New H6 influenza virus reassortment strains isolated from Anser fabalis in Anhui Province, China

Abstract

Background: H6 subtype avian influenza viruses are globally distributed and, in recent years, have been isolated with increasing frequency from both domestic and wild bird species as well as infected humans. Many reports have examined the viruses in the context of poultry or several wild bird species, but there is less information regarding their presence in migratory birds.

Methods: Hemagglutination and hemagglutination inhibition tests were used to measure HA activity for different HA subtypes. Whole viral genomes were sequenced and analysed using DNAstar and MEGA 6 to understand their genetic evolution. Pathogenicity was evaluated using a mouse infection model.

Results: We isolated 13 strains of H6 virus from faecal samples of migratory waterfowl in Anhui Province of China in 2014. Phylogenetic analysis showed gene reassortment between Eurasian and North American lineages. Five of the identified H6 strains had the ability to infect mice without adaptation.

Conclusion: Our findings suggest that regular surveillance of wild birds, especially migratory birds, is important for providing early warning and control of avian influenza outbreaks.

Keywords: Anser fabalis; Avian influenza virus; H6 subtype.

Fig. 1

Phylogenetic analysis based on maximum likelihood of surface genes H6 (a), N1 (b) and N2 (c) of AIVs isolated in Anhui Province, 2014. Phylogenetic trees were generated with the MAGE 6.0 software package. The phylogenetic trees for HA (1A) were rooted to A/Turkey/Canada/1/1963 (H6N8). The phylogenetic tree for the N1 genes were rooted to A/Brevig_Mission/1/18(H1N1) and N2 genes were rooted to turkey/Massachussetts/3740/65(H6N2). The genomic sequences of the viruses listed in black and blue were downloaded from available databases; the viruses listed in red, yellow, and/or purple was sequenced in this study. The sequences shown in cyan and italics in Fig 1c were obtained from a paper by Wang GJ. Abbreviations: CK, chicken; DK, duck; GS, goose; SW, swine; ML, mallard; GT, green-winged teal; PD, Pacific black duck; WDK, wild duck; MLDK, mallard duck; WB, wild bird; EN, environment

Fig. 2

Phylogenetic analysis by maximum likelihood of inner genes of H6 subtype AIVs isolated in Anhui Province, 2014: (a) PB2, (b) PB1, (c) PA, (d) NP, (e) M, and (f) NS. The colours of the virus names listed in the PB2, PB1, PA, NP, M, and NS trees match those used in the genotype table. The phylogenetic trees of inner genes were rooted to A/Turkey/Canada/1/1963 (H6N8). The nucleotide sequences of the viruses listed in black were downloaded from GenBank; the viruses listed in red, yellow, or green were sequenced in this study. The sequences shown in cyan and italics were obtained from a paper by Wang GJ. Abbreviations: CK, chicken; DK, duck; GS, goose; SW, swine; WDK, wild duck; WB, wild bird; WGS, wild goose; EN, environment

Fig. 3

Changes in body weight. Body weight (a) and mortality (b) in BALB/c mice inoculated with strains A and B of H6 viruses. a Body weights and survival rates of mice were observed over 14 days after infection. b Lungs and trachea were collected at 3 days post infection (p.i.), and virus replication levels were measured by EID₅₀ values in specific-pathogen-free (SPF) eggs

Fig. 4

A simplified schematic showing the putative genomic composition of the novel H6N1 and H6N2 reassortant avian influenza viruses identified in this study. The eight gene segments (from top to bottom) in each virus are PB2, PB1, PA, HA, NP, NA, M, and NS. Each colour represents a separate source background. Eight genotypes are listed here. The six different colours represent separate virus backgrounds. The illustration is based on nucleotide-distance comparisons and phylogenetic analyses