human upper and lower respiratory tracts, which are anatomically separated by the larynx. Recent studies have shown that α(2,3)-linked sialic acids are present in tissues of the human lower respiratory tract (i.e., lung alveolar cells) (24, 35) as well as tissues of the human upper respiratory tract (24). Consistent with these findings, HPAI H5N1 viruses have been shown to attach to and infect tissues belonging to the lower respiratory tract (i.e., trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43) as well as tissues belonging to the upper respiratory tract (i.e., nasopharyngeal, adenoid, and tonsillar) (25). Glycans with α(2,6)-linked sialic acids are more widespread on epithelial cells of the upper airways than lung alveoli (24, 35). In accordance, human seasonal influenza viruses preferentially attach to and infect cells of the upper respiratory tract (6, 25, 35, 43). If cells with both types of receptors are present in the human respiratory tract, simultaneous infection of a person with both human and avian viruses could generate reassortant viruses.Although viruses derived by reassortment between avian H5N1 and human H3N2 progenitors have been generated in vitro (17), reassortment between these avian and human strains in a coinfected mammalian host has not been shown. Furthermore, our knowledge of the genetic and phenotypic repertoire of such reassortants generated in vivo and their potential for transmission to uninfected hosts is limited (2, 17). In the present study, we used the ferret model to better understand the generation of reassortant viruses in a host coinfected with contemporary avian (H5N1) and human (H3N2) viruses and the extent to which such reassortants replicate and transmit from animal to animal. The domestic ferret (Mustela putoris) serves as an ideal small-animal model for influenza because ferrets are susceptible to human and avian influenza viruses, including HPAI H5N1 viruses, and reflect the relative transmissibility of human and avian influenza viruses in humans (9, 17, 18, 31, 36, 39, 53). Our study revealed that coinfection of ferrets reproducibly generated reassortant viruses that could be recovered from tissues within and extending toward the upper respiratory tract. Although H5 reassortant viruses were recovered from the upper airways, they displayed no transmissibility to contact ferrets, suggesting that additional functional changes are required for these viral subtypes to become pandemic within human populations.     

Amplification and Spread of Viruses in a Growing Plaque

The two-dimensional propagation of viruses through a "lawn" of receptive hosts, commonly called plaque growth, reflects the dynamics of interactions between viruses and host cells. Here we treat the amplification of viruses during plaque growth as a reaction-diffusion system, where interactions among the virus, uninfected host cells, and virus-producing host-virus complexes are accounted for using rates of viral adsorption to and desorption from the host-cell surface, rates of reproduction and release of progeny viruses by lysis of the host, and by the coupling of these reactions with diffusion of free virus within the agar support. Numerical solution of the system shows the development of a traveling wave of reproducing viruses, where the velocity of the wave is governed by the kinetic and diffusion parameters. The model has been applied to predict the propagation velocity of a bacteriophage plaque. Different mechanisms may account for the dependence of this velocity on the host density during early stages of a growing plaque. The model provides a means to explore how changes in the virus-host interactions may be manifest in a growing plaque.     

Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model

The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.The neuraminidase (NA) inhibitors (orally administered oseltamivir and inhaled zanamivir) are currently an important class of antiviral drugs available for the treatment of seasonal and pandemic influenza. Although administration of NA inhibitors may significantly reduce influenza virus transmission, it risks the emergence of drug-resistant variants (16, 32). The impact of drug resistance would depend on the fitness (i.e., infectivity in vitro and virulence and transmissibility in vivo) of the resistant virus. If the resistance mutation only modestly reduces the virus'' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. The unexpected natural emergence and spread of oseltamivir-resistant variants (carrying the H274Y NA amino acid substitution) among seasonal H1N1 influenza viruses of the A/Brisbane/59/07 lineage demonstrated that drug-resistant viruses can be highly fit and transmissible in humans (11, 22, 29), although the fitness of these variants is not completely understood. They are hypothesized to have lower NA receptor affinity and more-optimal NA and hemagglutinin (HA) functional balance than do wild-type (WT) viruses (38). Fortunately, oseltamivir-resistant variants have rarely been reported to occur among the novel pandemic H1N1 influenza viruses that emerged in April 2009; therefore, initial data suggest that currently circulating wild-type viruses possibly possess greater fitness than drug-resistant viruses (45), although only retrospective epidemiological data can provide a conclusive answer. The key questions are whether the risk posed by NA inhibitor-resistant viruses can be assessed experimentally and what the most reliable approach may be.All NA inhibitor-resistant influenza viruses characterized to date have contained specific mutations in the NA molecule. Clinically derived drug-resistant viruses have carried mutations that are NA subtype specific and differ in accordance with the NA inhibitor used (12, 35). The most commonly observed mutations are H274Y and N294S in the influenza A N1 NA subtype, E119A/G/D/V and R292K in the N2 NA subtype, and R152K and D198N in influenza B viruses (35, 36). The fitness of NA inhibitor-resistant viruses has been studied in vitro and in vivo. Many groups have assessed their replicative capacity in MDCK cells, but this assay system can yield anomalous results (49), particularly in the case of low-passage clinical isolates. The mismatch between virus specificity and cellular receptors can be overcome by using cell lines engineered to express human-like α-2,6-linked sialyl cell surface receptors (MDCK-SIAT1) (15, 34) or a novel cell culture-based system that morphologically and functionally recapitulates differentiated normal human bronchial epithelial (NHBE) cells (24). Investigations in vivo typically compare replication efficiencies, clinical signs, and transmissibility levels between oseltamivir-resistant viruses and the corresponding wild-type virus. Initial studies found that NA inhibitor-resistant influenza viruses were severely compromised in vitro and in animal models (6, 17, 26) and thus led to the idea that resistant viruses will unlikely have an impact on epidemic and pandemic influenza. However, clinically derived H1N1 virus with the H274Y NA mutation (18) and reverse genetics-derived H3N2 virus with the E119V NA mutation (46) were subsequently found to possess biological fitness and transmissibility similar to those of drug-sensitive virus in direct-contact ferrets. Recent studies in a guinea pig model showed that recombinant human H3N2 influenza viruses carrying either a single E119V NA mutation or the double NA mutation E119V-I222V were transmitted efficiently by direct contact but not by aerosol (5).There is limited information about the fitness of NA inhibitor-resistant H5N1 influenza viruses. Although they are not efficiently transmitted from human to human, their pandemic potential remains a serious public health concern because of their virulence in humans (1, 4, 7). H5N1 viruses isolated from untreated patients are susceptible to the NA inhibitors oseltamivir and zanamivir (21), although oseltamivir-resistant variants with the H274Y NA mutation have been reported to occur in five patients after (9, 30) or before (41) treatment with oseltamivir. The World Health Organization reported the isolation of two oseltamivir-resistant H5N1 viruses from an Egyptian girl and her uncle (44) after oseltamivir treatment. The virus was moderately resistant and possessed an N294S NA mutation. Preliminary evidence suggests that the resistance mutation existed before transmission of the virus from birds to the patients and thus before initiation of treatment (41). We previously showed that wild-type A/Vietnam/1203/04 (H5N1) influenza virus and recombinants carrying either the H274Y or the N294S NA mutation reached comparable titers in MDCK and MDCK-SIAT1 cells and caused comparable mortality rates among BALB/c mice (48). In contrast, clinically derived A/Hanoi/30408/05 (H5N1) influenza virus with the H274Y NA mutation reproduced to lower titers than the oseltamivir-sensitive virus in the lungs of inoculated ferrets (30).In a ferret model, we compared the fitness levels of two pairs of H5N1 viruses in the absence of selective drug pressure. One virus of each pair was the wild type, while the other carried the H274Y NA mutation conferring oseltamivir resistance. The two viruses used, A/Vietnam/1203/04 (HA clade 1) and A/Turkey/15/06 (HA clade 2.2), differ in their pathogenicity to ferrets. Virus fitness was evaluated by two approaches. Using the traditional approach, we compared clinical disease signs, relative inactivity indexes, weight and temperature changes, and virus replication levels in the upper respiratory tract (URT). We then used a novel competitive fitness approach in which we genetically analyzed individual virus clones after coinfection of ferrets with mixtures of oseltamivir-sensitive and -resistant H5N1 viruses; thus, we determined virus-virus interactions within the host. We observed no difference between the resistant and sensitive virus of each pair in clinical signs or virus replication in the URT; however, analysis of virus-virus interactions within the host showed that the H274Y NA mutation affected the fitness of the two viruses differently. The oseltamivir-resistant A/Vietnam/1203/04-like virus outgrew its wild-type counterpart, while the oseltamivir-resistant A/Turkey/15/06-like virus showed less fitness than its wild-type counterpart.     

Biological properties of a type C virus isolated from a human X mouse hybrid cell line.

The biological properties of the HMV-1 virus, spontaneously released from a human X C57BL/6 mouse hybrid cell line, were similar to those of RadLV, the prototype B-tropic virus of C57BL/6 mice. Both viruses replicated on B-type mouse cells and in the wild mouse cell line SC-1. The plaque-forming abilities of the two viruses were relatively low, but gradually increased after passage in new host cells. Both viruses were neutralized by AKR antisera but not by FMR antisera. HMV-1 virus could rescue the defective sarcoma genome from S+H- mouse cells. The pseudotype sarcoma virus so produced was deficient in "helper virus" activity. Newborn mice inoculated with HMV-1 virus remained tumor-free over a 1-yr observation period.     

Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract

Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have resulted in over 420 documented cases of human infection to date, have generally caused acute, often severe and fatal, respiratory illness (1, 50). While conjunctivitis following infection with H5N1 or human influenza viruses has been rare, most human infections associated with H7 subtype viruses have resulted in ocular and not respiratory disease (1, 9, 37, 38). Infrequent reports of human conjunctivitis infection following exposure to H7 influenza viruses date from 1977, predominantly resulting from laboratory or occupational exposure (21, 40, 48). However, in The Netherlands in 2003, more than 80 human infections with H7N7 influenza virus occurred among poultry farmers and cullers amid widespread outbreaks of HPAI in domestic poultry; the majority of these human infections resulted in conjunctivitis (14, 20). Additionally, conjunctivitis was documented in the two human infections resulting from an H7N3 outbreak in British Columbia, Canada, in 2004, as well as in H7N3- and H7N2-infected individuals in the United Kingdom in 2006 and 2007, respectively (13, 18, 29, 46, 51). The properties that contribute to an apparent ocular tropism of some influenza viruses are currently not well understood (30).Host cell glycoproteins bearing sialic acids (SAs) are the cellular receptors for influenza viruses and can be found on epithelial cells within both the human respiratory tract and ocular tissue (26, 31, 41). Both respiratory and ocular tissues additionally secrete sialylated mucins that function in pathogen defense and protection of the epithelial surface (5, 11, 22). Within the upper respiratory tract, α2-6-linked SAs (the preferred receptor for human influenza viruses) predominate on epithelial cells (26). While α2-3-linked SAs are also present to a lesser degree on respiratory epithelial cells, this linkage is more abundantly expressed on secreted mucins (2). In contrast, α2-3-linked SAs (the preferred receptor for avian influenza viruses) are found on corneal and conjunctival epithelial cells of the human eye (31, 41), while secreted ocular mucins are abundantly composed of α2-6 SAs (5). It has been suggested that avian influenza viruses are more suited to infect the ocular surface due to their general α2-3-linked SA binding preference, but this has not been demonstrated experimentally (30).The mouse model has been used previously to study the role of ocular exposure to respiratory viruses (6, 39). In mice, ocular inoculation with an H3N2 influenza virus resulted in virus replication in nasal turbinates and lung (39), whereas ocular infection with respiratory syncytial virus (RSV) resulted in detectable virus titers in the eye and lung (6). These studies have revealed that respiratory viruses are not limited to the ocular area following inoculation at this site. However, the ability of influenza viruses to replicate specifically within ocular tissue has not been examined.Despite repeated instances of conjunctivitis associated with H7 subtype infections in humans, the reasons for this apparent ocular tropism have not been studied extensively. Here, we present a murine model to study the ability of human and avian influenza viruses to cause disease by the ocular route. We found that highly pathogenic H7 and H5 influenza viruses were capable of causing a systemic and lethal infection in mice following ocular inoculation. These highly pathogenic viruses, unlike human H3N2 and H1N1 viruses, replicated to significant titers in the mouse corneal epithelium and primary human corneal epithelial cells (HCEpiCs). Identification of viruses well suited to infecting the ocular surface is the first step in better understanding the ability of influenza viruses of multiple subtypes to use this tissue as a portal of entry.     

Analysis of a model of a virus that replicates selectively in tumor cells

We consider a procedure for cancer therapy which consists of injecting replication-competent viruses into the tumor. The viruses infect tumor cells, replicate inside them, and eventually cause their death. As infected cells die, the viruses inside them are released and then proceed to infect adjacent tumor cells. This process is modelled as a free boundary problem for a nonlinear system of hyperbolic-parabolic differential equations, where the free boundary is the surface of the tumor. The unknowns are the densities of uninfected cells, infected cells, necrotic cells and free virus particles, and the velocity of cells within the tumor as well as the free boundary r=R(t). The purpose of this paper is to establish a rigorous mathematical analysis of the model, and to explore the reduction of the tumor size that can be achieved by this therapy. Mathematics Subject Classification (2000):35R35; 92A15     

Arenoviruses: Proposed Name for a Newly Defined Virus Group

The name "arenoviruses" is proposed for the newly defined taxonomic group containing lymphocytic choriomeningitis, Lassa, and the Tacaribe complex viruses.     

Isolation and Characterization of a Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Chaetoceros socialis

Diatoms are very significant primary producers in the world''s oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.Diatoms (Bacillariophyceae) account for a large part of the marine primary production, up to 35% in oligotrophic oceans and 75% in nutrient-rich systems (13). They play an important role in various marine systems as a food source for zooplankton and animal larvae. Moreover, diatoms are the primary oxygen producers for the atmosphere (25). Therefore, to understand diatom dynamics in nature is significant for biogeochemical science and fisheries studies. Phytoplankton population dynamics are the result of reproduction and losses. Losses include grazing, sinking, and natural mortality. Since the early 1990s, the importance of viruses infectious to microalgae is recognized as one of the principal causes of phytoplankton mortality. The direct evidence for the existence of diatom viruses was reported recently in 2004 (11). Since the discovery of the first diatom virus, the isolation and characterization of new viruses have been conducted. As a result, several new diatom viruses infecting ecologically important diatom members have been successfully isolated and reported.The first diatom virus, Rhizosolenia setigera RNA virus (RsRNAV), is a small icosahedral virus (32 nm) with a single-stranded RNA (ssRNA) genome at 8,877 nucleotides (nt), excluding a poly(A) tail (11, 15). Thereafter, two Chaetoceros-infecting single-stranded DNA (ssDNA) viruses were isolated and characterized: Chaetoceros salsugineum nuclear inclusion virus (CsNIV), a small (38-nm) virus harboring a covalently closed circular ssDNA (6,000 nt) and a segment of linear ssDNA (997 nt) (12) (H. Mizumoto, unpublished data), and Chaetoceros debilis DNA virus, whose partial genome sequence is highly similar to that of CsNIV (22). The genome analyses of the two ssDNA viruses showed that they are distinctive from previously reported viruses. The isolation of Chaetoceros nuclear inclusion virus (CspNIV) infectious to Chaetoceros cf. gracilis (a Chaetoceros sp. that looks like Chaetoceros gracilis) was also reported (1); however, its nucleic acid type is still unknown. A recent study reports the isolation of the second ssRNA diatom virus infectious to Chaetoceros tenuissimus (CtenRNAV). A phylogenetic analysis showed a putative RNA-dependent RNA polymerase (RdRp) domain from a genome sequence of CtenRNAV is highly similar to RsRNAV but less similar to other marine stramenopile organism viruses (16): Schizochytrium single-stranded RNA virus (SssRNAV) infecting a fungoid protist Aurantiochytrium sp. (formerly Schizochytrium sp.) (19) and Heterosigma akashiwo RNA virus (HaRNAV; Marnaviridae) infecting the bloom-forming raphidophyte H. akashiwo (7, 8). The ssRNA diatom viruses are unlike other known viruses at the family level. These reports suggest that the diatom viruses are an exclusively unique group distinct from previously described viruses where further study of diatom virus biology is significant to understand diatom ecology.Here we report the isolation and characterization of a new ssRNA virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) infecting Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko, one of the dominant phytoplankton species in the marine environments in especially productive areas during spring blooms; e.g., in the North Water polynya, the maximum concentration of C. socialis was as high as 3.0 × 10⁴ cells ml⁻¹ (2). Here, we also propose a new ssRNA virus family (Bacillariornaviridae), composed of three diatom-infecting ssRNA viruses based on phylogenetic analysis using the RdRp domain and other genomic characters.     

Nyamanini and Midway Viruses Define a Novel Taxon of RNA Viruses in the Order Mononegavirales

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.Nyamanini virus (NYMV) was first isolated in 1957 from a cattle egret (Bubulcus ibis) in South Africa (24). It has subsequently been isolated in Nigeria, Egypt, India, and Thailand from cattle egrets and Argas walkerae ticks (14, 16, 24). Although there has been no recognized human infection or disease associated with NYMV, suckling mice succumbed to NYMV infection 7 or 8 days after intracerebral inoculation (14). NYMV has not been definitively characterized or classified to date (10).Midway virus (MIDWV) was first isolated in 1966 from seabird ticks of two species [Ornithodoros (Alectorobius) spp.] collected on the Midway, Kure, and Manana islands in the Central Pacific and from northern Honshu, Japan. In addition, on Aomatsushima Island, nestling seabirds of two seabird species, Larus crassirostris and Nycticorax nycticorax, were found to have antibody to MIDWV. This virus is pathogenic for newborn Swiss mice but not 4-week-old Swiss mice injected intracranially. Also, the virus is cytopathic in BHK-21 cells and produces plaques in Vero cells. Efforts to classify MIDWV have revealed only that MIDWV is antigenically related to NYMV in cross-box complement fixation (CF) assays (22).To date, conventional approaches, such as serological analysis and electron microscopy (EM), have not yielded definitive characterization of MIDWV or NYMV. Heretofore, it was not known whether MIDWV and NYMV are DNA viruses or RNA viruses or to which virus family they belong. In this paper, we describe the application of unbiased high-throughput sequencing to define the genome sequences of NYMV and MIDWV. By analysis of their genomes, NYMV and MIDWV were determined to be negative-stranded RNA viruses highly divergent from all known viruses but most closely related to viruses in the order Mononegavirales. On the basis of the analysis presented herein, we propose that NYMV and MIDWV define a novel taxon within the order Mononegavirales.     

Isolation of Prasinoviruses of the Green Unicellular Algae Ostreococcus spp. on a Worldwide Geographical Scale

Ostreococcus spp. are extremely small unicellular eukaryotic green algae found worldwide in marine environments, and they are susceptible to attacks by a diverse group of large DNA viruses. Several biologically distinct species of Ostreococcus are known and differ in the ecological niches that they occupy: while O. tauri (representing clade C strains) is found in marine lagoons and coastal seas, strains belonging to clade A, exemplified by O. lucimarinus, are present in different oceans. We used laboratory cultures of clonal isolates of these two species to assay for the presence of viruses in seawater samples from diverse locations. In keeping with the distributions of their host strains, we found a decline in the abundance of O. tauri viruses from a lagoon in southwest France relative to the Mediterranean Sea, whereas in the ocean, no O. tauri viruses were detected. In contrast, viruses infecting O. lucimarinus were detected from distantly separated oceans. DNA sequencing, phylogenetic analyses using a conserved viral marker gene, and a Mantel test revealed no relationship between geographic and phylogenetic distances in viruses infecting O. lucimarinus.Viruses are the most abundant and genetically diverse biological entities in marine environments (48). The three ways most often used to assess eukaryotic algal virus diversity are (i) using a functional host-virus system to quantify viruses specific to one host strain (i.e., culture-based studies) (4), (ii) using PCR amplification and sequencing a conserved gene (10, 12-14, 28), and (iii) using whole-community genome sequencing (i.e., metagenomics) (6, 8, 31). Recently, the advent of sequencing techniques like shotgun sequencing or pyrosequencing (38) has led to an increase in the number of metagenomics projects. The Global Ocean Sampling (GOS) Expedition has provided a unique opportunity to investigate viral diversity in different environments within the size fraction of 0.1 to 0.8 μm (39). The GOS data revealed highly abundant viral sequences (at least 3% of the predicted proteins had a viral origin) (53). In another study, the analysis of marine viromes from four oceanic regions suggested that the composition of viral assemblages depends on their geographic locations, but these authors conclude that similar viruses are widespread throughout the oceans (2). Despite these new methods and different ways to analyze viral diversity, we still do not really know if “everything is really everywhere” (7).The present study addresses a specific part of this problem: are viruses infecting a single host strain present at geographically distant locations? If several viral strains are identified and characterized, how closely do these viruses resemble one another on a phylogenetic scale? In order to answer these questions, we focused on a microalgal (Prasinophyceae)-virus (Prasinovirus) system. The studied hosts belong to the genus Ostreococcus, a ubiquitous prasinophyte picoeukaryotic alga abundant throughout the oceanic euphotic zone (55, 56). Several strains from this genus were isolated and assigned to four distinct ecotypes according to their growth parameters under different light regimens (22, 36), which correspond to four well-defined phylogenetic clades in an internal transcribed spacer (ITS)-based phylogeny (clades A to D). The complete genome sequences of two Ostreococcus species have been described: O. tauri (19) and O. lucimarinus (35). In the present study, viruses infecting specific host species (Ostreococcus spp.) have been screened from a variety of locations around the world.Among viruses infecting Ostreococcus, the genome of a single strain (OtV5) has been fully sequenced (18), and the phylogenetic relationships among several virus strains have been investigated (4). These viruses belong to the Prasinovirus group, a genus of the Phycodnaviridae family. Many viruses infecting phytoplankton are members of the Phycodnaviridae; they have double-stranded DNA genomes and large polyhedral capsids (20). In the prasinophyte-Prasinovirus system, the hosts and viruses can be grown on solid medium and are easily maintained in the laboratory. Ostreococcus viral strains have been isolated and characterized by phylogenetic analysis based on their B-family DNA polymerase (DNA pol) partial gene sequence (4). This DNA polymerase is a useful marker for phylogenetic analyses because its sequence is well conserved in all known members of nucleocytoplasmic large DNA viruses (NCLDVs) (26), including Phycodnaviridae. Furthermore, several previous studies have examined the abundance and the genetic diversity of marine eukaryotic viruses using environmental sequencing approaches and amplified DNA pol gene fragments (11, 12, 43-46), and Monier et al. (31) used this marker to describe the taxonomic distribution of large DNA viruses from the GOS data.The first stage of this study was to isolate Ostreococcus viruses from different worldwide geographic locations, by culturing on various host strains. In a second stage, these viral strains were characterized via the sequencing of their pol sequence (encoding a part of their DNA polymerase gene), and their specificity toward different host strains was assessed in order to assess the potential host range of the viral strains isolated and to gain a better understanding of their population dynamics and distribution. Finally we compared these new Prasinovirus DNA sequences with metagenomic sequence data (obtained from sampling all around the world) and environmental sequence data identified using BLAST similarity to assess the global distribution of similar Ostreococcus viruses.     

Modifications to the Hemagglutinin Cleavage Site Control the Virulence of a Neurotropic H1N1 Influenza Virus

A key determinant of influenza virus pathogenesis is mutation in the proteolytic cleavage site of the hemagglutinin (HA). Typically, low-pathogenicity forms of influenza virus are cleaved by trypsin-like proteases, whereas highly pathogenic forms are cleaved by different proteases (e.g., furin). Influenza virus A/WSN/33 (WSN) is a well-studied H1N1 strain that is trypsin independent in vitro and has the ability to replicate in mouse brain. Previous studies have indicated that mutations in the neuraminidase (NA) gene allow the recruitment of an alternate protease (plasminogen/plasmin) for HA activation. In this study we have identified an additional mutation in the P2 position of the WSN HA cleavage site (S328Y) that appears to control virus spread in a plasmin-dependent manner. We reconstructed recombinant WSN viruses containing tyrosine (Y), phenylalanine (F), or serine (S) in the P2 position of the cleavage site. The Y328 and F328 viruses allowed plaque formation in the absence of trypsin, whereas the S328 virus was unable to form plaques under these conditions. In mice, Y328 and F328 viruses were able to efficiently spread following intracranial inoculation; in contrast, the S328 virus showed only limited infection of mouse brain. Following intranasal inoculation, all viruses could replicate efficiently, but with Y328 and F328 viruses showing a limited growth defect. We also show that wild-type HA (Y328) was more efficiently cleaved by plasmin than S328 HA. Our studies form the foundation for a more complete understanding of the molecular determinants of influenza virus pathogenesis and the role of the plasminogen/plasmin system in activating HA.For all viruses, the infectious cycle begins with the penetration of the host cell (27). Enveloped viruses penetrate cells via a membrane fusion event mediated by a spike protein present in the virus envelope, with fusion triggered by conformational changes in the spike protein following exposure to low pH and/or receptor (45). In the case of influenza virus, the viral spike protein hemagglutinin (HA) mediates both receptor binding and membrane fusion (46). Many viral fusion proteins are activated following cleavage by host cell proteases (9, 20, 21), and this has been most extensively documented for influenza viruses, where cleavage is directly related to exposure of the fusion peptide and fusion activation (38). For proteases, a general nomenclature for the cleavage site positions of the substrate has been designated, with cleavage occurring between P1 and P1′ and with the position numbers increasing in the N-terminal direction relative to the cleaved peptide bond (P2, P3, P4, etc.). Low-pathogenicity influenza virus strains contain an HA cleavage site with a single arginine residue at the P1 position and are thus described as having monobasic cleavage sites. These viruses can utilize trypsin (or other trypsin-like serine proteases) for activation, with the tissue distribution of the activating protease typically restricting infection to the respiratory and/or intestinal organs. The presence of a polybasic cleavage site is critical for the systemic spread and increased virulence associated with highly pathogenic avian influenza (HPAI) viruses (33). In the case of HPAI viruses such as H5N1 and H7N7, it is well established that mutations in the region of the HA cleavage site lead to an insertion of several arginine or lysine residues in addition to the P1 arginine (specifically in the P2 to P6 cleavage site positions) that can be recognized by furin—an intracellular serine protease found in many cell types—allowing a widening of the cell tropism of the virus (18).Influenza virus is currently of major biomedical interest, both due to annual morbidity and the threat of new pandemic viruses. Influenza viruses exist as many different subtypes (H1 to H16), with H1 and H3 viruses currently infecting humans (10, 30). Normally, H1 viruses are considered to have low pathogenicity and have a monobasic cleavage site. However, two H1 isolates A/WSN/33 (WSN) and A/NWS/33 (NWS) have been selected to propagate in mouse brain and are thus considered to be highly pathogenic, neurovirulent viruses in mice (39, 40). The A/WSN/33 virus in particular has been used extensively for studies on influenza virus replication and pathogenesis, in part because this virus forms plaques in the absence of trypsin and serves as a model of highly pathogenic influenza virus. The HA of the A/WSN/33 virus was originally shown to be cleaved by plasmin, following activation of serum plasminogen in MDBK cells (22). The virulence properties of A/WSN/33 were subsequently linked to the neuraminidase (NA) gene (37) and the absence of a glycosylation site at position 130 of NA (25). In addition, the presence of a C-terminal lysine on NA was shown to be critical for the virulence properties of A/WSN/33, with the viral NA binding and sequestering of plasminogen on the cell surface, leading to increased cleavage of HA (15, 16). It has also been suggested that the HA of A/WSN/33 can be cleaved by an endosomal serine protease in MDBK cells (7).Based on the recent pandemic status of novel H1N1 viruses and the known importance of the HA cleavage site in viral pathogenicity (30), we assessed the presence of cleavage site changes, in addition to the conventional monobasic/polybasic cleavage sites, in the context of the virulence properties of H1 influenza viruses. We characterize the role of the bulky hydrophobic residues tyrosine and phenylalanine, found in the P2 cleavage positions of only A/WSN/33 and A/NWS/33 HA, respectively, and show that these residues are major virulence determinants for these viruses, allowing efficient use of plasmin for spread in vitro and in vivo.     

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

Background

Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.

Methods and Findings

Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.

Conclusion

In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.     

Henipaviruses: Gaps in the Knowledge of Emergence

Over the past 10 years many new viruses have been identified in Australia and the Asian region. The viruses have been isolated from, and/or identified in, a range of animals; some of these viruses are of veterinary and medical importance while others are new threats to biodiversity. Of these viruses, Hendra and Nipah viruses have emerged as significant zoonotic agents belonging to the family Paramyxoviridae and genus Henipavirus. These agents cause fatalities in a range of animals including horses, pigs, and humans. Both viruses have been isolated from flying foxes (genus Pteropus; suborder Megachiroptera), which are accepted as their natural hosts. Although some research has been undertaken on the viruses and their hosts, little is known about how these viruses emerge. We have attempted to investigate the current knowledge of the bat-Henipavirus ecology by discussing the range of bat viruses that exist (viral assemblage), the significance of evolving viruses, possible functional role(s) of viruses, the ecology of viruses and their hosts, and identifying possible drivers (selection pressures) that may culminate with the overlap of new potential viral hosts thereby facilitating the replication of fit viral populations in a new host–virus continuum(s). By undertaking such an analysis, we have attempted to identify key questions, which should be investigated if the factors involved in driving Henipavirus emergence are to be understood.     

Dinoflagellates,diatoms, and their viruses

Since the first discovery of the very high virus abundance in marine environments, a number of researchers were fascinated with the world of "marine viruses", which had previously been mostly overlooked in studies on marine ecosystems. In the present paper, the possible role of viruses infecting marine eukaryotic microalgae is enlightened, especially summarizing the most up-to-the-minute information of marine viruses infecting bloom-forming dinoflagellates and diatoms. To author's knowledge, approximately 40 viruses infecting marine eukaryotic algae have been isolated and characterized to different extents. Among them, a double-stranded DNA (dsDNA) virus "HcV" and a single-stranded RNA (ssRNA) virus "HcRNAV" are the only dinoflagellate-infecting (lytic) viruses that were made into culture; their hosts are a bivalve-killing dinoflagellate Heterocapsa circularisquama. In this article, ecological relationship between H. circularisquama and its viruses is focused. On the other hand, several diatom-infecting viruses were recently isolated and partially characterized; among them, one is infectious to a pen-shaped bloom-forming diatom species Rhizosolenia setigera; some viruses are infectious to genus Chaetoceros which is one of the most abundant and diverse diatom group. Although the ecological relationships between diatoms and their viruses have not been sufficiently elucidated, viral infection is considered to be one of the significant factors affecting dynamics of diatoms in nature. Besides, both the dinoflagellate-infecting viruses and diatom-infecting viruses are so unique from the viewpoint of virus taxonomy; they are remarkably different from any other viruses ever reported. Studies on these viruses lead to an idea that ocean may be a treasury of novel viruses equipped with fascinating functions and ecological roles.     

Analysis by Single-Gene Reassortment Demonstrates that the 1918 Influenza Virus Is Functionally Compatible with a Low-Pathogenicity Avian Influenza Virus in Mice

The 1918-1919 "Spanish" influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight "7:1" chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.