Kanta Subbarao

Genetic characterization of H3N2 influenza viruses isolated from pigs in North America, 1977–1999: evidence for wholly human and reassortant virus genotypes

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw/ONT/97 virus, which did not spread from pig-to-pig, lacks 11 of these changes.

Molecular Correlates of Influenza A H5N1 Virus Pathogenesis in Mice

ABSTRACT Highly pathogenic avian influenza A H5N1 viruses caused an outbreak of human respiratory illness in Hong Kong. Of 15 human H5N1 isolates characterized, nine displayed a high-, five a low-, and one an intermediate-pathogenicity phenotype in the BALB/c mouse model. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this model.

Aged BALB/c Mice as a Model for Increased Severity of Severe Acute Respiratory Syndrome in Elderly Humans

ABSTRACT Advanced age has repeatedly been identified as an independent correlate of adverse outcome and a predictor of mortality in cases of severe acute respiratory syndrome (SARS). SARS-associated mortality may exceed 50% for persons aged 60 years or older. Heightened susceptibility of the elderly to severe SARS and the ability of SARS coronavirus to replicate in mice led us to examine whether aged mice might be susceptible to disease. We report here that viral replication in aged mice was associated with clinical illness and pneumonia, demonstrating an age-related susceptibility to SARS disease in animals that parallels the human experience.

Intercontinental Circulation of Human Influenza A(H1N2) Reassortant Viruses during the 2001–2002 Influenza Season

Reassortant influenza A viruses bearing the H1 subtype of hemagglutinin (HA) and the N2 subtype of neuraminidase (NA) were isolated from humans in the United States, Canada, Singapore, Malaysia, India, Oman, Egypt, and several countries in Europe during the 2001-2002 influenza season. The HAs of these H1N2 viruses were similar to that of the A/New Caledonia/20/99(H1N1) vaccine strain both antigenically and genetically, and the NAs were antigenically and genetically related to those of recent human H3N2 reference strains, such as A/Moscow/10/99(H3N2). All 6 internal genes of the H1N2 reassortants examined originated from an H3N2 virus. This article documents the first widespread circulation of H1N2 reassortants on 4 continents. The current influenza vaccine is expected to provide good protection against H1N2 viruses, because it contains the A/New Caledonia/20/99(H1N1) and A/Moscow/10/99(H3N2)-like viruses, which have H1 and N2 antigens that are similar to those of recent H1N2 viruses.

Immunohistochemical and In Situ Hybridization Studies of Influenza A Virus Infection in Human Lungs

Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans.

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian species and were isolated from humans in Hong Kong and Guangdong province, China in 1999 raising concern of their potential for pandemic spread. We generated a high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic reassortment, for evaluation as a potential vaccine candidate in humans. Pathogenicity studies showed that the G9/PR8 reassortant was not highly pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus vaccine induced hemagglutination inhibiting antibodies and conferred complete protection against challenge with G9 and the antigenically distinct H9N2 A/Hong Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the high growth G9/PR8 reassortant has properties that are desirable in a vaccine seed virus and is suitable for evaluation in humans for use in the event of an H9 pandemic.

Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine.

Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.

Molecular aspects of avian influenza (H5N1) viruses isolated from humans.

In 1997, 18 human infections with H5N1 influenza type A were identified in Hong Kong and six of the patients died. There were concomitant outbreaks of H5N1 infections in poultry. The gene segments of the human H5N1 viruses were derived from avian influenza A viruses and not from circulating human influenza A viruses. In 1999 two cases of human infections caused by avian H9N2 virus were also identified in Hong Kong. These events established that avian influenza viruses can infect humans without passage through an intermediate host and without acquiring gene segments from human influenza viruses. The likely origin of the H5N1 viruses has been deduced from molecular analysis of these and other viruses isolated from the region. The gene sequences of the H5N1 viruses were analysed in order to identify the molecular basis for the ability of these avian viruses to infect humans. Copyright

Encephalitis Associated with Influenza B Virus Infection in 2 Children and a Review of the Literature

Two children with influenza B-associated encephalitis (IBAE) presented to our hospital during the winter of 2000-2001, both of whom had cases notable for mutism in association with encephalitis. A review of the literature identified 13 additional reports consistent with IBAE that contained sufficient data for analysis. Eleven of 15 reported cases occurred in children aged <or=18 years; of these, more than one-half occurred in children <11 years of age. Neurologic symptoms appeared within the first 4 days of illness in 13 cases. Speech abnormalities were observed in 4 patients and consisted of mutism in 3. Although the majority of patients recovered fully, 3 were left with neurologic sequelae, and 1 died. These cases reveal the spectrum of IBAE and its potential for long-term sequelae. Clinicians caring for children should remain vigilant for this rare complication of influenza B virus infection.

Influenza Vaccines: Present and Future

Immunization is the most feasible method for preventing influenza. Vaccination against influenza is recommended for everyone 65 years of age and older and for persons less than 65 years of age who are at risk for developing complications of influenza. Immune correlates of protection have been established, and a global network is in place to monitor the appearance and circulation of antigenic variants of influenza viruses, as well as the appearance of novel subtypes of influenza A. Antigenic and genetic analyses of circulating viruses and testing of serum from vaccine recipients guide vaccine composition updates. The efficacy of influenza vaccines depends in part on the closeness of the antigenic match between the vaccine strain and the epidemic strain. Currently licensed influenza vaccines are trivalent, formalin-inactivated, egg-derived vaccines; their efficacy ranges from 70 to 90% in young, healthy populations when there is a close antigenic match between vaccine strains and epidemic strains. Development of intranasally administered alternative vaccines and improvement of the existing vaccine are areas of active research. A trivalent, ca live vaccine is the most promising LAIV candidate. In a field trial, efficacy rates of LAIV in young children were 96% against influenza A (H3N2) and 91% against influenza B. However, few data are available to compare this formulation of the trivalent ca live vaccine with the trivalent, inactivated vaccine. Influenza vaccine recommendations will most likely be revised on licensure of LAIV; each vaccine may offer distinct advantages in specific populations.