Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30
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PROVIDER: PRJNA209214 | ENA |
REPOSITORIES: ENA
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Project description:Gene transcription effects of mutations in the infuenza virus A/Hong Kong/1/1968(H3N2) nonstructural 1 NS1 gene in infected human A549 (lung epithilium) cells Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30 (manuscript submitted to Virology Journal). Human cells were infected with influenza viruses mutants with specific gain of function mutations in the NS1 gene in order to assess the affects of each mutation on host gene expression. Human (A549) and mouse (M1) cells were infected at a multiplicity of infection of 2 (infectious viruses/cell) and incubated for 8 hr before collection of total RNA and microarray anlaysis using the Affymetrix platforms. Samples were compared in triplicate to mock PBS infected (uninfected) cells to detecte dysregulated genes for A/HK/1/1968(H3N2) wt, and the following NS1 gene mutants: F103L, M106I, M106V, and F103L + M106I. Background: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997(H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. Methods: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein proprieties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, effect on host gene transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. Results: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was partially or totally restored by the F103L or M106I mutations respectively. Conclusions: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 viruses to switch hosts and cause severe disease in mammals.
2013-06-22 | GSE48200 | GEO
Project description:Gene transcription effects of mutations in the infuenza virus A/Hong Kong/1/1968(H3N2) nonstructural 1 NS1 gene in infected human A549 (lung epithilium) cells Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30 (manuscript submitted to Virology Journal). Human cells were infected with influenza viruses mutants with specific gain of function mutations in the NS1 gene in order to assess the affects of each mutation on host gene expression. Human (A549) and mouse (M1) cells were infected at a multiplicity of infection of 2 (infectious viruses/cell) and incubated for 8 hr before collection of total RNA and microarray anlaysis using the Affymetrix platforms. Samples were compared in triplicate to mock PBS infected (uninfected) cells to detecte dysregulated genes for A/HK/1/1968(H3N2) wt, and the following NS1 gene mutants: F103L, M106I, M106V, and F103L + M106I. Background: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997(H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. Methods: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein proprieties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, effect on host gene transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. Results: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-M-NM-2 promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was partially or totally restored by the F103L or M106I mutations respectively. Conclusions: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 viruses to switch hosts and cause severe disease in mammals. Triplicate biological replicates of mock PBS treated (uninfected) cells to detecte dysregulated genes for A/HK/1/1968(H3N2) wt, and the following NS1 gene mutants: F103L, M106I, M106V, and F103L + M106I. Cells were infected at a multiplicty of infection of 2 and cells were incubated for 8 hr at 37 C for 8 hrs before RNA extraction and analysis of 3 biological replicates relative to mock PBS infected cells.
2013-06-22 | E-GEOD-48200 | biostudies-arrayexpress
Project description:The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-? promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.
| S-EPMC3733596 | biostudies-literature
Project description:BACKGROUND: The underlying basis for the seasonality of influenza A viruses is still uncertain. Phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: 281 complete hemagglutinin (HA) and neuraminidase (NA) sequences were obtained from influenza A(H3N2) viruses, collected over 10 years (1997-2006) from Hong Kong. These dated sequences were analyzed with influenza A(H3N2) vaccine strain sequences (Syd/5/97, Mos/10/99, Fuj/411/02, Cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V). Before 2001, the Hong Kong HA and NA sequences clustered more closely with the older vaccine sequences (Syd/5/97, Mos/10/99) than did sequences from elsewhere. After 2001, this trend reversed with significant clusters containing HA and NA sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza A(H3N2) seasonality during this 10-year period. However, at least one example from Hong Kong was found suggesting that in some years, influenza A(H3N2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. None of these Hong Kong influenza A(H3N2) NA sequences contained any of the known NAI-resistance associated mutations. CONCLUSIONS/SIGNIFICANCE: The seasonality of influenza A(H3N2) may be largely due to global migration, with similar viruses appearing in different countries at different times. However, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. Naturally occurring NAI resistance mutations were absent or, at least, very rare in this population.
| S-EPMC2481298 | biostudies-literature
Project description:Pilaira anomala RSA 1997+ Resequencing
| PRJNA535044 | ENA
Project description:The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?
| S-EPMC136834 | biostudies-literature
Project description:Sentinel laboratory surveillance from one hospital and passive discharge diagnosis (Clinical Management System, CMS) data from all public Hospital Authority (HA) hospitals were used to estimate disease burden and incidence of rotavirus in hospitalised Hong Kong children over 14 rotavirus seasons (1 July 1997 to 31 March 2011). A primary diagnosis of a gastroenteritis-related disorder was noted in 9.8% of children aged below 5 years, and a primary or secondary diagnosis in 11.8%. Any CMS diagnosis of rotavirus (ICD 008.61) was initially used to derive incidence estimates of rotavirus by age group. Rotavirus was recorded as any primary or any secondary diagnosis in 1.6% of children below 5 years of age. The unadjusted incidence rates per 100,000 person-years based on any CMS diagnosis of rotavirus were: 249 (0 to <1m); 612 (1 to <2m); 1066 (2 to <6m); 1383 (6 to <11m); 959 (1 to <2y); 406 (2 to <3y); 233 (3 to <4y); 124 (4 to <5y). Overall the rotavirus incidence was 1071 in children below 2 years and 542 in children below 5 years of age, with the incidence rates trending up during the time period (p=0.001). A similar but less marked upward trend (p=0.046) was noted for the incidence of all-cause gastroenteritis. Laboratory results from a single surveillance hospital (1 July 2000 to 31 March 2011) were then linked to these CMS codes to derive adjustment factors for possible over- and under-diagnosis of rotavirus based on CMS codes alone. This analysis suggested that a CMS diagnosis of rotavirus alone likely under-reported true incidence by a factor of between 1.59 and 2.02 in children below 5 years of age. Despite the availability of rotavirus vaccines in the private sector since 2006, no reduction in the incidence of hospitalisation for either rotavirus or all-cause gastroenteritis was noted in Hong Kong children below 5 years of age over 14 rotavirus seasons (1997-2011).
| S-EPMC7115370 | biostudies-literature
Project description: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.
| S-EPMC124404 | biostudies-literature
Project description:From January 2004 through June 2008, surveillance of dead wild birds in Hong Kong, People's Republic of China, periodically detected highly pathogenic avian influenza (HPAI) viruses (H5N1) in individual birds from different species. During this period, no viruses of subtype H5N1 were detected in poultry on farms and in markets in Hong Kong despite intensive surveillance. Thus, these findings in wild birds demonstrate the potential for wild birds to disseminate HPAI viruses (H5N1) to areas otherwise free from the viruses. Genetic and antigenic characterization of 47 HPAI (H5N1) viruses isolated from dead wild birds in Hong Kong showed that these isolates belonged to 2 antigenically distinct virus groups: clades 2.3.4 and 2.3.2. Although research has shown that clade 2.3.4 viruses are established in poultry in Asia, the emergence of clade 2.3.2 viruses in nonpasserine birds from Hong Kong, Japan, and Russia raises the possibility that this virus lineage may have become established in wild birds.
| S-EPMC2666293 | biostudies-literature
Project description:We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses, which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different combinations of "internal" genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A, C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1, NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly selected in mice.
| S-EPMC149508 | biostudies-literature