Comparisons of Highly Virulent H5N1 Influenza A Viruses Isolated from Humans and Chickens from Hong Kong

Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393–395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate’s gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all seven viruses were highly pathogenic in chickens and caused similar lesions in experimental inoculations.     

Characterization of H5N1 Influenza Viruses That Continue To Circulate in Geese in Southeastern China

The H5N1 influenza virus, which killed humans and poultry in 1997, was a reassortant that possibly arose in one type of domestic poultry present in the live-poultry markets of Hong Kong. Given that all the precursors of H5N1/97 are still circulating in poultry in southern China, the reassortment event that generated H5N1 could be repeated. Because A/goose/Guangdong/1/96-like (H5N1; Go/Gd) viruses are the proposed donors of the hemagglutinin gene of the H5N1 virus, we investigated the continued circulation, host range, and transmissibility of Go/Gd-like viruses in poultry. The Go/Gd-like viruses caused weight loss and death in some mice inoculated with high virus doses. Transmission of Go/Gd-like H5N1 viruses to geese by contact with infected geese resulted in infection of all birds but limited signs of overt disease. In contrast, oral inoculation with high doses of Go/Gd-like viruses resulted in the deaths of up to 50% of infected geese. Transmission from infected geese to chickens occurred only by fecal contact, whereas transmission to quail occurred by either aerosol or fecal spread. This difference is probably explained by the higher susceptibility of quail to Go/Gd-like virus. The high degree of susceptibility of quail to Go/Gd (H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in quail support the hypothesis that quail were the host of origin of the H5N1/97 virus. The ease of transmission of Go/Gd (H5N1)-like viruses to land-based birds, especially quail, supports the wisdom of separating aquatic and land-based poultry in the markets in Hong Kong and the need for continued surveillance in the field and live-bird markets in which different types of poultry are in contact with one another.     

Novel Genotypes of H9N2 Influenza A Viruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.     

Continued Evolution of H5N1 Influenza Viruses in Wild Birds,Domestic Poultry,and Humans in China from 2004 to 2009

Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51 representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to 2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition, antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in nature have complex biological characteristics and pose a continued challenge for disease control and pandemic preparedness.The highly pathogenic H5N1 influenza viruses that emerged over a decade ago in southern China have evolved into over 10 distinct phylogenetic clades based on their hemagglutinin (HA) genes. The viruses have spread to over 63 countries and to multiple mammalian species, including humans, resulting in 498 cases of infection and 294 deaths by 6 May 2010 according to the World Health Organization (WHO) (http://www.who.int). To date, none of the different H5N1 clades has acquired the ability to consistently transmit among mammalian species. The currently circulating H5N1 viruses are unique in that they continue to circulate in avian species. All previous highly pathogenic H5 and H7 viruses have naturally “burned out” or were stamped out because of their high pathogenicity in domestic poultry. While there is growing complacency about the potential of H5N1 “bird flu” to attain consistent transmissibility in humans and develop pandemicity, it is worth remembering that we have no knowledge of the time that it took the 1918 Spanish, the 1957 Asian, the 1968 Hong Kong, and the 2009 North American pandemics to develop their pandemic potentials. We may therefore currently be witnessing in real time the evolution of an H5N1 pandemic influenza virus.H5N1 avian influenza viruses (AIVs) were first detected in sick geese in Guangdong province in 1996, and both nonpathogenic and highly pathogenic (HP) H5N1 viruses were described (18). In 1997, H5N1 reassortant viruses that derived the HA gene from A/goose/Guangdong/1/96 (GS/GD/1/96)-like viruses and the other genes from H6N1 and/or H9N2 viruses caused lethal outbreaks in poultry and humans in Hong Kong (6, 7). Since then, long-term active surveillance of influenza viruses in domestic poultry has been performed, and multiple subtypes of influenza viruses have been detected in chickens and ducks in China (16, 19, 37). H5N1 influenza viruses have been repeatedly detected in apparently healthy ducks in southern China since 1999 (4, 13) and were also detected in pigs in Fujian province in 2001 and 2003 (39).Since the beginning of 2004, there have been significant outbreaks of H5N1 avian influenza virus infection involving multiple poultry farm flocks in more than 20 provinces in China (2). H5N1 viruses resulted in the deaths of millions of domestic poultry, including chickens, ducks, and geese, as the result of infection or of culling and the deaths of thousands of wild birds (5, 20). Thirty-eight human cases of HP H5N1 infection with 25 fatalities have been associated with direct exposure to infected poultry (WHO; http://www.who.int). Since 2004, the vaccination of domestic poultry has been used for the control of HP H5N1 influenza virus in China. While this strategy has been effective at reducing the incidence of HP H5N1 in poultry and at markedly reducing the number of human cases, it is impossible to vaccinate every single bird due to the enormous poultry population. Outbreaks of H5N1 influenza virus still continue to occur in poultry although at a reduced frequency.A previous study by Smith et al. reported that a “Fujian-like” H5N1 influenza virus emerged in late 2005 and predominated in poultry in southern China (26). Those authors suggested that vaccination may have facilitated the selection of the “Fujian-like” sublineage. Here, we analyzed 51 representative H5N1 viruses that were isolated from wild birds, domestic poultry, and humans from 2004 to 2009 in China and described their genetic evolution and antigenicity profiles. Our results indicate that H5N1 influenza viruses in southern China, including the “Fujian-like” viruses, are complicated reassortants, which could be well protected against by GS/GD/1/96 virus-based vaccines. We documented the emergence of the latest variant of H5N1 (A/chicken/Shanxi/2/06 [CK/SX/2/06]) that broke through existing poultry vaccines. We show that this variant is less pathogenic in mice and ducks than the earlier strains and propose that the variant was not selected by the use of vaccines.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Spatiotemporal Structure of Molecular Evolution of H5N1 Highly Pathogenic Avian Influenza Viruses in Vietnam

Background

Vietnam is one of the countries most affected by outbreaks of H5N1 highly pathogenic avian influenza viruses. First identified in Vietnam in poultry in 2001 and in humans in 2004, the virus has since caused 111 cases and 56 deaths in humans. In 2003/2004 H5N1 outbreaks, nearly the entire poultry population of Vietnam was culled. Our earlier study (Wan et al., 2008, PLoS ONE, 3(10): e3462) demonstrated that there have been at least six independent H5N1 introductions into Vietnam and there were nine newly emerged reassortants from 2001 to 2007 in Vietnam. H5N1 viruses in Vietnam cluster distinctly around Hanoi and Ho Chi Minh City. However, the nature of the relationship between genetic divergence and geographic patterns is still unclear.

Methodology/Principal Findings

In this study, we hypothesized that genetic distances between H5N1 viruses in Vietnam are correlated with geographic distances, as the result of distinct population and environment patterns along Vietnam''s long north to south longitudinal extent. Based on this hypothesis, we combined spatial statistical methods with genetic analytic techniques and explicitly used geographic space to explore genetic evolution of H5N1 highly pathogenic avian influenza viruses at the sub-national scale in Vietnam. Our dataset consisted of 125 influenza viruses (with whole genome sets) isolated in Vietnam from 2003 to 2007. Our results document the significant effect of space and time on genetic evolution and the rise of two regional centers of genetic mixing by 2007. These findings give insight into processes underlying viral evolution and suggest that genetic differentiation is associated with the distance between concentrations of human and poultry populations around Hanoi and Ho Chi Minh City.

Conclusions/Significance

The results show that genetic evolution of H5N1 viruses in Vietnamese domestic poultry is highly correlated with the location and spread of those viruses in geographic space. This correlation varies by scale, time, and gene, though a classic isolation by distance pattern is observed. This study is the first to characterize the geographic structure of influenza viral evolution at the sub-national scale in Vietnam and can shed light on how H5N1 HPAIVs evolve in certain geographic settings.     

Phylogenetic and Pathogenic Analyses of Avian Influenza A H5N1 Viruses Isolated from Poultry in Vietnam

Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature.     

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.     

广东地区流感H3N2毒株血凝素基因特征、进化和变异分析

为揭示广东地区2007～2010年甲型H3N2毒株血凝素(HA)基因特征和变异,采用时空抽样方法抽样,检测广东2007～2010年甲型H3N2毒株HA基因核苷酸序列,同时检索全球HA基因序列作为对照,采用Lasergene 7.1和Mega 5.05软件对HA基因核苷酸序列进行比对和分析;并结合流行病学资料,对变异毒株进行进化速度分析;同时进行抗原分析。结果发现,广东2007～2010年H3N2毒株HA基因同义进化(Ks)和错义进化(Ka)速度分别为2.06×1E-3～2.23×1E-3核苷酸/年和1.05×1E-3～1.21×1E-3核苷酸/年,HA1较HA2的错义突变速率要高3.13倍。与疫苗株A/Perth/16/2009的HA基因比较,2009年广东毒株同源性达到98.8%～99.7%、2010年同源性达到98.0%～98.4%。在广东2007～2010年毒株中,HA1五个抗原表位均有氨基酸位点变异,尤其是2010年毒株B区(N160K)和D区(K174R/N)的变异;此外,广东2010年毒株受体结合部位(RBS)还发生K189E/N/Q和T228A置换变异;两个糖基化位点变异影响到抗原性;目前使用的H3N2疫苗株与目前流行毒株的抗原性有差异。广东地区2007～2010年的毒株中,血凝抑制抗体的抗原分析结果有差异。结果提示,目前广东乃至全球甲型H3N2毒株HA1B区和D区均有氨基酸位点变异,RBS的两个位点发生置换,糖基化位点变异影响到表位A区和B区抗原性;与WHO推荐2011年流感H3N2毒株疫苗株比较,目前流行毒株HA基因有抗原位点变异。     

The N-Terminal Fragment of a PB2 Subunit from the Influenza A Virus (A/Hong Kong/156/1997 H5N1) Effectively Inhibits RNP Activity and Viral Replication

Background

Influenza A virus has a RNA-dependent RNA polymerase (RdRp) that is composed of three subunits (PB1, PB2 and PA subunit), which assemble with nucleoproteins (NP) and a viral RNA (vRNA) to form a RNP complex in the host nucleus. Recently, we demonstrated that the combination of influenza ribonucleoprotein (RNP) components is important for both its assembly and activity. Therefore, we questioned whether the inhibition of the RNP combination via an incompatible component in the RNP complex could become a methodology for an anti-influenza drug.

Methodology/Principal Findings

We found that a H5N1 PB2 subunit efficiently inhibits H1N1 RNP assembly and activity. Moreover, we determined the domains and important amino acids on the N-terminus of the PB2 subunit that are required for a strong inhibitory effect. The NP binding site of the PB2 subunit is important for the inhibition of RNP activity by another strain. A plaque assay also confirmed that a fragment of the PB2 subunit could inhibit viral replication.

Conclusions/Significance

Our results suggest that the N-terminal fragment of a PB2 subunit becomes an inhibitor that targets influenza RNP activity that is different from that targeted by current drugs such as M2 and NA inhibitors.     

1株含H9N2内部基因的H5N1亚型基因重配禽流感病毒的全基因测序及遗传进化分析

在对华东地区家养水禽中流感病毒的带毒状况的流行病学监测过程中,从表观健康家鸭体内分离到一株H5N1亚型禽流感病毒A/duck/Shandong/009/2008(简称Dk/SD/009/08)。为了解该毒株的基因组构成,对该分离株进行全基因测序。测序结果显示:该毒株HA裂解位点处的氨基酸序列为PLRERRRK-R/GL,符合高致病性禽流感病毒的分子特征,且参照H5N1国际统一命名准则,Dk/SD/009/08的HA基因属于2.3.4进化支。BLAST结果显示,HA、NA、NP及NS基因均与H5N1亚型病毒的核苷酸一致性最高,而RNA聚合酶基因(PB2、PB1、PA)及M基因则与H9N2亚型病毒的亲缘关系最近,故推测该分离株可能是一株天然重组病毒;遗传进化分析进一步表明,流行于华南地区鹌鹑中的G1-like H9N2亚型病毒可能为该分离株提供部分的内部基因。     

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins, referred to as “antigenic drift” and “antigenic shift.” To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged 50 times in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum. The resulting mutant acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected, in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to that of the wild type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA segment could be generated; however, generation of this recombinant virus required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent, even though the HPAIV polybasic hemagglutinin cleavage site was still present. Overall, the variants reported here are the first HPAIV H5N1 strains without a functional neuraminidase shown to grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the role of neuraminidase in virus replication and pathogenicity.     

Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses

Background and Objectives

Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes.

Methods and Results

Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8_LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8_LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates.

Conclusion

This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.