Characterization of an H10N8 influenza virus isolated from Dongting lake wetland

All 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A virus have been isolated from wild birds [1, 2]. Therefore, wild birds, especially those in wetlands and aquatic environments, are considered to be natural reservoirs of avian influenza viruses[2]. It is accepted that water is an important component in the transmission cycle of avian influenza virus, because shedding of virus into the water leads to transmission among wild birds and poultry via the indirect fecal-oral route [2, 3].

Dongting Lake wetland is an important habitat and over-wintering area for East Asian migratory birds, and is located at 28°30'-30°20' N and 111°40'-113°40' E in the Northeastern part of Hunan Province, China. In 2007, an influenza virus A/environment/Dongting Lake/Hunan/3-9/07 (H10N8) was isolated from water from Dongting Lake wetland. The whole genome of the isolated virus was sequenced, the phylogenetic trees of each gene segment were generated, and the pathogenicity of the strain for mice and SPF White Leghorn Chickens was studied. To study further its potential pathogenicity for mammals, the virus was passaged in mouse lung, and the pathogenicity and corresponding amino acid variations of the mouse-lung-adapted virus from passages 2, 4 and 6 (P2, P4 and P6) were compared with those of wild-type virus (P0).

Among all 16 HA and 9 NA subtypes of influenza A viruses, the highly pathogenic avian influenza viruses are restricted to subtypes H5 and H7, although not all H5 and H7 viruses are virulent. However, low-pathogenicity viruses previously have been shown to be precursors of highly pathogenic viruses [5, 6]. The H10N8 strain isolated in the present study replicated efficiently in mouse lung without prior adaptation. Its pathogenicity for mice increased rapidly during lung adaptation, and even after 2 passages, it became lethal for mice. It has been reported that H11N9 subtype virus can be transmitted directly from wild ducks to waterfowl hunters[7]. Therefore, when emphasis is placed on H5, H7 and H9 subtype avian influenza viruses, the other subtypes should not be ignored, because they might also be a potential threat to public health.

Migratory birds that carry avian influenza virus might shed virus into the environment along their migratory route. After the birds leave an area, environmental persistence of the virus could play an important ecological role in virus transmission [8, 9]. Shedding of the virus into water could lead to infection of any waterfowl that are dabbling in the same area, via the direct or indirect fecal-oral route[2]. Animals that utilize an area in which viruses persist might experience increased viral exposure, and therefore, greater potential for viral infection and reassortment [8].

Phylogenic analysis showed that all the gene segments of environment/DT/Hunan/3-9/07 belonged to the Eurasian lineage, but some gene segment of the virus had different origin. It is believed that all 16 subtypes of HA and 9 subtypes of NA are perpetuated in the aquatic bird population, and reassorted with each other with a high frequency [1, 2]. It is assumed that, when viruses of different origin are mixed somewhere in the habitats or aggregation sites along the migration route, gene reassortment takes place [10]. The virus strain isolated in the present study could have been resulted from multiple gene segments reassortment between different viruses, including H5 and H7 subtypes.

The virus strain isolated in this study replicated effectively in mouse lung without prior adaptation. During adaptation, the virus demonstrated extrapulmonary spread and enhanced replication in the mouse, and the viruses were recovered from multiple organs, including the brain. The virulence of the strain in mice increased rapidly and became lethal after only 2 lung-to-lung passages. The host specificity and pathogenicity of influenza A virus have always been considered as being determined by multiple genes [11, 12]. However, the genetic basis for virulence of influenza A virus is largely unknown [13]. During 6 passages of the H10N8 strain in mouse lung, amino acid substitutions were observed at 22 sites in the viral genome (Table 4). These demonstrated that multiple amino acid substitutions were likely to have been involved in the adaptation of the virus to mice. It has been reported that the amino acid substitution from E to K at site 627 of the PB2 gene is the first step in virus adaptation in mammals, and that this substitution is host-dependent [14, 15]. Therefore, we deduced that the PB2-E627K substitution significantly enhanced the pathogenicity of the H10N8 strain for mice. However, after 2 lung-to-lung passages, viral pathogenicity was also enhanced and caused death, compared with the wild-type virus, but there was no amino acid substitution at the 627 site in the PB2 gene of P2 virus, which indicated that the amino acid substitutions at other sites in the viral genome were also involved in the increased virulence of mouse-lung-adapted virus strains. It has also been shown that molecular changes at specific sites of PA and PB1 genes are associated with high pathogenicity of the H5N1 virus [16]. However, no amino acid substitution was observed in PB1 gene during virus adaptation, whereas the amino acids 247 and 611 of PA were substituted. The amino acid at site 479 of the NP gene of the virus strain isolated in the present study was substituted from L to F during passage in murine lung, which might influence NP oligomerization [13, 17]. The activity-enhancing mutations of the viral polymerase complex that consists of PB2, PB1, PA and NP might be a prerequisite for adaptation to a new host [17, 18].

The amino acids at 5 sites of the HA gene were substituted during passage of the virus in mouse lung. In the H5N1 subtype viruses, the multiple basic acids adjacent to the cleavage site of the HA gene are a prerequisite for lethality in mice and chickens [19]. The pathogenicity of the H10N8 virus isolated in this study increased rapidly during passage in mouse lung, although no amino acid substitutions were observed near the cleavage sites of its HA gene. The balance between neuraminidase activity of the NA gene and receptor-binding activity of the HA gene is closely associated with replication of influenza virus in the host [20]. Studies have shown that M1 gene mutation during passage in mouse lung might enhance virus replication, which results in enhanced pathogenicity [21]. The amino acid substitutions at sites 53 and 192 of the M1 gene might have close relationship with viral pathogenicity. NS1 protein plays an important role in counteracting the host interferon system [22], and is closely related to viral pathogenicity and host specificity [23, 24]. In the present study, the amino acids at sites 54, 89 and 155 of the NS1 gene were substituted. It should be noticed that the substitution from Y to H at site 89 might be closely related to pathogenicity and adaptation of influenza A virus, because the same mutation has been observed at the same site during H9N2 virus adaptation in mouse lung [12]. Amino acid substitutions were observed at multiple sites of the genomes of the H10N8 strain during adaptation in mouse lung. Comparison of the genomic amino acid sequence of P0, P2, P4 and P6 viruses are helpful in understanding the molecular mechanism of pathogenicity of influenza A virus.

When the virus was passaged in the mouse lung from P0 to P6, 22 amino acid substitutions appeared. Some of these substitutions might be introduced randomly and maintained, whereas others are selected during adaptation of the virus in mice. Some substitutions such as the PB2-E627K, NP-L479F and NS1-Y89H have been found during the other influenza virus adaptation in mouse lung [12, 13, 17]. However, whether these amino acid substitutions lead to increased virus virulence in chickens remains unknown. The wild-type H10N8 strain showed no significant pathogenicity towards SPF chickens, but the infected chickens had shed virus through the respiratory tract and cloaca. The H10N8 virus isolated in present study possesses internal genes of both H5 and H7 subtype origin, which might provide gene segments for further gene reassortment between various influenza A viruses. It is assumed that the wider the circulation of low-pathogenicity avian influenza virus in poultry, the higher the chance that mutation to high-pathogenicity virus will occur [6]. Low-pathogenicity viruses previously have been shown to be the precursors of high-pathogenicity viruses [5, 6].If such a virus is allowed to circulate in poultry or wild birds, mutations may merge, and the low-pathogenicity virus could become more pathogenic by gene mutation or reassortment.

Influenza A viruses have been maintained in waterfowl populations by water-borne transmission [25]. Shedding of the virus into the water is a major threat for epidemics in poultry [2]. Therefore, water persistence of viruses might play an important ecological role in virus transmission. Monitoring the water at aggregation and breeding sites of migratory waterfowl, mainly wetland, is very important for early detection of avian influenza virus [3]. Dongting Lake wetland is an important habitat and overwintering area along the migration route of migratory birds in East Asia. In the wetland, domestic ducks often share with wild waterfowl the same water area for dabbling and habitat, which provides ample opportunity for influenza virus to infect domestic ducks and other domestic poultry. Thus, investigation of water in Donting Lake wetland for avian influenza virus is of greater significance and convenience for understanding the route and mechanism of virus transmission between domestic fowl and migratory birds.

In the wetland, water persistence of viruses might play an important ecological role in virus transmission. The avian influenza viruses might be transmitted among wild and domestic waterfowls through waterway. It should be noted that the H10N8 subtypes of avian influenza viruses might evolve to pose a potential threat to mammals and multiple amino acid substitutions are likely to be involved in the adaptation of H10N8 influenza virus to mice.