Kennedy F. Shortridge

Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2–4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003–04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.

Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus

BACKGROUND Human infection with an avian influenza A virus (subtype H5N1) was reported recently in Hong Kong. We describe the clinical presentation of the first 12 patients and options for rapid viral diagnosis. METHODS Case notes of 12 patients with virus-culture-confirmed influenza A H5N1 infection were analysed. The clinical presentation and risk factors associated with severe disease were defined and the results of methods for rapid virus diagnosis were compared. FINDINGS Patients ranged from 1 to 60 years of age. Clinical presentation was that of an influenza-like illness with evidence of pneumonia in seven patients. All seven patients older than 13 years had severe disease (four deaths), whereas children 5 years or younger had mild symptoms with the exception of one who died with Reyes syndrome associated with intake of aspirin. Gastrointestinal manifestations, raised liver enzymes, renal failure unrelated to rhabdomyolysis, and pancytopenia were unusually prominent. Factors associated with severe disease included older age, delay in hospitalisation, lower-respiratory-tract involvement, and a low total peripheral white blood cell count or lymphopenia at admission. An H5-specific reverse-transcription PCR assay (RT-PCR) was useful for rapid detection of virus directly in respiratory specimens. A commercially available enzyme immunoassay was more sensitive than direct immunofluorescence for rapid viral diagnosis. Direct immunofluorescence with an H5-specific monoclonal antibody pool was useful for rapid exclusion of H5-subtype infection. INTERPRETATION Avian Influenza A H5N1 virus causes human influenza-like illness with a high rate of complications in adults admitted to hospital. Rapid H5-subtype-specific laboratory diagnosis can be made by RT-PCR applied directly to clinical specimens.

Human infection with influenza H9N2

We report the clinical features of two cases of human infection with influenza A virus subtype H9N2 in Hong Kong, and show that serum samples from blood donors in Hong Kong had neutralising antibody suggestive of prior infection with influenza H9N2.

Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?

BACKGROUND In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role. METHODS We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor alpha (TNF alpha) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-alpha response. FINDINGS The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta. The concentration of TNF-alpha protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF alpha induced by the virus. INTERPRETATION The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF alpha. This characteristic may contribute to the unusual severity of human H5N1 disease.

The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses

In an attempt to assess the importance of the nucleoprotein (NP) in the determination of host specificity, a series of experiments was performed on influenza A viruses of the H3N2 subtype. We have examined rescue of mutants of A/FPV/Rostock/34 with temperature-sensitive (ts) lesions in the nucleoprotein (NP) gene by double infection of chick embryo cells with H3N2 strains isolated from different species. The ts mutants could be rescued by all avian H3N2 strains but not by any of the human H3N2 isolates. Only two of the swine H3N2 strains tested were able to rescue our mutants. The NP gene of these two swine isolates resembled the NP gene of the avian strains genetically in the hybridization test. However, their NPs reacted differently with a set of monoclonal antibodies when compared with NPs of avian H3N2 strains. Concerning multiplication in ducks they behaved like the other swine and human strains. The phosphopeptide fingerprints of all swine isolates tested were alike and were different from those of human or avian origin. Our observations are compatible with the idea that human H3N2 strains might not be able to cross the species barrier to birds directly, and possibly also not the other way around, without prior reassortment in pigs, which seem to have a broader host range concerning the compatibility of the NP gene in reassortants.

Cocirculation of Avian H9N2 and Contemporary “Human” H3N2 Influenza A Viruses in Pigs in Southeastern China: Potential for Genetic Reassortment?

ABSTRACT Pigs are permissive to both human and avian influenza viruses and have been proposed to be an intermediate host for the genesis of pandemic influenza viruses through reassortment or adaptation of avian viruses. Prospective virological surveillance carried out between March 1998 and June 2000 in Hong Kong, Special Administrative Region, Peoples Republic of China, on pigs imported from southeastern China, provides the first evidence of interspecies transmission of avian H9N2 viruses to pigs and documents their cocirculation with contemporary human H3N2 (A/Sydney/5/97-like, Sydney97-like) viruses. All gene segments of the porcine H9N2 viruses were closely related to viruses similar to chicken/Beijing/1/94 (H9N2), duck/Hong Kong/Y280/97 (H9N2), and the descendants of the latter virus lineage. Phylogenetic analysis suggested that repeated interspecies transmission events had occurred from the avian host to pigs. The Sydney97-like (H3N2) viruses isolated from pigs were related closely to contemporary human H3N2 viruses in all gene segments and had not undergone genetic reassortment. Cocirculation of avian H9N2 and human H3N2 viruses in pigs provides an opportunity for genetic reassortment leading to the emergence of viruses with pandemic potential.

Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR

Although A/Hong Kong/156/97 (H5N1/97)-like viruses associated with the “bird flu” incident in Hong Kong SAR have not been detected since the slaughter of poultry in 1997, its putative precursors continue to persist in the region. One of these, Goose/Guangdong/1/96 (H5N1 Gs/Gd)-like viruses, reassorted with other avian viruses to generate multiple genotypes of H5N1 viruses that crossed to chickens and other terrestrial poultry from its reservoir in geese. Whereas none of these recent reassortants had acquired the gene constellation of H5N1/97, these events provide insight into how such a virus may have been generated. The recent H5N1 reassortants readily infect and kill chicken and quail after experimental infection, and some were associated with significant mortality of chickens within the poultry retail markets in Hong Kong. Some genotypes are lethal for mice after intra-nasal inoculation and spread to the brain. On this occasion, the early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5N1 viruses provide cause for pandemic concern.

H9N2 Influenza Viruses Possessing H5N1-Like Internal Genomes Continue To Circulate in Poultry in Southeastern China

ABSTRACT The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.

Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans?

ABSTRACT A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical “mixing vessel.” Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus.

Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?

ABSTRACT In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized.