Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015

Abstract

Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

Keywords: Vietnam; antigenicity; evolution; highly pathogenic avian influenza; reassortment; vaccine selection.

FIG 1

Maximum likelihood tree of the HA1 genes of H5 highly pathogenic avian influenza A(H5) viruses. The nearest reassortant WHO candidate vaccine viruses (CVV) for each clade are colored red. Amino acid differences at branch nodes indicate shared HA1 substitutions relative to the reference strains. Mutations listed to the right of each strain name indicate amino acid changes found only in that individual virus. Mutations highlighted with underlining indicate previously recognized molecular markers. Branches on the tree with HA sequences from human cases are colored red. Bootstraps with values greater than 50 generated from 1,000 replicates are shown at branch nodes. The scale bar represents the number of nucleotide substitutions per site.

FIG 2

Schematic diagram of genome constellations of H5 viruses in Vietnam, 2012 to 2015. Surface and/or internal protein gene derivatives of clade 1.1.2 viruses are shown in red, clade 2.3.2.1a viruses in light green, clade 2.3.2.1b viruses in brown, clade 2.3.2.1c viruses in purple, clade 2.3.4.4 H5N6 viruses in blue, clade 7.2 viruses in dark green, H6N6 LPAI PB2 viruses in black, H7N7 LPAI PB2 viruses in light blue, and N6 with a stalk deletion in orange.

FIG 3

Spatial distribution of H5 virus clades and genotypes in Vietnam. (a) 2012 to 2013. (b) 2014. (c) 2015. Maps drawn using ArcGIS software (Esri, Redlands, CA).

FIG 4

HA1 substitutions identified at putative antigenic sites on the HA trimeric structure. Amino acid differences between the consensus sequence of each clade and the sequence of the nearest poultry vaccine virus are displayed for clade 1.1.2 versus A/Vietnam/1203/2004 (A), clade 2.3.2.1c versus A/duck/Guangdong/S1322/2010 (Re-6) (B), clade 2.3.4.4 versus A/Anhui/1/2005 (Re-5) (C), and clade 7.2 versus A/chicken/Shanxi/2/2006 (Re-4) (D).

FIG 5

Antigenic mapping for clades 1.1.2 and 7.2 (A), clade 2.3.2.1c (B), and clade 2.3.4.4 (C). Each grid indicates 1 unit of antigenic distance, a 2-fold dilution in HI titer.