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.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Evaluation of Replication and Cross-Reactive Antibody Responses of H2 Subtype Influenza Viruses in Mice and Ferrets

H2 influenza viruses have not circulated in humans since 1968, and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The development of an H2 pandemic influenza virus vaccine candidate should therefore be considered a priority in pandemic influenza preparedness planning. We selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2), and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination inhibition and microneutralization assays. These data suggested that these three viruses may be suitable candidates for development as live attenuated H2 pandemic influenza virus vaccines.Influenza pandemics occur when a novel influenza virus enters a population with little preexisting immunity (36). During the pandemics of the last century, novel influenza viruses were introduced either directly from an avian reservoir (34) or were the result of reassortment between contemporaneously circulating human, avian, and swine influenza viruses (5, 29, 36). Due to the lack of preexisting immunity to the novel virus, morbidity and mortality rates are typically higher than in epidemics caused by seasonal influenza viruses (4).Although pandemic preparedness planning has largely focused on the highly pathogenic H5 and H7 avian influenza virus subtypes, the recent emergence of the 2009 pandemic H1N1 viruses underscores the need to consider other influenza virus subtypes as well. Of the 16 hemagglutinin (HA) influenza A virus subtypes that have been identified to date, H1, H2, and H3 have been known to cause influenza pandemics (7, 27), suggesting that these viruses are capable of sustained transmission and can cause disease in humans. While the H1 and H3 subtypes have cocirculated in humans since 1977, H2 influenza viruses have not circulated in humans since 1968 (36) and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The 1957 H2 pandemic virus was a reassortant that derived the HA, neuraminidase (NA), and PB1 genes from an avian virus and the remaining gene segments from the circulating H1N1 virus (15, 30). As H2 subtype viruses continue to circulate in avian reservoirs worldwide (12, 17, 18, 22, 33), they remain a potential pandemic threat. The development of an H2 influenza virus vaccine candidate should therefore be considered a priority in future pandemic influenza preparedness planning.Given the low likelihood that a previously selected vaccine virus will exactly match the pandemic virus, the ability to elicit a broadly cross-reactive antibody response to antigenically distinct viruses within a subtype is an important consideration in the selection of a pandemic influenza vaccine candidate. Previous studies have examined the ability of inactivated H2 influenza viruses to provide cross-protection against mouse-adapted variants of reassortant human viruses and an avian H2 influenza virus from 1978 (9, 14). Given the potential for live attenuated influenza virus vaccines to confer a great breadth of heterologous cross-protection (1, 2, 6, 35), we recently conducted a study evaluating cold-adapted A/Ann Arbor/6/1960 (AA CA), an H2 influenza virus used as the backbone of the seasonal live attenuated influenza A virus vaccine currently licensed in the United States (3). However, as H2 influenza virus continues to circulate widely and appear in migratory birds (10, 24, 26), in poultry markets (20), and in swine (21), with evidence of interregional gene transmission (19, 22), a more extensive evaluation of recent isolates may be warranted in the selection of a potential H2 pandemic vaccine candidate.H2 influenza viruses fall into three main lineages: a human lineage, a North American avian lineage, and a Eurasian avian lineage (29). In addition to viruses whose replicative ability in mammals has previously been established (11, 21, 23, 25), we selected a group of geographically and temporally diverse H2 influenza viruses from each lineage. We evaluated the kinetics of replication of each of these viruses in mice and ferrets and compared the abilities of these viruses to induce a broadly cross-reactive antibody response to determine which of these viruses would be suitable for further development as an H2 pandemic influenza vaccine candidate.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

A 27-Amino-Acid Deletion in the Neuraminidase Stalk Supports Replication of an Avian H2N2 Influenza A Virus in the Respiratory Tract of Chickens

The events and mechanisms that lead to interspecies transmission of, and host adaptation to, influenza A virus are unknown; however, both surface and internal proteins have been implicated. Our previous report highlighted the role that Japanese quail play as an intermediate host, expanding the host range of a mallard H2N2 virus, A/mallard/Potsdam/178-4/83 (H2N2), through viral adaptation. This quail-adapted virus supported transmission in quail and increased its host range to replicate and be transmitted efficiently in chickens. Here we report that of the six amino acid changes in the quail-adapted virus, a single change in the hemagglutinin (HA) was crucial for transmission in quail, while the changes in the polymerase genes favored replication at lower temperatures than those for the wild-type mallard virus. Reverse genetic analysis indicated that all adaptive mutations were necessary for transmission in chickens, further implicating quail in extending this virus to terrestrial poultry. Adaptation of the quail-adapted virus in chickens resulted in the alteration of viral tropism from intestinal shedding to shedding and transmission via the respiratory tract. Sequence analysis indicated that this chicken-adapted virus maintained all quail-adaptive mutations, as well as an additional change in the HA and, most notably, a 27-amino-acid deletion in the stalk region of neuraminidase (NA), a genotypic marker of influenza virus adaptation to chickens. This stalk deletion was shown to be responsible for the change in virus tropism from the intestine to the respiratory tract.Of the 16 known hemagglutinin (HA) subtypes, only 3 (H1, H2, and H3) have established stable lineages in humans. The H2N2 virus caused a pandemic in 1957 and circulated in the human population until reassortment of the H2N2 virus with an avian H3 virus resulted in the H3N2 pandemic of 1968 (36). Since then, H2N2 viruses have been absent from the human population; however, the H2 subtype has been repeatedly isolated in wild-bird surveillance, and its HA has been found to be antigenically similar to the H2 pandemic virus HA (23, 25, 36). An H2 influenza virus containing human-like receptor specificity was recently isolated as an H2N3 avian-swine reassortant. This virus caused disease and was transmitted in swine and ferrets (24), indicating that this subtype continues to circulate and mutate and can cross the species barrier to mammals. The repeat introduction of a novel H1N1 pandemic this past year (12, 37) highlights the need to understand the mechanisms of introduction, adaptation, and transmission of avian H2N2 influenza viruses in terrestrial birds and potentially mammalian species.Our previous study built on reports that Japanese quail (Coturnix coturnix) play an important role as an intermediate host in the adaptation of avian influenza viruses to land-based birds (38). Japanese quail are typically more susceptible to aquatic influenza viruses than other terrestrial poultry. These viruses establish infection in the respiratory tract, and shedding occurs via aerosol (2, 19, 26, 34, 38, 43). Quail have been implicated in the transmission of avian influenza viruses, such as H5N1 and H9N2 viruses, which have crossed the species barrier to infect humans (9, 14, 15, 22, 28). The susceptibility of quail to multiple subtypes and their role in interspecies transmission led to their removal from live-bird markets in Hong Kong in 2000; however, they continue to be an integral part of live-bird markets throughout the world. Their role as potential intermediate hosts requires further study to identify important molecular markers in the adaptation via quail of avian viruses to other terrestrial poultry, and possibly to humans.The molecular determinants of the host range and pathogenesis of influenza A viruses have been linked to multiple regions of the 11 genes, most notably those encoding the viral surface glycoproteins (HA and neuraminidase [NA]) and the polymerase proteins (PB2, PB1, PA, and NP). However, a comprehensive map of the various determinants remains incomplete, and the molecular mechanisms involved are unclear. In our previous report, we demonstrated that through the use of quail as an intermediate host, a mallard H2N2 influenza virus, A/mallard/Potsdam/178-4/83 (mall/178), which in its wild-type (wt) form was unable to be transmitted in quail or to establish an efficient infection in chickens, was able, in its adapted form (qa-mall/178), not only to be transmitted to sentinel quail but also to replicate to efficient levels in the chicken intestinal tract and to be transmitted to sentinel cagemates via the fecal-oral route. This adaptation was the result of six serial passages of lung homogenates in quail that led to six amino acid changes in four genes (38). Here we present data confirming the role that Japanese quail play in the transmission of this mall/178 H2N2 virus in land-based birds. Reverse genetics studies confirmed that the amino acid changes produced during the adaptation in quail were necessary for the infection of chickens with this virus and for its transmission in chickens. Further adaptation of the qa-mall/178 H2N2 virus in chickens, aimed at establishing replication in the respiratory tract, resulted in a deletion in the stalk region of the NA, which supported replication in the chicken trachea and lung. This 27-amino-acid deletion in the stalk region of the N2 NA is characteristic of the adaptation of aquatic influenza viruses to domestic poultry, particularly chickens (3, 5, 29). Our work indicates that through the use of quail as an intermediate host, this mallard H2N2 virus is able to further adapt within an additional terrestrial poultry species, potentially improving its chances of expanding its host range further.     

Neuraminidase Receptor Binding Variants of Human Influenza A(H3N2) Viruses Resulting from Substitution of Aspartic Acid 151 in the Catalytic Site: a Role in Virus Attachment?

Changes in the receptor binding characteristics of human H3N2 viruses have been evident from changes in the agglutination of different red blood cells (RBCs) and the reduced growth capacity of recently isolated viruses, particularly in embryonated eggs. An additional peculiarity of viruses circulating in 2005 to 2009 has been the poor inhibition of hemagglutination by postinfection ferret antisera for many viruses isolated in MDCK cells, including homologous reference viruses. This was shown not to be due to an antigenic change in hemagglutinin (HA) but was shown to be the result of a mutation in aspartic acid 151 of neuraminidase (NA) to glycine, asparagine, or alanine, which caused an oseltamivir-sensitive agglutination of RBCs. The D151G substitution was shown to cause a change in the specificity of NA such that it acquired the capacity to bind receptors, which were refractory to enzymatic cleavage, without altering its ability to remove receptors for HA. Thus, the inhibition of NA-dependent agglutination by the inclusion of oseltamivir carboxylate in the assay was effective in restoring the anti-HA specificity of the hemagglutination inhibition (HI) assay for monitoring antigenic changes in HA. Since the NA-dependent binding activity did not affect virus neutralization, and virus populations in clinical specimens possessed, at most, low levels of the “151 mutant,” the biological significance of this feature of NA in, for example, immune evasion is unclear. It is apparent, however, that an important role of aspartic acid 151 in the activity of NA may be to restrict the specificity of the NA interaction and its receptor-destroying activity to complement that of HA receptor binding.A characteristic feature of human influenza viruses is their frequent antigenic change to evade host immunity and cause recurrent annual epidemics of disease. As a consequence, available vaccines do not confer long-term immunity, and their composition is regularly reviewed by the WHO Global Influenza Surveillance Network (GISN) and updated to reflect changes in the antigenic characteristics of circulating viruses (2, 43).The two surface glycoproteins of the virus, hemagglutinin (HA) and neuraminidase (NA), perform clearly defined complementary roles in virus infection. Virus HA is responsible for the attachment of virus to sialic acid-containing glycoconjugates on susceptible cells, and it is antibody to HA, which neutralizes virus infectivity, that is of prime importance in immunity (37). Antibody to NA also contributes to the suppression of disease (3, 16). NA is responsible for destroying receptors for HA by removing the terminal sialic acid moieties from, and thereby inactivating, potentially inhibitory molecules such as mucins in the respiratory tract and from receptors on the surface of virus-infected cells to promote the release of progeny virus, thereby aiding virus transmission (1, 21, 26, 34). Thus, since NA may also cleave receptors from target cells, the maintenance of a balance between the receptor binding and receptor-destroying properties of HA and NA, respectively, is important in optimizing their respective functions in virus replication and maintaining epidemic potential (29, 41).Virus neutralization is principally the result of the inhibition of the attachment of HA to its receptor (9), and the hemagglutination inhibition (HI) assay is a simple and generally robust surrogate assay for monitoring antigenic relationships among viruses and is the principal basis for changes in vaccine composition recommended by the WHO (2, 43). Many mutations resulting in antibody escape cause amino acid substitutions close to the HA receptor binding site, which may influence receptor binding affinity and/or specificity as well as antigenicity (7, 37, 44). In turn, changes in receptor avidity and binding characteristics of HA, possibly associated with antigenic changes, may influence the effectiveness of the antibody inhibition of the agglutination of red blood cells (RBCs) in the standard HI assay and thereby complicate the interpretation of antigenic relationships (8, 12, 44).Antigenic drift among H3N2 viruses has been more muted in recent years. Whereas the antigenic drift of viruses between 1992 and 1997 required four changes in the H3N2 vaccine component, there was little progressive antigenic change in the HAs of A/Sydney/5/97(H3N2)-like viruses during the subsequent 5 years prior to the emergence of the A/Fujian/411/2002(H3N2)-like viruses (20) or among the more recently isolated A/Wisconsin/67/2005(H3N2)-like viruses between 2005 and 2009 (see below). Changes in the receptor binding characteristics of HA have been apparent from changes in the spectrum of RBCs agglutinated by the viruses, e.g., the loss of agglutination of chicken RBCs by H3N2 viruses circulating in the early 1990s (28, 31) and more recently by the poorer growth characteristics following the emergence of A/Fujian/411/2002-like viruses, particularly in embryonated eggs (22), which has “hampered” the selection of suitable vaccine candidates. Amino acid substitutions in residues 190 and 226 in the HA receptor binding site were implicated in the changes in hemagglutination (28, 31), while changes that increased the receptor binding of HA or decreased the enzyme activity of NA were shown to increase the growth of virus in eggs (22). Studies of differences among H3N2 viruses in their relative abilities to bind to and elute from RBCs of different species led Gulati et al. (11) to conclude that the more recently isolated Fujian/411/2002-like viruses bound different forms of sialic acid, which were not cleaved by the virus enzyme. However, studies using glycan arrays failed to identify any differences in receptor binding specificities, or in the amino acid sequences, of HAs that correlated with differences in hemagglutination (18).Another peculiar feature of many MDCK cell isolates of A/Wisconsin/67/2005-like viruses, isolated between 2005 and 2009, has been the poor inhibition of agglutination of turkey (and guinea pig) RBCs by reference postinfection ferret antisera, with the consequent difficulty in interpreting antigenic relationships from HI data (as reported herein). Here we describe the results of a series of experiments that demonstrate that this phenomenon is not due to changes in antigenicity or simply to changes in HA receptor binding but is the result of the selection in MDCK cells of changes in NA that promote NA-dependent, NA inhibitor-sensitive hemagglutination, which is refractory to inhibition by anti-HA antibody. The replacement of aspartic acid 151 of NA by glycine, which did not affect significantly the activity of the enzyme or its ability to remove receptors for HA, was shown to alter the specificity of NA, resulting in the attachment of virus via its NA to sialic acid receptors refractory to catalytic cleavage.     

Eukaryotic Viruses in Wastewater Samples from the United States

Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments.Millions of viruses and bacteria are excreted in human fecal matter (5, 17, 82), and current methods of sewage treatment do not always effectively remove these organisms (74, 76-78). The majority of treated wastewater, as well as untreated sewage, drains into the marine environment (1) and has the potential to threaten environmental (e.g., nutrients and chemicals) (45) and public (e.g., pathogen exposure via swimming and seafood consumption) (1, 24, 28, 29, 33, 44, 57, 63) health. Currently, the U.S. Environmental Protection Agency (EPA) mandates the use of bacterial indicators such as fecal coliforms and enterococci to assess water quality (75). Although monitoring of these bacteria is simple and inexpensive, it has been shown that fecal-associated bacteria are not ideal indicators of fecal pollution.Since fecal-associated bacteria are able to live in sediments in the absence of fecal pollution (18, 32, 55), their resuspension into the water column can result in false-positive results and mask correlations between their concentrations and the extent of recent fecal pollution. Another unfavorable characteristic of current bacterial indicators is their inability to predict or correlate with the presence of pathogenic viruses (25, 40, 41, 64, 80). Human-pathogenic viruses associated with feces are generally more robust than enteric bacteria and are not as easily eliminated by current methods of wastewater treatment (43, 80). For example, adenoviruses are more resilient to tertiary wastewater treatment and UV disinfection than are bacterial indicators of fecal pollution (74). Since bacterial indicators cannot accurately depict the risks to human health from fecal pollution, several studies have proposed the use of a viral indicator of wastewater contamination (35, 41, 61).While it is impractical to monitor the presence of all viral pathogens related to wastewater pollution, the development of an accurate viral indicator of sewage contamination is needed for enhanced water quality monitoring. Enteric viruses (including viruses belonging to the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae) are transmitted via the fecal-oral route and are known to be abundant in raw sewage. These viruses have been used to identify fecal pollution in coastal environments throughout the world (27, 35, 39, 40, 48, 50, 56, 57, 63, 64, 67-69, 71, 80). To determine which viruses are effective indicators of fecal pollution, it is first necessary to establish a broad, baseline understanding of the many diverse groups of eukaryotic viruses in raw sewage. Several studies have identified adenoviruses, noroviruses, reoviruses, rotaviruses, and other enteroviruses (e.g., polioviruses, coxsackie viruses, and echoviruses) in raw sewage in Australia, Europe, and South Africa (30, 47, 58, 76-78). However, no broad baseline data on the presence of eukaryotic viruses in raw sewage in the United States currently exist.This study determined the presence of 10 viral groups (adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses) in raw sewage samples collected throughout the United States. All viral groups that were detected in raw sewage were then examined further to determine if they were also present in final treated wastewater effluent. These 10 viral groups were chosen because of their potential to be transmitted via the fecal-oral route, suggesting that they might be found in raw sewage. Many of these viruses (excluding adenoviruses, enteroviruses, noroviruses, reoviruses, and rotaviruses) have not been studied in sewage despite their likely presence. Picobirnaviruses have been detected in individual fecal samples (12, 70, 79, 82); however, their presence has never been analyzed in collective waste, nor have they been proposed to be potential markers of fecal pollution. This study identified potential viral indicators of fecal pollution and will have important applications to water quality monitoring programs throughout the country.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.Since the first detection of the Asian lineage of highly pathogenic avian influenza (HPAI) virus (H5N1) in southern China in 1996, H5N1 virus infection in birds has continued for 13 years in Asia, acquiring pathogenicity not only in birds but also in mammals. In 1997, the H5N1 Hong Kong isolates caused illness and death in a variety of terrestrial birds and even in humans (9, 37, 48, 49). In 2001, emerging H5N1 Hong Kong isolates were more pathogenic to chickens and the mean death time (MDT) was 2 days without any prior clinical signs (12). In 2003 to 2004, the H5N1 epizootic occurred simultaneously in East Asian countries (22, 30). The 2003/2004 H5N1 isolates caused death in taxonomically diverse avian species, including domestic ducks (46, 47, 51), and humans (7, 55). Furthermore, the first indication of wild aquatic bird involvement occurred at recreational parks in Hong Kong in late 2002 to 2003 (46), and then migratory aquatic bird die-off occurred in 2005 at Qinghai Lake in China (6, 24). The broad host spectrum and increased pathogenicity of H5N1 viruses to diverse bird species raise serious concerns about the worldwide spread of the virus by migratory birds.According to the international criteria, HPAI viruses are defined by over 75% mortality in 4- to 8-week-old chickens following an intravenous pathogenicity test or an intravenous pathogenicity index (IVPI) of more than 1.2 in 6-week-old chickens (34); however, there are some variations in pathogenicity intensity among the HPAI viruses in chickens (1, 3, 5, 12, 15, 28, 31, 48, 50-52, 57). Most of the HPAI viruses that were isolated before 1996 cause severe clinical signs (e.g., ruffled feathers, depression, labored breathing, and neurological signs) and severe gross lesions (e.g., head and face edema, cyanosis, subcutaneous hemorrhages in combs and leg shanks, and necrosis of combs and wattles) in chickens (1, 3, 15, 31, 50, 52, 57). These viruses usually kill chickens 3 to 6 days after intranasal inoculation. On the other hand, the recently emerged Asian H5N1 HPAI viruses are more virulent and kill chickens within 1 to 2 days without causing typical clinical signs and gross lesions (5, 12, 27, 33, 48, 51), although some Asian H5N1 viruses, such as A/Goose/Guangdong/2/96 (23), A/goose/Hong Kong/437-10/99 (17), and A/chicken/Indonesia/7/03 (58), were less virulent. To successfully control HPAI in poultry, it is important to better understand the mechanisms of increased pathogenicity of recent H5N1 HPAI viruses in chickens.The Asian H5N1 HPAI virus has another important characteristic, which is its capability of crossing host-species barriers. It was reported that the H5N1 virus can infect and cause death in mammals such as mice (5, 9, 12, 14, 29), cats (21), tigers (2), ferrets (11, 26), monkeys (40), and humans (7, 49, 55). High-level inductions of proinflammatory cytokines in mammals infected with the H5N1 viruses, referred to as “cytokine storms,” have been hypothesized to contribute to the severity of pathological changes and ultimate death (4, 7, 13, 45, 55). Cytokine and chemokine dysregulation was detected in clinical cases of H5N1-infected humans (8, 13, 36) and also in monkeys experimentally infected with the H1N1 Spanish flu strain (20). In a mouse model, lymphocyte apoptosis and cytokine dysregulation have been proposed to contribute to the severity of the disease caused by H5N1 (56). Investigations with transgenic mice deficient in each cytokine gene suggest that tumor necrosis factor alpha (TNF-α) may contribute to morbidity and interleukin-1 (IL-1) may be important for virus clearance (53). However, mice deficient in TNF-α or IL-6 succumb to infection with H5N1, and cytokine inhibition treatment does not prevent death (42), suggesting that therapies targeting the virus rather than cytokines may be preferable. Thus, the significance of elevated proinflammatory cytokine responses in the pathogenesis of H5N1-infected mammals requires further studies.In contrast, little is known about proinflammatory cytokine responses and their roles in pathogenicity in chickens infected with HPAI viruses, including the recent Asian H5N1 viruses. It was reported that infection with an HPAI virus results in upregulation of gene expression of gamma interferon (IFN-γ) and inducible nitric oxide synthase (58). However, the roles of proinflammatory cytokines in disease severity and outcomes in chickens infected systemically with HPAI viruses are largely unknown. The less-virulent Asian H5N1 virus, which causes severe clinical signs and gross lesions in chickens (17, 23, 27, 58), would be a valuable tool for investigating the role of proinflammatory cytokines in chickens infected with HPAI viruses, as well as for exploring the pathogenesis of the more-virulent Asian H5N1 HPAI virus, because of the antigenic and molecular similarities between them.In this study, we compared the pathogenicities in chickens of the less-virulent and more-virulent Asian H5N1 HPAI viruses based on MDT, fever, cytokine responses, and viral replication. Our results suggest that the shift in the Asian H5N1 virus to increased virulence may be associated with efficient and rapid replication of the virus in chickens, accompanied by early destruction of host immune responses and followed by peracute death before fever can be induced. Finally, we discuss candidate genes that may account for the high pathogenicity of Asian H5N1 HPAI viruses in chickens.     

Probing the Spatial Organization of Measles Virus Fusion Complexes

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.Paramyxoviruses infect cells through fusion of the viral envelope with target cell membranes. For all members of the Paramyxovirinae subfamily, this involves the concerted action of two envelope glycoproteins, the fusion (F) and attachment (H, HN, or G, depending on the Paramyxovirinae genus) proteins. Both proteins feature short lumenal tails, a single transmembrane domain, and large ectodomains. The F protein, in type I orientation, forms homotrimers, while homodimers or homotetramers have been suggested as functional units for attachment proteins of different Paramyxovirinae subfamily members (7, 14, 28, 41, 49, 50, 66). For entry, upon receptor binding, the attachment protein is considered to initiate a series of conformational rearrangements in the metastable prefusion F protein (15, 77), which ultimately brings together transmembrane domains and fusion peptides and, thus, donor and target membranes (3, 32, 45, 53, 80).Multiple studies have demonstrated that specific interactions between compatible F and attachment proteins of paramyxovirinae are imperative for the formation of functional fusion complexes (6, 29, 36, 42, 43, 56, 75). However, the molecular nature of these interactions and the spatial organization of functional glycoprotein hetero-oligomers remain largely unknown. Individual ectodomain and partial ectodomain crystal structures have been obtained for different paramyxovirus F (13, 76, 77) and attachment (8, 14, 17, 28, 35, 79) proteins, respectively. For F, a stabilized human parainfluenza virus type 5 (HPIV5) ectodomain that is believed to represent a prefusion conformation folds into a globular head structure that is attached to the transmembrane domains through a helical stalk consisting of the membrane-proximal heptad repeat B (HR-B) domains (77). For the attachment protein, a globular head that harbors the receptor binding sites is considered to be connected to the transmembrane region through extended stalk domains (34, 78). Crystal structures of isolated head domains have been solved for several paramyxovirus attachment proteins, including measles virus (MeV) H, and reveal the six-blade propeller fold typical of sialidase structures (8, 14, 17, 28, 79). However, morbilliviruses recognize proteinaceous receptors (for MeV, the regulator of complement activation [CD46] and/or signaling lymphocytic activation molecule [SLAM], depending on the virus strain) (21, 40, 46, 51, 64, 65). X-ray data do not extend to the stalk domains, but circular dichroism analysis (78) and structure predictions (36, 78) support an α-helical coiled-coil configuration of the stalk.The nature of individual residues that engage in specific intermolecular interactions between glycoproteins of paramyxovirinae prior to refolding has been studied most extensively for the attachment protein. The stalk domains of several paramyxovirus HN proteins have been implicated in mediating specificity for their homotypic F proteins (18, 20, 43, 63, 70, 72). We have found that this extends to MeV and canine distemper virus H and, thus, to paramyxovirinae recognizing proteinaceous receptors (36), supporting the general hypothesis that F-interacting residues may reside in the stalk region of the attachment protein (30, 78).Considerably less information concerning the nature of F microdomains that mediate attachment protein specificity is available. Among the few exceptions are peptides derived from Newcastle disease virus (NDV) and Sendai virus F HR-B domains, which interact with soluble variants of the respective HN proteins in vitro (25, 67). Multiple domains have been suggested to mediate specificity of HPIV2 F for its HN (69). However, a conclusive N-glycan shielding study (43) and structural information (77, 78) argue against direct contacts between NDV F HR-B domains and HN in native glycoprotein complexes. Thus, the role of individual HPIV2 F residues in HN binding is unclear (25, 43).Building on the observation that MeV H is able to engage in productive heterotypic interactions with F proteins derived from some but not all isolates of closely related canine distemper virus, we have recently identified residues in morbillivirus F (MeV F residue 121) and H (H stalk residues 110 to 114) that interdependently contribute to physical MeV glycoprotein interaction and F triggering for fusion (36). While these residues could mediate reciprocal glycoprotein specificity through long-range effects, molecular modeling of the MeV H stalk in an α-helical conformation has posited F residue 121 at the same level above the viral envelope as H residues 110 to 114, making direct contacts structurally conceivable (36). This spatial organization of functional fusion complexes furthermore provides a comprehensive explanation for previous demonstrations of a specific role for attachment protein stalk domains of paramyxovirinae in functional and physical interactions with F (18, 43, 63, 70, 72). However, this “staggered-head” model mandates positioning the globular head of the attachment protein above the prefusion F trimer (36), as opposed to a suggested “parallel-head” alignment of the glycoproteins (31, 47). The latter is mostly based on transmission electron microscopy micrographs of viral particles apparently showing glycoprotein spikes of equal length (33). Unfortunately, these images lack the resolution for an identification of the molecular nature of the spikes (attachment or F protein) or the distinguishing between densely packaged H and F head domains of different heights and laterally aligned head domains. Indeed, a recent single-particle reconstruction based on cryo-electron microscopy images of HPIV5 particles revealed that defined spikes correspond to F in a postfusion conformation, which was interpreted as a product of possible premature F refolding (38). These two-dimensional images of heavy-metal-stained particles did not reveal F spikes in a prefusion conformation. Rather, a dense surface layer was considered to correspond to prefusion glycoprotein hetero-oligomers (38). In addition to further-advanced image reconstructions, biochemical assessment of alternative docking modes is imperative for the elucidation of the organization of functional fusion complexes of paramyxovirinae.In this study, we subjected central predictions of the hypothetical alignment models to experimental analysis. By employing carbohydrate shielding, directed mutagenesis, and variation of the length of the H stalk domain, we examined the proximity of different regions of the H stalk to F, probed a role of individual residues around the previously identified H stalk section from positions 110 to 114 in the formation of functional fusion complexes, tested the effect of varying the length of the H stalk membrane proximal and membrane distal to this section, and explored the general possibility of whether specific contacts between the prefusion F and H head domains are required for F triggering. Experimental data were interpreted in the light of a working model of MeV glycoprotein hetero-oligomers prior to receptor binding.     

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.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 × 10⁶ to 10 × 10⁶ viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.Despite years of intense research, a truly protective AIDS vaccine is far away. Suboptimal immunogenicity, inadequate antigen presentation, and inappropriate immune system activation are believed to have contributed to these disappointing results. However, several lines of evidence suggest that the control or prevention of infection is possible. For example, despite repeated exposures, some individuals escape infection or delay disease progression after being infected (1, 14, 15). Furthermore, passively infused neutralizing antibodies (NA) (28, 42, 51) or endogenously expressed NA derivatives (29) have been shown to provide protection against intravenous simian immunodeficiency virus challenge. On the other hand, data from several vaccine experiments suggest that cellular immunity is an important factor for protection (6, 32). Therefore, while immune protection against human immunodeficiency virus (HIV) and other lentiviruses appears feasible, the strategies for eliciting it remain elusive.Because of its crucial role in viral replication and infectivity, the HIV envelope (Env) is an attractive immunogen and has been included in nearly all vaccine formulations tested so far (28, 30, 31). Env surface (SU) and transmembrane glycoproteins (gp) are actively targeted by the immune system (9, 10, 47), and Env-specific antibodies and cytotoxic T lymphocytes (CTLs) are produced early in infection. The appearance of these effectors also coincides with the decline of viremia during the acute phase of infection (30, 32). Individuals who control HIV infection in the absence of antiretroviral therapy have Env-specific NA and CTL responses that are effective against a wide spectrum of viral strains (14, 23, 35, 52, 60). At least some of the potentially protective epitopes in Env appear to interact with the cellular receptors during viral entry and are therefore highly conserved among isolates (31, 33, 39, 63). However, these epitopes have complex secondary and tertiary structures and are only transiently exposed by the structural changes that occur during the interaction between Env and its receptors (10, 11, 28). As a consequence, these epitopes are usually concealed from the immune system, and this may explain, at least in part, why Env-based vaccines have failed to show protective efficacy. Indeed, data from previous studies suggested that protection may be most effectively triggered by nascent viral proteins (22, 28, 30, 48, 62).We have conducted a proof-of-concept study to evaluate whether presenting Env to the immune system in a manner as close as possible to what occurs in the context of a natural infection may confer some protective advantage. The study was carried out with feline immunodeficiency virus (FIV), a lentivirus similar to HIV that establishes persistent infections and causes an AIDS-like disease in domestic cats. As far as it is understood, FIV evades immune surveillance through mechanisms similar to those exploited by HIV, and attempts to develop an effective FIV vaccine have met with difficulties similar to those encountered with AIDS vaccines (25, 37, 66). In particular, attempts to use FIV Env as a protective immunogen have repeatedly failed (13, 38, 58). Here we report the result of one experiment in which specific-pathogen-free (SPF) cats primed with a DNA immunogen encoding FIV Env and feline granulocyte-macrophage colony-stimulating factor (GM-CSF) and boosted with viable, autologous T lymphocytes ex vivo that were transduced to express Env and feline interleukin 15 (IL-15) showed a remarkable level of protection against challenge with ex vivo FIV. Consistent with recent findings indicating the importance of NA in controlling lentiviral infections (1, 59, 63), among the immunological parameters investigated, only the titers of NA correlated inversely with protection. Collectively, the findings support the notion that Env is a valuable vaccine immunogen but needs to be administered in a way that permits the expression of its full protective potential.     

The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model

Adaptation of influenza A viruses to a new host species usually involves the mutation of one or more of the eight viral gene segments, and the molecular basis for host range restriction is still poorly understood. To investigate the molecular changes that occur during adaptation of a low-pathogenic avian influenza virus subtype commonly isolated from migratory birds to a mammalian host, we serially passaged the avirulent wild-bird H5N2 strain A/Aquatic bird/Korea/W81/05 (W81) in the lungs of mice. The resulting mouse-adapted strain (ma81) was highly virulent (50% mouse lethal dose = 2.6 log₁₀ 50% tissue culture infective dose) and highly lethal. Nonconserved mutations were observed in six viral genes (those for PB2, PB1, PA, HA, NA, and M). Reverse genetic experiments substituting viral genes and mutations demonstrated that the PA gene was a determinant of the enhanced virulence in mice and that a Thr-to-Iso substitution at position 97 of PA played a key role. In growth kinetics studies, ma81 showed enhanced replication in mammalian but not avian cell lines; the PA_97I mutation in strain W81 increased its replicative fitness in mice but not in chickens. The high virulence associated with the PA_97I mutation in mice corresponded to considerably enhanced polymerase activity in mammalian cells. Furthermore, this characteristic mutation is not conserved among avian influenza viruses but is prevalent among mouse-adapted strains, indicating a host-dependent mutation. To our knowledge, this is the first study that the isoleucine residue at position 97 in PA plays a key role in enhanced virulence in mice and is implicated in the adaptation of avian influenza viruses to mammalian hosts.Migratory waterfowl are the natural reservoir of influenza A viruses (11, 53). The viruses replicate efficiently in their natural hosts but replicate poorly if at all in other species (53). However, these viruses can undergo adaptation or genetic reassortment to infect other hosts (43, 44, 53), including humans. Since 1997, the World Health Organization has documented more than 400 laboratory-confirmed cases of human infection with H5N1 avian influenza virus (54).The molecular basis of influenza virus host range restriction and adaptation to a new host species is poorly understood. Mutations associated with cross-species adaptation are thought to be associated with increased virulence (30). Therefore, studies in animal models have attempted to identify the viral molecular determinants of virulence in specific hosts. Reverse genetics (Rg) methods have also identified genetic differences that affect virus virulence and host range, including changes in the viral internal proteins. Experimental infection of mouse lungs is an effective approach for understanding influenza virus virulence and adaptation (reviewed by A. C. Ward [51]). To acquire virulence in mice, influenza A viruses usually must adapt to these hosts over several consecutive generations (serial passages) in the lungs or brain (1, 25, 30). Previous studies have found that the acquisition of virulence during adaptation in the mouse model is associated with mutations in the HA, NP, NA, M, and NS genes and one or more polymerase genes (2, 3, 18, 36, 42, 51). The polymerase basic protein 2 (PB2) gene is a particularly well-characterized polymerase subunit (7, 23, 40, 46). The PB1 and polymerase acidic protein (PA) genes have been implicated in mouse lung virulence (5, 18, 36, 39, 49) but have shown no evidence of having acquired mutations during adaptation (52). However, the many studies conducted to date have focused mainly on highly pathogenic avian influenza (HPAI) viruses such as the H1N1, H5N1, and H7N7 subtypes (7, 23, 48, 50).Various low-pathogenic avian influenza (LPAI) viruses are considered to be potential genetic contributors to the next pandemic strain. Lee et al. (2009) recently reported the presence of avian-like LPAI H5N2 viruses in a number of Korean swine and proposed that the efficient transmissibility of the swine-adapted H5N2 virus could facilitate spread of the virus. They suggested that this adapted virus could potentially serve as a model for pandemic outbreaks of HPAI (e.g., H5N1 and H7N7) virus or could become a pandemic strain itself (21). These findings prompted our interest in the adaptation of an LPAI virus often harbored by wild migratory birds of South Korea. In our ongoing surveillance from 2004 to 2008, approximately 27% of the viruses isolated were of the H5N2 subtype (unpublished data). Studies show that influenza viruses with different genetic backgrounds can acquire different mutations during adaptation in mice. Therefore, we sought to determine whether this common H5N2 virus (nonlethal in mice) would undergo changes different from those observed in highly virulent viruses during adaptation in mice. Wild-bird influenza virus strain A/Aquatic bird/Korea/W81/05 (W81) was adapted in mice over 11 passages and became highly virulent. To identify molecular determinants of this adaptation and altered virulence, we used Rg-generated recombinant viruses to compare the parental and mouse-adapted strains. Here we show that the PA subunit of the polymerase complex, independently of PB2, contributed to adaptation and increased virulence in our mammalian model.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).