Geographic distributions of avian influenza viruses
Based on RT-PCR, the overall positivity rate for influenza A was 15.51% (Fig 1-a). The positivity rates were particularly high (17.21-44.94%) in 11 provinces in five of the seven regions tested: Central China (Hunan and Hubei provinces), Eastern China (Fujian, Jiangxi and Zhejiang provinces), Southern China (Guangxi Autonomous Region and Guangdong Province), Southwest China (Si Chuan, Chong Qing municipality and Guizhou), and Northwest China (Gan Su).
Among the influenza A viruses detected, the H5 subtype was detected in an average of 3.17% of samples (Fig 1-b). The positivity rates were significantly high (9.74%-14.66%) in three provinces and one municipality in three regions: Southwest China (Chong Qing municipality), Central China (Hunan and Jiangxi provinces), and Northwest China (Gansu Province).
The mean positivity rate for the H7 subtype was 1.64% (Fig 1-c), with rates of up to 4% being detected in three regions: Eastern China (Jiangsu, Fujian, Zhejiang and Jiangxi provinces), South China (Guangdong Province) and Central China (Hunan Province). During the study period, of the H7 subtypes, only H7N9 was detected.
The highest mean positivity rate for the H9 subtype 9.74% (Fig 1-d). Fifteen provinces, autonomous regions and municipalities displayed high positivity rates (10%-26.82%).
Seasonality of avian influenza viruses in different environments
The monthly nucleic acid positivity rates for influenza A and the subtypes H5, H7, and H9 in the poultry-related environmental samples are shown in Fig 2. The positivity rates in China showed obvious seasonality and were highest in December and January and lowest from May to September.
Variations in total influenza A, H5, H9 and H7 positivity rates among the sampling sites
During 2014-2017, samples with the highest total influenza A and H5, H9 and H7 positivity rates were collected from LPMs (29.91%, 5.19%, 16.85%, 5.21% on average, respectively), followed by slaughterhouses (21.25%, 3.45%, 11.9%, 2.17% on average, respectively). In contrast, poultry farms, backyards, and wild bird habitats had influenza A positivity rates of 3.26%, 3.36%, and 1.17% on average, respectively, while the H5, H9 and H7 positivity rates were all less than 1%. The statistical analysis indicated that LPMs and slaughterhouses were associated with significantly higher positivity rates for total influenza A and the H5, H9, and H7 subtypes than all other sites during the study period (p < 0.05; Table 1).
Influenza A and subtype H5, H9 and H7 positivity rates among different sample types
Environmental samples that were collected from sewage and chopping boards had significantly higher positivity rates for influenza A and the subtypes H5, H7, and H9 than those collected from faeces, cages, and feeding troughs (p < 0.05). Furthermore, the positivity rates of subtype H9 in samples originating from sewage and chopping boards were significantly higher than those of subtypes H5 and H7 (p < 0.05; Table 2).
Multiple subtypes of influenza A viruses were detected in poultry-related environments
In total, 9 HA subtypes and 7 NA subtypes were detected during the study period, including the HA subtypes H1, H3, H4, H5, H6, H7, H9, H10, and H11 and the NA subtypes N1, N2, N3, N6, N7, N8, and N9. The H5, H7, and H9 subtypes of influenza A virus accounted for the majority of positive samples (Supplemental table S2). The H5 subtype of influenza A virus was present in 23.1-41.52% of the influenza A-positive samples, the H7 subtype was present in 6.86-27.04%, and the H9 subtype was present in 35.19-59.97%. The other subtypes, including H3 (6.3%), H4 (1.04%), and H6 (4.02%), were present in only small proportions of samples (Supplemental table S2).
Based on the virus isolation data, H5, H9 and H7 were the major subtypes. The H5 subtypes comprised H5N1, H5N6, H5N2, H5N8 and H5N9. The proportion of subtype H5N6 increased more than threefold from 11% in 2014 to 34% in 2016 and then decreased to 12.14% in 2017. The H5N1 subtype showed a declining trend from 2014-2017. The proportions of H5N1 were 8.74% (2014), 10.53% (2015), 5.54% (2016) and 5.94% (2017). The H5N2, H5N8 and H5N9 subtype proportions were much lower than the H5N1 and H5N6 subtype proportions. The proportion of H7N9 reached 23% in 2017, which was approximately four times that in 2014 (6%), while the proportion of H9N2 decreased from 2014 to 2017, with proportions of 59%, 51%, 35% and 36%, respectively (Fig 3 and supplementary tableS2). GraphPad Prism 5 was used to construct the Fig 3.
In approximately 90% of the samples, only a single subtype of influenza A virus was detected. A small proportion of samples (1.5%) contained a combination of different subtypes; for example, subtype H9 was detected with H7, H5 and other subtypes in the same sample, further proving the co-circulation of multiple subtypes.