Juan Pu

Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008.

H9N2 influenza viruses have been circulating in China since 1994, but a systematic investigation of H9N2 in northern China has not been undertaken since 2004. Here, using the sequences of 22 viruses we isolated from poultry and pigs in northern China during 2003-2008, in combination with sequences available in a public database, we analyzed the evolution of H9N2 influenza viruses in China from 1994 to 2008. Our findings demonstrated that the H9N2 viruses in China underwent extensive reassortment, and novel genotypes continued to emerge. Among 330 viruses, 54 genotypes were observed including 19 novel genotypes that have not been recognized before, and major genotypes were further divided into five series (BJ/94-, G1-, BG-, F/98- and Aq-series). Different epidemiological and biological features among these series were recognized. The BJ/94- and F/98-series viruses were circulating in both southern and northern China, while the other three series viruses were mainly detected in southern China. BJ/94-series influenza viruses predominated in China before 2000 and were gradually replaced by F/98-series viruses that became the predominant viruses since 2004. At least five antigenic groups could be identified over the study period, during which a significant antigenic drift likely occurred between 2002 and 2003. Animal experiments demonstrated that F/98-series viruses were able to replicate and transmit more effectively in chickens than BJ/94-series viruses. The continuing evolution of H9N2 influenza viruses in China emphasizes the importance of H9N2 influenza virus surveillance throughout this region to aid pandemic prediction and prevention.

H5N1 avian influenza re-emergence of Lake Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation.

Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.

Natural and experimental infection of dogs with pandemic H1N1/2009 influenza virus

Evidence of H1N1/2009 influenza virus infection was identified in two domestic dogs in China in November 2009. Virus isolation and sequence analysis of all eight genes of the two isolates showed that they were related closely to the H1N1/2009 influenza virus circulating in humans, indicating that they were probably acquired from humans. To determine the pathogenicity and transmissibility of H1N1/2009 influenza virus in dogs, experimental infection and transmission were performed. Inoculated dogs were able to shed virus in nasal secretions, but symptoms were very mild. Uninoculated dogs were co-mingled to determine the transmissibility of the isolate, and one of three exposed dogs was shown to develop infection. The present findings indicate that human H1N1/2009 can infect dogs, but is transmitted inefficiently between dogs.

Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence.

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.

Emergence of European Avian Influenza Virus-Like H1N1 Swine Influenza A Viruses in China

ABSTRACT During swine influenza surveillance from 2007 to 2008, 10 H1N1 viruses were isolated and analyzed for their antigenic and phylogenetic properties. Our study revealed the emergence of avian-origin European H1N1 swine influenza virus in China, which highlights the necessity of swine influenza surveillance for potential pandemic preparedness.

Novel swine influenza virus reassortants in pigs, China.

During swine influenza virus surveillance in pigs in China during 2006–2009, we isolated subtypes H1N1, H1N2, and H3N2 and found novel reassortment between contemporary swine and avian panzootic viruses. These reassortment events raise concern about generation of novel viruses in pigs, which could have pandemic potential.

Evaluation of the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 influenza viruses in chickens

Despite the long-term vaccination programs implemented in China, H9N2 avian influenza viruses (AIVs) continue to persist in chicken populations, even in vaccinated flocks. We previously demonstrated that H9N2 AIV isolated from chickens in China also underwent antigenic drift and evolved into distinct antigenic groups (C, D and E). To understand whether antigenic drift of viruses away from the vaccine strain partially contributed to the circulation of H9N2 AIV in China, we evaluated the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 AIV. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group C viruses, but not those of the more recent groups D and E. Vaccinated chickens, even those with vaccine-induced HI titers of 1:1024, shed virus after being infected with A/chicken/Shandong/ZB/2007, a representative virus of antigenic group D. Genetic analysis showed that the representative viruses of antigenic groups D and E possessed greater numbers of amino acid substitutions in the hemagglutinin protein compared to the vaccine strain and the antigenic group C virus, and many of which were located in antigenic sites. Our results indicated that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection, and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection. Furthermore, the avian influenza vaccination policy also needs to be re-assessed, and increased veterinary biosecurity on farms, rather than vaccine application alone, should be implemented to prevent and control avian influenza.

A Single Amino Acid at the Hemagglutinin Cleavage Site Contributes to the Pathogenicity and Neurovirulence of H5N1 Influenza Virus in Mice

ABSTRACT H5 influenza viruses containing a motif of multiple basic amino acids at the hemagglutinin (HA) cleavage site (HACS) are highly pathogenic in chicken but display different virulence phenotypes in mammals. Previous studies have shown that multiple basic amino acids of H5N1 influenza virus are a prerequisite for lethality in mice. However, it remains unclear which specific residue at the cleavage site affects the pathogenicity of H5N1 in mammals. A comprehensive genetic analysis of the H5N1 HACS showed that residues at P6 (position 325, by H3 numbering) were the most polymorphic, including serine (S), arginine (R), deletion (*), glycine (G), and isoleucine (I). To determine whether a single residue at P6 could affect virulence, we introduced different mutations at P6 of an avirulent clade 7 H5N1 strain, rg325G, by reverse genetics. Among the recombinant viruses, the rg325S virus showed the highest cleavage efficiency in vitro. All these viruses were highly pathogenic in chicken but exhibited different virulences in mice. The rg325S virus exhibited the highest pathogenicity in terms of unrestricted organ tropism and neurovirulence. Remarkably, the HA-325S substitution dramatically increased the pathogenicity of H5N1 viruses of other clades, including clades 2.2, 2.3.2, and 2.3.4, indicating that this residue impacts genetically divergent H5N1 viruses. An analysis of predicted structures containing these mutations showed that the cleavage site loop with 325S was the most exposed, which might be responsible for the efficient cleavage and high virulence. Our results demonstrate that an amino acid substitution at the P6 cleavage site alone could modulate the virulence of H5N1 in mice.

Guinea pig model for evaluating the potential public health risk of swine and avian influenza viruses.

Background The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed. Methodology/Principal Findings We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung. Conclusions/Significance We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.

Rapid detection of avian influenza virus a and subtype H5N1 by single step multiplex reverse transcription-polymerase chain reaction.

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.