Guohua Deng

Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model

ABSTRACT We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.

Properties and Dissemination of H5N1 Viruses Isolated during an Influenza Outbreak in Migratory Waterfowl in Western China

ABSTRACT H5N1 influenza A viruses are widely distributed among poultry in Asia, but until recently, only a limited number of wild birds were affected. During late April through June 2005, an outbreak of H5N1 virus infection occurred among wild birds at Qinghai Lake in China. Here, we describe the features of this outbreak. First identified in bar-headed geese, the disease soon spread to other avian species populating the lake. Sequence analysis of 15 viruses representing six avian species and collected at different times during the outbreak revealed four different H5N1 genotypes. Most of the isolates possessed lysine at position 627 in the PB2 protein, a residue known to be associated with virulence in mice and adaptation to humans. However, neither of the two index viruses possessed this residue. All of the viruses tested were pathogenic in mice, with the exception of one index virus. We also tested the replication of two viruses isolated during the Qinghai Lake outbreak and one unrelated duck H5N1 virus in rhesus macaques. The Qinghai Lake viruses did not replicate efficiently in these animals, producing no evidence of disease other than transient fever, while the duck virus replicated in multiple organs and caused symptoms of respiratory illness. Importantly, H5N1 viruses isolated in Mongolia, Russia, Inner Mongolia, and the Liaoning Province of China after August 2005 were genetically closely related to one of the genotypes isolated during the Qinghai outbreak, suggesting the dominant nature of this genotype and underscoring the need for worldwide intensive surveillance to minimize its devastating consequences.

A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice

ABSTRACT In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.

H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet

H7N9 Adaptation Puzzling and alarming reports of an outbreak in early 2013 of human infections by a low-pathogenicity avian influenza virus has rocked the poultry industry in central eastern China and brought fears of initiating a human pandemic. Over 130 human cases have been reported with 37 deaths until closure of poultry markets accompanied a near-cessation of human case reports. From surveillance sampling of >10,000 isolates obtained during April 2013, Zhang et al. (p. 410, published online 18 July) took 37 isolates of avian origin H7N9 and compared them to human H7N9 isolates. The majority of H7N9 isolates came from live poultry markets, although some originated in pigeons. Sequence analysis indicated that the chicken isolates had retained the avian characteristics at sites on the influenza genes for PB2 and the surface hemagglutinin HA, where adaptive mutations have been observed before. Sequence analysis also showed a higher variability in the internal genes than in HA and neuraminidase NA. By using glycan arrays, it was shown that avian and human isolates bound to human, but also to some extent to avian, receptors. As expected, the virus replicated well in chickens without causing disease, whereas in mice only the human isolates were highly pathogenic. The human virus, but not the avian, transmitted between ferrets through the air. A large survey of H7N9 influenza viruses in China reveals a spectrum of mutation and virulence. A newly emerged H7N9 virus has caused 132 human infections with 37 deaths in China since 18 February 2013. Control measures in H7N9 virus–positive live poultry markets have reduced the number of infections; however, the character of the virus, including its pandemic potential, remains largely unknown. We systematically analyzed H7N9 viruses isolated from birds and humans. The viruses were genetically closely related and bound to human airway receptors; some also maintained the ability to bind to avian airway receptors. The viruses isolated from birds were nonpathogenic in chickens, ducks, and mice; however, the viruses isolated from humans caused up to 30% body weight loss in mice. Most importantly, one virus isolated from humans was highly transmissible in ferrets by respiratory droplet. Our findings indicate nothing to reduce the concern that these viruses can transmit between humans.

Identification of Amino Acids in HA and PB2 Critical for the Transmission of H5N1 Avian Influenza Viruses in a Mammalian Host

Since 2003, H5N1 influenza viruses have caused over 400 known cases of human infection with a mortality rate greater than 60%. Most of these cases resulted from direct contact with virus-contaminated poultry or poultry products. Although only limited human-to-human transmission has been reported to date, it is feared that efficient human-to-human transmission of H5N1 viruses has the potential to cause a pandemic of disastrous proportions. The genetic basis for H5N1 viral transmission among humans is largely unknown. In this study, we used guinea pigs as a mammalian model to study the transmission of six different H5N1 avian influenza viruses. We found that two viruses, A/duck/Guangxi/35/2001 (DKGX/35) and A/bar-headed goose/Qinghai/3/2005(BHGQH/05), were transmitted from inoculated animals to naïve contact animals. Our mutagenesis analysis revealed that the amino acid asparagine (Asn) at position 701 in the PB2 protein was a prerequisite for DKGX/35 transmission in guinea pigs. In addition, an amino acid change in the hemagglutinin (HA) protein (Thr160Ala), resulting in the loss of glycosylation at 158–160, was responsible for HA binding to sialylated glycans and was critical for H5N1 virus transmission in guinea pigs. These amino acids changes in PB2 and HA could serve as important molecular markers for assessing the pandemic potential of H5N1 field isolates.

Newcastle Disease Virus-Based Live Attenuated Vaccine Completely Protects Chickens and Mice from Lethal Challenge of Homologous and Heterologous H5N1 Avian Influenza Viruses

ABSTRACT H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.

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

ABSTRACT 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.

A Naturally Occurring Deletion in Its NS Gene Contributes to the Attenuation of an H5N1 Swine Influenza Virus in Chickens

ABSTRACT In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.

H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet.

Influencing Influenza Currently, there is anxiety that the avian H5N1 influenza virus will reassort with the highly transmissible and epidemic H1N1 subtype to trigger a virulent human pandemic. Y. Zhang et al. (p. 1459, published online 2 May) used reverse genetics to make all possible reassortants between a virulent bird H5N1 with genes from a human pandemic H1N1. Virulence was tested in mice and transmissibility was tested between guinea pigs, which have both avian- and human-like airway influenza virus receptors. To assess what is happening to the receptor-ligand interactions as a result of these mutations, W. Zhang et al. (p. 1463, published online 2 May) probed the structure of both wild-type and mutant hemagglutinin of H5 in complex with analogs of the avian and human receptor types. Certain mutations in the receptor-binding site changed binding affinity. Some reassortants between H5N1 and H1N1 influenza viruses are transmissible by respiratory droplet among mammals. In the past, avian influenza viruses have crossed species barriers to trigger human pandemics by reassorting with mammal-infective viruses in intermediate livestock hosts. H5N1 viruses are able to infect pigs, and some of them have affinity for the mammalian type α-2,6-linked sialic acid airway receptor. Using reverse genetics, we systematically created 127 reassortant viruses between a duck isolate of H5N1, specifically retaining its hemagglutinin (HA) gene throughout, and a highly transmissible, human-infective H1N1 virus. We tested the virulence of the reassortants in mice as a correlate for virulence in humans and tested transmissibility in guinea pigs, which have both avian and mammalian types of airway receptor. Transmission studies showed that the H1N1 virus genes encoding acidic polymerase and nonstructural protein made the H5N1 virus transmissible by respiratory droplet between guinea pigs without killing them. Further experiments implicated other H1N1 genes in the enhancement of mammal-to-mammal transmission, including those that encode nucleoprotein, neuraminidase, and matrix, as well as mutations in H5 HA that improve affinity for humanlike airway receptors. Hence, avian H5N1 subtype viruses do have the potential to acquire mammalian transmissibility by reassortment in current agricultural scenarios.

Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes

Inactivated whole avian influenza virus (AIV) vaccine provides protection against homologous haemagglutinin (HA) subtype virus, but poor protection against a heterologous HA virus. Moreover, it induces chickens to produce antibodies to cross-reactive antigens, especially nucleoprotein, which is limits AIV serological surveillance. In this study, a recombinant fowlpox virus co-expressing HA (H5 subtype) and NA (N1 subtype) genes of AIV was evaluated for its ability to protect chickens against intramuscular challenge with a lethal dose of highly pathogenic (HP) AIV. Susceptible chickens were also vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus were protected, while the chickens vaccinated with either the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 100% mortality following challenge. This protection was accompanied by the high levels of specific antibody to the respective components of the recombinant vaccine. The above results showed that rFPV-HA-NA could be a potential vaccine to replace current inactivated vaccines for preventing AI.