Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.Influenza virus infections continue to represent a major public health threat. Epidemics caused by influenza A viruses (FLUAV) occur regularly, often leading to excess mortality in susceptible populations, and may result in devastating pandemics for humans (37). An avian FLUAV originating from Asia and currently circulating among domestic birds in many countries has the potential to infect and kill people. If further adaptation to humans occurs, this virus strain might become the origin of a future pandemic (57). Although influenza viruses are well characterized, the molecular determinants governing cross-species adaptation and enhanced virulence of emerging virus strains in humans are presently not well understood. The known viral virulence factors are the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), the nonstructural proteins NS1 and PB1-F2, and the polymerase complex. HA and NA are of key importance for host specificity and virulence because they determine specific receptor usage and efficient cell entry, as well as formation and release of progeny virus particles. NS1 is a multifunctional protein with interferon-antagonistic activity able to suppress host innate immune responses (11, 15). The small proapoptotic protein PB1-F2 induces more-severe pulmonary immunopathology and increases susceptibility to secondary bacterial pneumonia (3, 30). Recent evidence indicates that the polymerase complex consisting of the three subunits PA, PB1, and PB2 is also a determinant of virulence. Analyses of the 1918 pandemic virus showed that PB1 contributed to the high virulence of this deadly strain (38, 54, 56). Likewise, PB1 also contributed to the unusually high virulence of the pandemic viruses of 1957 and 1968 (23, 47). Interestingly, in recent avian-to-human transmissions of H5N1 and H7N7 viruses, the PB2 subunit was found to play a critical role (32, 40). Molecular studies revealed that an E-to-K exchange at position 627 of PB2 facilitates efficient replication of avian viruses in human cells (24, 33) and determines pathogenicity in mammals (18, 32, 51). Furthermore, recent analyses of highly pathogenic H5N1 viruses demonstrated that PA is involved in high virulence of these avian strains for both avian and mammalian hosts (21, 27).Moderately pathogenic FLUAV strains can be rendered more pathogenic by repeated passages in experimentally infected animals (2, 13, 16, 49, 55). During such adaptations, the evolving viruses frequently seem to acquire virulence-enhancing mutations in the polymerase genes. We recently characterized a virus pair with strikingly different virulences in mice and showed that the virulence-enhancing mutations of the highly virulent strain mapped to the HA, NA, and polymerase genes (13). The two A/Puerto Rico/8/34 (A/PR/8/34) strains are referred to here as high-virulence A/PR/8/34 (hvPR8) and low-virulence A/PR/8/34 (lvPR8). Interestingly, hvPR8 is also highly virulent in mice that carry functional alleles of the Mx1 resistance gene (17), most likely because it replicates rapidly enough to evade the innate immune response of naïve hosts (13).Here, we systematically analyzed which mutations in the three viral polymerase genes contribute to enhanced virulence of hvPR8. We found that two conservative mutations, one in PB2 (I504V) and one in PA (I550L), account for the high-virulence phenotype and that each single mutation considerably increases the activity of the reconstituted polymerase complex. Interestingly, in a new mouse adaptation experiment, the same I504V mutation in PB2 was acquired again by a highly virulent isolate as the only change in the polymerase complex. In contrast, another virulent, mouse-adapted isolate acquired two different mutations in PA and PB1. In this case, the change in PA had a greater impact on both enhanced polymerase activity and enhanced virulence than the mutation in PB1. These data demonstrate that increased polymerase activity contributes to high virus virulence and that human FLUAV have a range of options to achieve this goal.(This work was conducted by Thierry Rolling, Iris Koerner, and Petra Zimmermann in partial fulfillment of the requirements for an M.D. degree from the Medical Faculty [T.R.] or a Ph.D. degree from the Faculty of Biology [I.K. and P.Z.] of the University of Freiburg, Germany.)     

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.     

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.In April 2009, the H1N1 influenza A virus of swine origin was detected in humans in North America (9, 12, 42). Evidence for its origin came from analyses of the viral genome, with six gene segments displaying the closest resemblance to American “triple-reassortant” swine viruses and two to “Eurasian-lineage” swine viruses (13, 42). After this first detection in humans, the virus spread rapidly around the globe, starting the first influenza pandemic of the 21st century. The 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively mild, with a spectrum of disease ranging from subclinical infections or mild upper respiratory tract illness to sporadic cases of severe pneumonia and acute respiratory distress syndrome (3, 11, 27, 29, 30, 37). Overall, the case-fatality rate during the start of the pandemic was not significantly higher than in seasonal epidemics in most countries. However, a marked difference was observed in the case-fatality rate in specific age groups, with seasonal influenza generally causing highest mortality in elderly and immunocompromised individuals, and the pdmH1N1 affecting a relatively large proportion of (previously healthy) young individuals (3, 11, 27, 29, 30, 37).Determinants of influenza A virus virulence have been mapped for a wide variety of zoonotic and pandemic influenza viruses to the polymerase genes, hemagglutinin (HA), neuraminidase (NA), and nonstructural protein 1 (NS1). Such virulence-associated substitutions generally facilitate more efficient replication in humans via improved interactions with host cell factors. Since most of these virulence-associated substitutions were absent in the earliest pdmH1N1s, it has been speculated that the virus could acquire some of these mutations, potentially resulting in the emergence of more pathogenic viruses. Such virulence markers could be acquired by gene reassortment with cocirculating influenza A viruses, or by mutation. The influenza virus polymerase genes, in particular PB2, have been shown to be important determinants of the virulence of the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the transmission of the 1918 H1N1 Spanish influenza virus (17, 26, 34, 51). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is generally found in avian influenza viruses, while human viruses have a lysine (K), and this mutation was described as a determinant of host range in vitro (48). Given that all human and many zoonotic influenza viruses of the last century contained 627K, it was surprising that the pdmH1N1 had 627E. In addition, an aspartate (D)-to-asparagine (N) substitution at position 701 (D701N) of PB2 has previously been shown to expand the host range of avian H5N1 virus to mice and humans and to increase virus transmission in guinea pigs (26, 46). Like E627K, D701N was absent in the genome of pdmH1N1. Thus, the pdmH1N1 was the first known human pandemic virus with 627E and 701D, and it has been speculated that pdmH1N1 could mutate into a more virulent form by acquiring one of these mutations or both. Recently, it was shown that neither E627K nor D701N in PB2 of pdmH1N1 increased its virulence in ferrets and mice (18). The PB1-F2 protein has previously also been associated with high pathogenicity of the 1918 H1N1 and HPAI H5N1 viruses (8). The PB1-F2 protein of the pdmH1N1 is truncated due to premature stop codons. However, restoration of the PB1-F2 reading frame did not result in viruses with increased virulence (15). The NS1 protein of pdmH1N1 is also truncated due to a stop codon and, as a result, does not contain a PDZ ligand domain that is involved in cell-signaling pathways and has been implicated in the pathogenicity of 1918 H1N1 and HPAI H5N1 viruses (5, 8, 21). Surprisingly, restoration of a full-length version of the NS1 gene did not result in increased virulence in animal models (16). Mutations affecting virulence and host range have further frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with α2,3- or α2,6-linked sialic acids (SAs), the virus receptors on host cells (17, 32, 35, 50). The HA gene of previous pandemic viruses incorporated substitutions that allow efficient attachment to α2,6-SAs—the virus receptor on human cells—compared to ancestral avian viruses that attach more efficiently to α2,3-SAs (35, 47, 50).To search for mutations of potential importance to public health, numerous laboratories performed genome sequencing of pdmH1N1s, resulting in the real-time accumulation of information on emergence of potential virulence markers. Of specific interest were reports on amino acid substitutions from aspartic acid (D) to glycine (G) at position 222 (position 225 in H3) in HA of pdmH1N1. This substitution was observed in a fatal case of pdmH1N1 infection in June 2009 in the Netherlands (M. Jonges et al., unpublished data). Between July and December 2009, viruses from 11 (18%) of 61 cases with severe disease outcome in Norway have also been reported to harbor the D222G substitution upon direct sequencing of HA in clinical specimens. Such mutant viruses were not observed in any of 205 mild cases investigated, and the frequency of detection of this mutation was significantly higher in severe cases than in mild cases (23). In Hong Kong, the D222G substitution was detected in 12.5% (6) and 4.1% (31) of patients with severe disease and in 0% of patients with mild disease, in two different studies without prior propagation in embryonated chicken eggs. In addition to Norway and Hong Kong, the mutation has been detected in Brazil, Japan, Mexico, Ukraine, and the United States (56). Thus, D222G in HA could be the first identified “virulence marker” of pdmH1N1. pdmH1N1 with D222G in HA have not become widespread in the population, although they were detected in several countries. However, D222G in HA is of special interest, since it has also been described as the single change in HA between two strains of the “Spanish” 1918 H1N1 virus that differed in receptor specificity (47). Furthermore, upon propagation in embryonated chicken eggs, pdmH1N1 can acquire the mutation rapidly, presumably because it results in virus adaptation to avian (α2,3-SAs) receptors (49). The presence of the substitution in pdmH1N1s in the human population and its potential association with more severe disease prompted us to test its effect on pdmH1N1 receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models.     

Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.Influenza A viruses contain eight negative-stranded RNA segments that encode 11 known viral proteins. The 11th viral protein was originally found in a search for unknown peptides during influenza A virus infection recognized by CD8⁺ T cells. It was termed PB1-F2 and is the second protein that is alternatively translated by the same PB1 gene (8). PB1-F2 can be encoded in a large number of influenza A viruses that are isolated from various hosts, including human and avian hosts. The size of PB1-F2 ranges from 57 to 101 amino acids (aa) (41). While strain PR8 (H1N1) contains a PB1-F2 with a length of 87 aa, PB1-F2 is terminated at amino acid position 57 in most human H1N1 viruses and is thus a truncated form compared with the length in PR8. Human H3N2 and most avian influenza A viruses encode a full-length PB1-F2 protein, which is at least 87 aa (7). Many cellular functions of the PB1-F2 protein, and especially the protein of the PR8 strain, have been reported (11, 25). For example, PR8 PB1-F2 localizes to mitochondria in infected and transfected cells (8, 15, 38, 39), suggesting that PB1-F2 enhances influenza A virus-mediated apoptosis in human monocytes (8). The phosphorylation of the PR8 PB1-F2 protein has been suggested to be one of the crucial causes of the promotion of apoptosis (30).The rates of synonymous and nonsynonymous substitutions in the PB1-F2 gene are higher than those in the PB1 gene (7, 20, 21, 37, 42). Recent work has shown that both PR8 PB1-F2 and H5N1 PB1-F2 are important regulators of influenza A virus virulence (1). Additionally, the expression of the 1918 influenza A virus (H1N1) PB1-F2 increases the incidence of secondary bacterial pneumonia (10, 28). However, PB1-F2 is not essential for viral replication because the knockout of PB1-F2 in strain PR8 has no effect on the viral titer (40), suggesting that PB1-F2 may have cellular functions other than those that were originally thought (29).PB1-F2 was translated from the same RNA segment as the PB1 protein, whose function is strongly related to virus RNP activity, which is responsible for RNA chain elongation and which exhibits RNA-dependent RNA polymerase activity (2, 5) and endonuclease activity (9, 16, 26). Previous research has already proved that the knockout of PR8 PB1-F2 reduced virus RNP activity, revealing that PR8 PB1-F2 contributes to virus RNP activity (27), even though PB1-F2 has no effect on the virus growth rate (40). In the present study, not only PR8 PB1-F2 but also H5N1 PB1-F2 and putative full-length swine-origin influenza A virus (S-OIV) PB1-F2 contributed to virus RNP activity. However, PR8 PB1-F2 and H5N1 PB1-F2 exhibit different biological behaviors, including different levels of expression, cellular localizations, and apoptosis enhancements. The molecular determinants of the different localizations were also addressed. The function of the putative PB1-F2 derived from S-OIV was also studied. The investigation described here reveals that PB1-F2 proteins derived from various viral strains exhibited distinct functions, possibly contributing to the variation in the virulence of influenza A viruses.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

A Nine-Segment Influenza A Virus Carrying Subtype H1 and H3 Hemagglutinins

Influenza virus genomic RNAs possess segment-specific packaging signals that include both noncoding regions (NCRs) and adjacent terminal coding region sequences. Using reverse genetics, an A/Puerto Rico/8/34 (A/PR/8/34) virus was rescued that contained a modified PB1 gene such that the PB1 packaging sequences were exchanged for those of the neuraminidase (NA) gene segment. To accomplish this, the PB1 open reading frame, in which the terminal packaging signals were inactivated by serial synonymous mutations, was flanked by the NA segment-specific packaging sequences including the NCRs and the coding region packaging signals. Next, the ATGs located on the 3′ end of the NA packaging sequences of the resulting PB1 chimeric segment were mutated to allow for correct translation of the full-length PB1 protein. The virus containing this chimeric PB1 segment was viable and able to stably carry a ninth, green fluorescent protein (GFP), segment flanked by PB1 packaging signals. Utilizing this method, we successfully generated an influenza virus that contained the genes coding for both the H1 hemagglutinin (HA) from A/PR/8/34 and the H3 HA from A/Hong Kong/1/68 (A/HK/1/68); both subtypes of HA protein were also incorporated into the viral envelope. Immunization of mice with this recombinant virus conferred complete protection from lethal challenge with recombinant A/PR/8/34 virus and with X31 virus that expresses the A/HK/1/68 HA and NA. Using the described methodology, we show that a ninth segment can also be incorporated by manipulation of the PB2 or PA segment-specific packaging signals. This approach offers a means of generating a bivalent influenza virus vaccine.Influenza viruses possess segmented, negative-sense RNA genomes and belong to the family of Orthomyxoviridae. Three types of influenza viruses have been identified: A, B, and C (24). Based on the two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), type A viruses are further divided into different subtypes; there are now 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) of influenza A viruses (24). Current influenza A viruses circulating in humans include the H1N1 and H3N2 subtypes.The genomes of influenza A and B viruses consist of eight RNAs, while C viruses have only seven segments. Influenza virus genomic RNAs associate with nucleoprotein (NP) and three viral polymerase subunits (PB2, PB1, and PA), to form the ribonucleoprotein (RNP) complexes within virions (24). Previous data indicated that each segment of the influenza A/WSN/33 (H1N1) virus possesses segment-specific RNA packaging signals that include both the 3′ and 5′ noncoding regions (NCRs), as well as coding sequences at the two ends of each open reading frame (ORF) (4, 5, 10, 11, 13, 15, 22, 23, 28; and see Fig. 47.23 in reference 24). In addition, an electron microscopy study showed that the wild-type influenza A virus contains exactly eight RNPs within the virions, with seven RNPs surrounding a central one (19). These results suggest that influenza virus genome packaging is a specific process, with each particle containing eight unique RNA segments. Additional evidence supporting a specific packaging theory came from studies of defective interfering (DI) RNAs which contain internal deletions in the coding sequences. These short RNAs can be incorporated into the virus particles despite the fact that they do not encode full-length functional proteins. The finding that incorporation of DI RNAs interferes with the parent full-length RNAs in a segment-specific manner (1, 16, 17) also suggests that influenza virus genome packaging is a specific process.However, there are also data arguing that influenza virus RNA packaging can be nonspecific. First, studies showed that the two different RNA segments of influenza virus can be engineered to share the same set of 3′ and 5′ NCRs, which are important components of the influenza virus RNA packaging signals (18, 31). In addition, under specific circumstances, influenza virus is able to contain nine RNA segments, in which two of them share identical NCRs and partially identical coding region sequences (2, 29). Titrations of the nine-segment virus revealed a linear relationship between dilutions and plaque numbers, suggesting an influenza virus virion can incorporate more than eight segments (2).Herein, we describe a novel approach for the generation of nine-segment influenza viruses based on the manipulation of the segment-specific packaging signals. When the packaging sequences of the PB1 (or PB2 or PA) segment were replaced by those of the NA segment, influenza A/PR/8/34 virus was able to stably incorporate a ninth segment flanked by the PB1 (or PB2 or PA) packaging signals. Using this property, we successfully generated influenza viruses encoding two full-length HA glycoproteins: a subtype H1 A/PR/8/34 HA and a subtype H3 A/HK/1/68 HA. Immunization of mice with the virus carrying two HAs protected them from the lethal challenge with either A/PR/8/34 or X31 virus, the latter of which carries the HA and NA genes of A/HK/1/68. This approach can be used to construct live attenuated influenza vaccine viruses targeting two heterologous strains.     

Receptor Specificity of Influenza A H3N2 Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs

Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.Influenza A viruses are generally isolated and propagated in embryonated chicken eggs or in cultures of cells of mammalian origin. Human influenza viruses were previously noted to acquire mutations in the hemagglutinin (HA) gene upon isolation and culture in the allantoic sac of embryonated chicken eggs (herein simply referred to as “eggs”) compared to the sequences of those isolated in mammalian cell substrates (herein referred to as “cells”) (29, 30, 44, 53, 58). These mutations resulted in amino acid substitutions that were found to mediate receptor specificity changes and improved viral replication efficiency in eggs (37). In general, cell-grown viruses are assumed to be more similar than their egg-grown counterparts to the viruses present in respiratory secretions (30, 56). Since their emergence in 1968, influenza A (H3N2) viruses have evolved and adapted to the human host while losing their ability to be efficiently isolated and replicate in eggs, particularly after 1992 (37, 42, 48). The rate of isolation of H3N2 clinical specimens after inoculation into eggs can be up to ∼30 times lower than that in mammalian cell cultures, highlighting the strong selective pressure for the emergence of sequence variants (77).Virtually all influenza vaccines for human use were licensed decades ago by national regulatory authorities, which used a product manufactured from influenza viruses isolated and propagated exclusively in eggs; therefore, cell culture isolates have been unacceptable for this purpose (41, 71). The antigen composition of influenza vaccines requires frequent updates (every 2 years, on average) to closely match their antigenic properties to the most prevalent circulating antigenic drift variant viruses (51). The limited availability of H3N2 viruses isolated in eggs has on one or more occasions delayed vaccine composition updates and may have reduced the efficacy of vaccination against new antigenically drifted viruses (3, 34, 37).Entry of influenza viruses into host cells is mediated by HA, which binds to sialic acid containing glycoconjugates on the surface of epithelial cells in the upper respiratory tract (2, 13). The nature of the linkage between sialic acid and the vicinal sugar (usually galactose) varies in different host species and tissues and may therefore determine whether an influenza virus binds to and infects avian or human cells (40, 46, 59, 62, 72-75). Human influenza viruses preferentially bind to α2-6-linked sialic acids, and avian viruses predominantly bind to α2-3-linked sialic acids (59). Previous studies with chicken embryo chorioallantoic membranes revealed differential lectin binding, suggesting that α2-3-linked but not α2-6-linked sialosides are present on the epithelial cells (28). Human H3N2 viruses isolated in cell culture were reported to bind with a high affinity to α2-6-linked sialosides, while viruses isolated in eggs often had increased specificity for α2-3-linked sialosides (19, 20, 28). The functional classification of avian and mammalian influenza virus receptors is further complicated since in vitro and tissue-binding assays have led to new working hypotheses involving glycan chain length, topology, and the composition of the inner fragments of the carbohydrate chain as additional receptor specificity determinants (9, 17, 65, 66, 82). However, the significance of these in vitro properties remains unknown, since the structures of the natural sialosides on host cells that are used for infectious virus entry are undefined.The techniques most widely used to study the interactions of the influenza virus with host cell receptors employ animal cells in various assay formats (36, 57, 59, 64, 69). To overcome the problems of cell-based techniques, new assays that rely on labeled sialyl-glycoproteins or polymeric sialoglycans have been developed (18). However, these assays are limited by having only a few glycans available in polymeric form and offer low throughput. In contrast, glycan microarrays can assess virus binding to multiple well-defined glycans simultaneously. Previous work with influenza live or β-propiolactone (BPL)-inactivated virions as well as recombinantly produced HAs revealed a good correlation with receptor specificity compared to that achieved by other methods of analysis (4, 11, 57, 58, 65-68).Here we have compared paired isolates derived in eggs or cell cultures from the single clinical specimen to better understand their receptor binding specificity and its implications for vaccine production. We examined the differences in the sequences of the HAs between egg- and cell-grown isolates and analyzed their receptor binding profiles using glycan microarrays. Sequence analysis of the HA and glycan binding results revealed two distinct groups of viruses, with many egg isolates showing unexpectedly reduced levels of binding to α2-3 and α2-6 sialosides compared to the levels for the viruses isolated in mammalian cells. Furthermore, these studies highlighted that specific glycans may be important for H3N2 virus growth in eggs.     

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.     

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.     

Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses

Since dendritic cells may play a key role in defense against influenza virus infection, we examined the effects of recombinant hemagglutinin (HA) proteins derived from mouse-adapted H1N1 (A/WSN/1933), swine-origin 2009 pandemic H1N1 (A/Texas/05/2009), and highly pathogenic avian influenza H5N1 (A/Thailand/KAN-1/2004) viruses on mouse myeloid dendritic cells (mDCs). The results reveal that tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12) p70, and major histocompatibility complex class II (MHC-II) expression was increased in mDCs after treatment with recombinant HA proteins of H1N1 and H5N1. The specificity of recombinant HA treatments for mDC activation was diminished after proteinase K digestion. HA apparently promotes mDC maturation by enhancing CD40 and CD86 expression and suppressing endocytosis. No significant differences in mDC activation were observed among recombinant proteins of H1N1 and H5N1. The stimulation of mDCs by HA proteins of H1N1 and H5N1 was completely MyD88 dependent. These findings may provide useful information for the development of more-effective influenza vaccines.Influenza viruses trigger seasonal epidemics or pandemics of contagious diseases with mild to severe consequences in human and poultry populations worldwide (28). Members of the Orthomyxoviridae family, influenza viruses consist of single-stranded, eight-segment, negative-sense genomic RNAs, helical viral ribonucleoprotein (RNP) complexes (RNA segments, NP, PB2, PB1, and PA) and four viral envelope proteins (hemagglutinin [HA], neuraminidase [NA], and M1 and M2 matrix proteins). Type A influenza viruses are further classified into various serotypes based on the antigenic characteristics of HA and NA glycoproteins (14).In 2009, a swine-origin H1N1 strain emerged from the genetic reassortants of existing human, avian, and swine influenza viruses, resulting in a global pandemic marked by symptoms more severe than those associated with seasonal influenza virus (3, 24). According to comparative pathology in macaque monkeys, H5N1 induces greater cytokinemia, tissue damage, and interference with immune regulatory mechanisms than H1N1 infection (2). The HA spike protein of influenza virus is believed to play important roles in viral receptor binding, fusion, transmission, host range restriction, virulence, and pathogenesis (13, 27-30).Dendritic cells (DCs), considered the most potent professional antigen-presenting cells, serve as links between innate and adaptive immunity (31). Upon encountering microbial pathogens, endogenous danger signals, or inflammatory mediators, DCs mature and elicit rapid and short-lived innate immune responses before migrating to secondary lymphoid organs and enhancing adaptive immunity (17). Two major subsets of DCs are recognized in mice and humans: (i) myeloid DCs (mDCs, also called conventional DCs), which participate most directly in antigen presentation and activation of naïve T cells, and (ii) plasmacytoid DCs (pDCs), which produce type I interferons in response to viral infection (16, 42) and are also capable of inducing immunotolerance under some conditions (9). mDCs and pDCs also comprise different heterologous subsets, with unique localizations, phenotypes, and functions (36). Due to their key role in immune regulation, DCs have been developed for immunotherapeutic agents or prophylactic or therapeutic vaccines for cancer, infectious diseases, and immune system-related diseases (32, 34).DCs are essential in controlling the innate and adaptive immune responses against influenza virus infection (21). Viral RNA is recognized by various pattern recognition receptors (PRRs), including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs). TLRs play an especially important role in detecting virus invasion and activating DCs (18, 35). However, the mechanisms causing DC activation and maturation in response to influenza viruses are not clear. HA has been described as playing an important role in modulating influenza virus virulence and host immune responses (29). In this study, we examined the effects of several recombinant HA proteins (rHAs) derived from rHA of H1N1 (rH1HA) (A/WSN/1933) and (A/Texas/05/2009) and rHA of H5N1 (rH5HA) (A/Thailand/KAN-1/2004) viruses on the activation and maturation of the mDC subset derived from mouse bone marrow.     

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.Streptococcus sanguinis is a member of the viridans group of streptococci and is a primary colonizer of teeth (8). The viridans species and, in particular, S. sanguinis (15, 18) are a leading cause of infective endocarditis, a serious infection of the valves or lining of the heart (48). Damage to the heart resulting from rheumatic fever or certain congenital heart defects dramatically increases the risk of developing endocarditis (48, 71). The damage is thought to result in the formation of sterile cardiac “vegetations” composed of platelets and fibrin (48) that can be colonized by certain bacteria during periods of bacteremia. This view is supported by animal studies in which formation of sterile vegetation by cardiac catheterization is required for the efficient establishment of streptococcal endocarditis (17). Prevention of infective endocarditis currently relies upon prophylactic administration of antibiotics prior to dental or other surgical procedures that are likely to produce bacteremia. The growing realization that oral bacteria such as S. sanguinis can enter the bloodstream through routine daily activities such as eating has led the American Heart Association (71) and others (57) to question the value of using antibiotic prophylaxis for dental procedures. Clearly, a better understanding of the bacterial virulence factors that contribute to endocarditis could lead to better preventive measures, such as a vaccine that could potentially afford continuous protection to high-risk patients (71).In a previous study, we used the signature-tagged mutagenesis (STM) technique to search for endocarditis virulence factors of S. sanguinis in a rabbit model (53). This study identified a number of housekeeping enzymes that contribute to endocarditis. Because these proteins are not likely to be surface localized, they hold little promise as vaccine candidates. One class of streptococcal surface proteins that is rich in both virulence factors (4, 7, 25, 33, 38, 60) and promising vaccine candidates (6, 39, 42, 51, 70) is the lipoproteins. Lipoprotein activities that have been suggested to contribute to streptococcal virulence include adhesion (4, 7, 63), posttranslational modification (25, 29, 51), and ATP-binding cassette (ABC)-mediated transport (33, 52, 60). In the last instance, lipoproteins anchored to the cell membrane by their lipid tails appear to serve the same transport function as the periplasmic substrate-binding proteins of gram-negative bacteria (66). STM studies performed with Streptococcus pneumoniae (26, 41, 55) and Streptococcus agalactiae (34) have identified multiple lipoprotein mutants among collections of reduced virulence mutants. In an attempt to determine the cumulative contribution of streptococcal lipoproteins to virulence, some investigators have created mutations in the lgt or lspA genes, encoding lipoprotein-processing enzymes (12, 25, 27, 36). The lgt gene encodes prolipoprotein diacylglyceryl transferase, which catalyzes the transfer of a diacylglycerol lipid unit to a cysteine in the conserved N-terminal “lipobox” of lipoproteins, while lspA encodes the signal peptidase II enzyme that cleaves the signal peptide of the prolipoprotein just prior to the conserved cysteine (59, 65). While mutation of these genes has been shown to be lethal in gram-negative bacteria (21, 73), many gram-positive bacterial species have been shown to tolerate such mutations, often with only minor effects on growth (3, 12, 13, 25, 27, 36, 54). Some of these studies indicated a deleterious effect on the virulence of the lgt (25, 54) or lspA (36) mutation, but others found no effect (12) or an enhancement of virulence (27). It is clear from these and other studies (3, 13) that neither the loss of acylation due to lgt inactivation nor the loss of signal peptidase II-mediated cleavage completely eliminates lipoprotein function, necessitating alternative approaches for assessing the global contribution of lipoproteins to virulence.We have used bioinformatic approaches to identify every putative lipoprotein encoded by S. sanguinis strain SK36. To determine the contribution of these lipoproteins to the endocarditis virulence of S. sanguinis, we have systematically mutagenized each of these genes, as well as the lgt and lspA genes, and evaluated these mutants for virulence by using STM in an animal model. Selected mutants were further examined for virulence in competitive index (CI) assays. A strain with a disrupted ssaB gene, which encodes a putative metal transport protein, was found to exhibit a profound defect in virulence that was far greater than that of any other strain tested, including the lgt or lspA mutant.     

Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.The new H1N1 swine-origin influenza virus (S-OIV) recently emerged to cause the first influenza pandemic in 40 years (2). The S-OIV presumably emerged from pigs, as its genome was shown to consist of six gene segments of “triple-reassortant” swine viruses and two of “Eurasian lineage” swine viruses (9). The start of the S-OIV pandemic has been relatively mild, with a clinical spectrum ranging from mild upper respiratory tract illness to sporadic cases of severe pneumonia leading to acute respiratory distress syndrome (22). As of 15 November 2009, worldwide, more than 206 countries have reported laboratory-confirmed cases of S-OIV infection, including over 6,770 deaths (32).In previous influenza pandemics, such as the Spanish influenza pandemic of 1918 and the Hong Kong influenza pandemic of 1968, a first wave of cases of relatively mild illnesses was followed by more severe subsequent waves (29). The reason for this increased severity has remained largely unknown, but one possible explanation could be that the pandemic viruses required further adaptation to the human host, resulting in the emergence of viruses that were more virulent than those of the first wave. Such adaptive changes could occur by gene reassortment between cocirculating influenza A viruses or by mutation.In the past decade, determinants of influenza A virus virulence have been mapped using reverse genetics with a variety of pandemic, epidemic, and zoonotic influenza viruses. Mutations affecting virulence and host range have frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with sialic acids, the virus receptors on host cells (11, 18, 30). Nonstructural protein 1 (NS1) has been implicated in the virulence of highly pathogenic avian influenza (HPAI) virus H5N1 and the 1918 H1N1 virus, as the NS1 proteins of these viruses were shown to act as strong antagonists of the interferon pathways (10, 25). Furthermore, the polymerase genes, in particular the PB2 gene, have been shown to be important determinants of virulence in the HPAI H5N1 and H7N7 viruses and of transmission in the 1918 H1N1 virus (11, 21, 31). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is found generally in avian influenza viruses, while human viruses have a lysine (K), and this mutation has been described as a determinant of the host range in vitro (28). When avian viruses lacking the E627K substitution were passaged in mice, the viruses acquired the mutation spontaneously upon a single passage (15, 17). In the HPAI H5N1 and H7N7 viruses, E627K was shown to be the prime determinant of pathogenesis in mice (11, 21, 23). Given that all human and many zoonotic influenza viruses of the last century contained 627K (1), it was surprising that the S-OIV had 627E.Additionally, the aspartate (D)-to-asparagine (N) mutation at position 701 of PB2, which was shown to compensate for the absence of E627K, has also not been detected in S-OIV (27). This D701N mutation has previously been shown to expand the host range of avian H5N1 to mice and humans (3, 15) and to increase virus transmission in guinea pigs (27). Thus, S-OIV was the first known human pandemic virus with 627E and 701D, and it has been speculated that S-OIV could mutate into a more virulent form by acquiring one of these mutations, or both.On 8 May 2009, the detection of another mutation in the PB2 gene of S-OIV, an E-to-glycine (G) mutation at position 667, was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090508.1722). It has previously been suggested that the E667G substitution in PB2 of HPAI H5N1 virus was under positive selection and possibly played a role in sustainable transmission in humans (14).On 28 September 2009, detection of the E627K mutation in PB2 of S-OIVs of two individuals in the Netherlands was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090928.3394) and raised concern about the possible enhanced replication of the S-OIV in humans, possibly associated with increased virulence. To date, the D701N mutation in PB2 has not been reported in any of the S-OIVs sequenced, and additional viruses with mutation E627K have not been recorded, either. In contrast, viruses with E677G have been reported from the United States, Canada, Germany, the United Kingdom, Norway, and France, according to the public sequence databases.Here, the effects of the E627K, D701N, and E677G mutations in the PB2 genes of S-OIVs was investigated using genetically engineered influenza viruses based on a prototype S-OIV, A/Netherlands/602/2009. Polymerase activity was measured in minigenome assays in human 293T cells, virus replication was analyzed in Madin-Darby Canine kidney (MDCK) cells, virulence was tested in mouse and ferret models, and transmission by aerosols or respiratory droplets was tested in ferrets. In contrast to the earlier assumptions based on experience with other influenza A viruses, S-OIVs with E627K, D701N, or E677G in PB2 did not show a marked increase in virulence or transmission compared to the wild-type virus.     

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 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.     

A Maraviroc-Resistant HIV-1 with Narrow Cross-Resistance to Other CCR5 Antagonists Depends on both N-Terminal and Extracellular Loop Domains of Drug-Bound CCR5

CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile.Small-molecule CCR5 antagonists are a relatively new class of drugs that block HIV entry into target cells, with the first member of this class, maraviroc (MVC), having been approved for the treatment of HIV-infected patients. These drugs bind to a hydrophobic pocket formed by the transmembrane helices of CCR5, inducing conformational changes in the extracellular loops (ECLs) of the receptor (18, 31, 39, 40, 58, 62, 64). These conformational changes can vary with different drugs, as evidenced by differential chemokine binding and HIV resistance profiles, and block the ability of HIV to use drug-bound CCR5 as a coreceptor for entry (59, 64).As with other antiretroviral agents, HIV can develop resistance to CCR5 antagonists. One pathway by which HIV can become resistant to CCR5 antagonists is via mutations in the viral envelope (Env) protein that enable it to recognize the drug-bound conformation of the coreceptor. Most of our information on this pathway has come from in vitro passaging of HIV-1 in the presence of increasing concentrations of inhibitor (2, 4, 5, 33, 41, 44, 61, 66). In most instances, the viral determinants of resistance are localized to the V3 loop of gp120 (5, 33, 41, 44, 46, 63, 66). This is as expected: the base of the V3 loop interacts with O-sulfated tyrosines in the N terminus of CCR5, while the tip of the V3 loop is thought to contact the ECLs of the receptor (14, 15, 17, 19, 26, 29, 37). Viral resistance to one CCR5 antagonist commonly results in cross-resistance to other drugs in this class, although this is not universally the case (33, 41, 60, 63, 66). Mechanistically, a number of CCR5 antagonist-resistant viruses have been shown to have increased dependence on the N-terminal domain of CCR5 (5, 34, 44, 45, 48), which is largely unaffected by drug binding and may allow viruses to tolerate drug-induced changes in ECL conformation.In contrast to several well-characterized viruses that have evolved resistance to CCR5 antagonists in vitro, few examples of patient-derived CCR5 antagonist-resistant viruses have been reported. One mechanism of resistance that has been described in patients is the outgrowth of CXCR4-tropic HIV isolates that were present at low frequencies prior to the initiation of therapy (22, 23, 35, 36, 42, 65). Due to this finding, patients undergo tropism testing prior to treatment with CCR5 antagonists, with only those harboring exclusively R5-tropic viruses considered candidates for therapy. Patient-derived viruses capable of using drug-bound CCR5 have been reported in studies using vicriviroc and aplaviroc (45, 60, 63). The aplaviroc-resistant viruses were determined to utilize the drug-bound form of the receptor by interacting primarily with the N terminus of CCR5, similar to the viruses derived by serial in vitro passaging (48).In the present study, we report the isolation of MVC-resistant Envs from a treatment-experienced patient who had a viral load rebound while on a regimen containing MVC. Viral Envs isolated from this patient at the time MVC therapy was initiated were fully sensitive to drug. However, resistance evolved over the course of 224 days, culminating in Envs that were completely resistant to inhibition but continued to use CCR5 for entry. The emergence of resistance was dependent upon changes within the V3 loop of the virus, while changes in the V4 loop modulated the magnitude of resistance. The MVC-resistant Envs studied here exhibited several unusual properties. First, while they were cross-resistant to TAK779, they remained sensitive to all other CCR5 antagonists tested, including vicriviroc and aplaviroc. Second, the Envs were particularly adept at utilizing low levels of CCR5 to mediate infection of cells. Third, and in contrast to several recent reports of CCR5 antagonist-resistant viruses, these Envs were dependent upon residues within both the N terminus and ECLs of CCR5 for efficient entry in the presence of drug. When considered in the context of other reports, our data suggest a model in which resistance to multiple CCR5 antagonists can arise if an Env protein becomes highly dependent upon the N-terminal domain of CCR5, the conformation of which appears to be unaffected by drug binding. A more narrow resistance profile results from changes in Env that enable it to use both the N-terminal domain of CCR5 as well as the drug-induced conformation of the CCR5 ECLs.