Genetic Reassortment and Virus Evolution
We performed phylogenetic analyses of the internal genes of H5N6 viruses
and classified them into different lineages according to tree topology
and Bayesian phylogenetic analyses [Figure S1][Figure S2]. Each
internal gene belongs to at least two lineages, one from circulating H5
AIVs and the other from LPAIVs, including H6 and H9N2/H7N9. In general,
the H5/Minor::N6/Major viruses and
H5/Major::N6/Major viruses have
nearly all of the internal genes derived from H5N6 viruses, 23 of 34 of
the H5/Major::N6/Minor H5N6 had H9N2/ H7N9-derived internal genes
[Figure 1(A)]. Some H5N6 isolates contained internal gene(s) from
LPAIVs circulating in waterfowl. According to lineage classification of
phylogenetic trees and Bayesian phylogenetic analyses, we classified all
of the H5N6 viruses into 20 genotypes [Figure
2][Table S1]. These genotypes
are G1(53), G1.1(4), G1.2(4), G1.3(1), G1.4(4), G1.5(3), G2(9),
G2.1(30), G2.2(3), G3(1), G3.1(17), G3.2(3), G3.3(3), G3.4(2), G3.5(4),
G3.6(3), G3.7(1), G3.8(1), G2.1.1(6), and G2.1.2(1)[Figure 2]. 81 of
153 comprised 15 genotypes have been isolated since 2016, 39 of the
viruses were classified into six genotypes (G1(28), G1.1(2), G1.2(1),
G1.3(1), G2.1(7)) and had been isolated during 2017, and 27 of 153 virus
comprised six genotypes (G1(17), G1.1(2), G1.2(3), G1.5(3), and G2.1(2))
and had been isolated during 2018. Only six viruses (G1(6)) were
isolated during 2019 [Figure 2]. The number of genotypes decreased
between 2016 and 2019 in terms of the genetic diversity of the viruses,
which means that the strain may have evolved to become more stable. The
G1 genotype HA-NA and the
combination of internal genes may be best suited to the current
adaptation of the strains.
Bi et al. showed that, from 2013 to 2016, a large number of H5N6
reassortment strains emerged(Bi et al., 2016). Our research confirmed
that the H5N6 reassortment strain types in 2016 were very numerous. By
contrast, the genotypes of this strain gradually decreased from 2017 to
2019 [Figure 2]. Influenza mainly occurs in winter, and we set July
2015 to June 2016 as one influenza season. It can be seen that all of
the strains of the G3 series mostly (30/33) occurred in this winter.
Many genotypes (G3.1, G3.2, G3.3, G3.4, G3.5, G3.6, G3.7, and G3.8)
emerged by the G3 strain reassortment with H9N2 and other subtypes of
AIVs[Table S1]. The frequency of G3.1 posed a major advantage. Only
three strains of G3 were isolated during the subsequent influenza
season. The G2 and G2.1 strain appeared in early 2016 and the winter of
2016. In the winter of 2016, two reassortment genotypes, G2.1.1 and
G2.1.2, emerged by the reassortment of G2.1 and H9N2 with other subtype
strains and G2 with H9N2. Four genotypes (G1.1, G1.2, G1.3, and G1.4)
emerged by G1 reassorted with H6 and other subtypes of the AIVs during
July 2017 and June 2018. Only the G1.4 genotype and the new G1.5
genotype were isolated in the winter of 2018 [Figure 2][Table
S1].