Scott Krauss

Large-scale sequence analysis of avian influenza isolates.

The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.

Eight-plasmid system for rapid generation of influenza virus vaccines.

The antigenic variation of influenza A virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins requires frequent changes in vaccine formulation. The classical method of creating influenza virus seed strains for vaccine production is to generate 6 + 2 reassortants that contain six genes from a high-yield virus, such as A/PR/8/34 (H1N1) and the HA and NA genes of the circulating strains. The techniques currently used are time-consuming because of the selection process required to isolate the reassortant virus. We generated the high-yield virus A/PR/8/34 (H1N1) entirely from eight plasmids. Its growth phenotype in embryonated chicken eggs was equivalent to that of the wild-type virus. By using this DNA-based cotransfection technique, we generated 6 + 2 reassortants that had the antigenic determinants of the influenza virus strains A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), A/teal/HK/W312 (H6N1), and A/quail/HK/G1/97 (H9N2). Our findings demonstrate that the eight-plasmid system allows the rapid and reproducible generation of reassortant influenza A viruses for use in the manufacture of vaccines.

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.

Evolution of Swine H3N2 Influenza Viruses in the United States

ABSTRACT During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851–8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4,382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.

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.

Lethality to Ferrets of H5N1 Influenza Viruses Isolated from Humans and Poultry in 2004

ABSTRACT The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.

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.

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

ABSTRACT 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.

Inefficient Transmission of H5N1 Influenza Viruses in a Ferret Contact Model

ABSTRACT The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with α2,3 or α2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess “avian-like” α2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 103 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for “human-like” α2,6-linked SA receptors in addition to their affinity for α2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two naïve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other naïve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.

Hemagglutinin–neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin–neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.