| 1.BackgroundThe hosts of the avian influenza virus(AIVs)are widely distributed.AIVs can break species barriers,not only infect wild birds and poultry but also have the ability to infect humans directly,causing a pandemic among the population.Recently,human infection cases with H5N1,H7N9,H5N6,H10N8 posed a great threat to public health.Wild birds are the natural host of the AIVs and considered as gene banks providing genetic diversity of the virus.The AIV has been in a state of dynamic evolutionary balance in wild waterfowl.Most cases infected with highly pathogenic AIV H5 subtypes have no obvious clinical symptoms,carring viruses for a long time and distribute to new areas,then indirectly pollute environmental water or directly infect poultry or humans by contact,which is a vital role in the spread of the virus.Studies have shown that there is still an epidemic of AIVs among healthy poultry in poultry farms and live poultry markets,and the positive rate of AIV detection in live poultry markets is higher than that of farms.China's poultry farming industry has a large amount of feeding and high density.Some infected poultry could not be treated in time due to no obvious symptoms,the virus emitted continued to pollute the environment due to the irregular implementation of the environmental regular disinfection system,and the vaccination was incompleted and limited,all of which led to the widespread presence of low-pathogenic AIV in poultry in farms.The live poultry market is the main exposure site for humans infected with the AIVs.Large-scale cross-regional live-poultry transport make the sources in the live poultry market complicated and difficult to trace,resulting in the contact with poultry in different regions that may carry different subtypes of the AIVs,which artificially increase the chance of re-allocation of the virus and the possibility of new influenza virus.The prevalence of AIVs in poultry farms and live poultry markets will inevitably increase the risk of infection for occupationally exposed workers,who could be a bridge for AIVs to spread to the general population.It is generally believed that occupationally exposed people engaged in poultry breeding,transaction processing,etc.are at high risk of being infected with AIVs.Pigs are considered to be mixing vessels of influenza viruses and play a special role in cross-species transmission.Therefore,the swine workers are also included in the scope of the study to analyze and evaluate the infection status of AIVs more comprehensively and the risk factors in occupational exposure populations.Epidemiological surveys have shown that most patients infected with AIVs have a history of direct or indirect contact with birds prior to onset and the centralized farming and trans-regional live bird trade in China also increase the risk of human infection.Current serological studies have shown that silent infections of subtypes such as H4,H5,H7,H9,H10,and H11 exist in poultry and swine occupational exposure populations.Therefore,it is urgent to conduct the dynamic monitoring surveilence on sentinel chickens,poultry farms,live poultry markets,occupational exposed workers and the general population,which provide new ideas and intervention measures to the prevention and control of avian influenza virus.2.ObjectivesIn this study,specific pathogen free chickens were selected as sentinel animals for monitoring at Taihu Lake in Wuxi city,Jiangsu province,aiming to clarify the rules for the transmission of AIVs from wild birds to poultry under natural conditions.We conducted a three-year monitoring of poultry and the environment in live poultry farms and trading markets in Wuxi City in order to clarify the prevalence and subtype distribution of local AIVs,master the biological characteristics of known or new AIVs,explore genetic evolution of virus.We conducted a follow-up study contained the occupational exposure population and the general population,in order to determine whether there were silent infections in occupationally exposed populations,calculate the infection rate of different subtypes of avian influenza virus and the infection risk factors in occupational exposed workers and the general population.Finally provide advices for the prevention and control of AIVs in the future.3.MethodsFrom January to October in 2014,based on SPF sentinel chickens,we regularly collected cloacal and pharyngeal swab specimens and photographed the process of contact with wild birds.We screened for influenza A positive specimens by real-time quantitative polymerase chain reaction(Real-time PCR),in which positive specimens were cultured using chick embryos,or complex infection specimens were isolated using plaque formation assays,and then obtained whole genome sequences by high-throughput sequencing for further genetic evolutionary analysis.From July 2013 to June 2016,we conducted active surveillance in the farms and markets in Wuxi City,collecting samples including poultry throat swabs and corresponding environments once a month.We screened positive samples of influenza A by Real-time PCR and typed them,and inoculated into SPF embryo or cell culture and isolation techniques,and then obtained sequence results by one-step reverse transcription PCR amplification of whole genome or direct high-throughput sequencing technology,in order to identify the infections in poultry and genetic evolutionary characteristics of AIVs.Occupational exposure populations in farms and live poultry markets were regard as major source and enrolled in the three-year cohort study.We collected demographic information and time-exposure methods,etc.through questionnaires each year,and collected serum for hemagglutination inhibition test and microneutralization assay to detect H7N9,H9N2,H5N1,and H5N6 subtype antibody levels to determine whether there were silent infections,infection rate and risk factors of occupational exposure population.4.Results1623 samples of pharyngeal swabs were collected in sentinel chickens,of which 364(22.4%)were positive for influenza A virus by Real-time PCR detection.We successfully isolated 2 strains of H7N2 strain and 1 strain of H5N6 strain and obtained the complete genome sequence of 3 samples,in which two samples contained the gene fragments of Influenza A H7,H9 and N2,suggesting the co-infection of H7N2 and H9N2 subtypes,and another sample contained the gene fragments of Influenza A H5 and N6.Phylogenetic analysis showed that H7N2 isolation was a low pathogenic strain with a reassortment betwwen H7N9 and H9N2 viruses,which may have increased ability to infect mammals and were sensitive to neuraminidase inhibitors.The H7N2 strain was a high pathogenic strain formed by the reassortment of the H5N1 and H6N6 strains of the 2.3.4 branch,which preferentially bound to birds receptors.The sentinel chickens contacted with wild birds and shared feed and water.The strains isolated from them were highly homologous to strains isolated from humans and poultry,suggesting that wild birds played an significant factor in noval AIVs transmission.During the period of the monitoring of poultry farms in Wuxi City from July 2013 to June 2016,we collected a total of 240 samples of poultry throat swabs and 284 environmental samples,in which 84(16.0%)samples were positive for influenza A virus including 24(4.6%)samples of H9 subtypes and 2(0.4%)samples of H5 subtypes.There were no co-infection.The positive rate in poultry was significantly higher than that in the corresponding environment(23.3% vs.9.9%,?2 = 17.54,P < 0.001).Through isolation and sequencing,four strains of H9N2 virus were finally obtained,of which the HA and NA genes belonged to the Y280 strain and were highly homologous to the H9N2 predominant strain widely prevalent in domestic poultry in mainland China.The HA protein has a mutation in the Q226L(H3 numbering)site and can bind to human receptors.The R292 K site of NA protein and the S31 N site of M2 protein were mutated,suggesting they were resistant to neuraminidase inhibitors and amantadine drugs.A monitor surveilence on poultry and environment in the live poultry market in Wuxi City was conducted with 3121 samples collected,of which 726(23.3%)were positive for influenza A virus,including 466/2010(23.4%)cloacal swabs,145/590(24.5%)environmental surface swabs and 115/521(22.0%)feces/sewage samples,with no statistically significant difference(?2 = 0.99,P = 0.61).In the 726 influenza A positive samples,229(31.5%),27(3.7%)25(3.4%)were H9,H7 H5 subtypes respectively,and 11 samples were H7 and H9 subtype coinfections,2 samples were H5 and H9 subtype coinfections and 1 sample was H5,H7 and H9 subtype coinfections.The gene sequence analysis revealed that 10 different AIV subtypes(H7N9,H9N2,H3N8,H2N2,H5N1,H5N2,H5N6,H5N8,H11N2 and H1N1)are involved in the prevalence in the live poultry market and the gene reassortment between poultry and wild birds,among different subtypes or different branches of the same subtype is common.All H5 subtypes in the live poultry market are highly pathogenic.The Q226L(H3 numbering)site of the HA protein of all four H7N9 strains and all H9N2 strains was mutated,but there was no E627 K or D701 N mutation in the PB2 protein,indicating virus were prefer to human receptors,but is not fully adapted mammal.All strains of H7,H9 and H5 subtypes have various degrees of deletion in the stem region of NA,which is associated with increased virulence and adaptation and transmission to poultry.All H9N2 viruses are resistant to neuraminidase inhibitors.Two strains of H5N1 virus and all H5N2,H1N1,H7N9 and H9N2 isolates were mutated at S31 N site in M2 protein with amantadine resistance.A sero-epidemiological cohort study of occupational exposure population conducted in the same period,a total of 1995 people met the inclusion criteria on baseline in July 2013,including 511 poultry workers,569 swine workers and 950 control subjects,and due to the high mobility for occupational workers,there were dynamic changes in the number of follow-ups during the three years.A total of 964,1079 and 1545 different subjects were involved in the cohort study respectively in the poultry exposed group,swine exposed group and control group,2070,2026 and 3436 human serums were collected.The positive rate of H7N9,H9N2,and H5N1 antibodies in poultry occupational exposures(0.93%,1.87%,1.87%)was higher than that of swine occupational exposures(0.19%,0.28%,0)and control group(0.26%,0.58%,0),with a statistically significant difference(P=0.01,P<0.001,P<0.001).In baseline and 3-year follow-up,the H7N9 antibody positive rate in the avian exposed group was basically stable at 0.5%,and the H9N2 antibody positive rate increased from the initial 0.2% to about 2%.H5N1 antibody-positive subjects were found only in poultry exposed workers,and the positive rate(0.20%)was lower at baseline and increased to 3.46% in the third year of follow-up.No H5N6 antibody-positive person was found in any of the survey subjects.A total of 30 subjects in the cohort had H4N9,H9N2,and H5N1 viruses with a 4-fold increase in MN titer.Overall,the seroconversion rate of the H7N9 virus in the poultry occupational exposure group was 0.43%,the swine occupational exposure group was 0%,and control group was 0.1%.A relatively high seroconversion rate(2.78%)of H9N2 virus was observed in the poultry occupational exposure group,compared with 0.58% and 0.39% for swine occupational exposure group and control group;the seroconversion rate of the H5N1 virus in the poultry occupational exposure group was only 1.5%;no H5N6 antibody sero-conversion was observed in all subjects.By multivariate logistic regression analysis,the risk factor for H5N1 antibody sero-conversion was contacting with pigeons during work(OR = 5.13;95% CI,1.14-23.21).Those exposed to pigeons at work(adjusted OR = 3.52;95% CI,1.02-12.14)and those exposed to chronic respiratory disease(adjusted OR = 21.92;95% CI,2.73-176.25)were more likely to be infected with the H9N2 virus.After adjustment for gender and age grouping,there was no significant difference in H7N9 virus infection(corrected IRR = 5.98,95% CI: 1.91-8.65)between the poultry occupational exposure group and the control group,but H9N2 and H5N1(P <0.001)virus infections were more likely to occur.5.ConclusionThe novel reconstituted H7N2 and H5N6 viruses were isolated from the SPF sentinel chickens,demonstrating that the AIVs transmitted from wild birds to poultry under natural conditions and may become a novel influenza virus through genetic reassortment.In Wuxi City located nearby Yangtze River Delta,poultry infection with AIVs is complicated.There are at least 10 different AIV subtypes in poultry and their environment,and the gene reassortment between poultry and wild birds,among different subtypes or different branches of the same subtype is common.There are silent infections of H7N9,H9N2 and H5N1 in occupationally exposed populations,but no H5N6 infection presented.However antibody positive rate and sero-conversion rate are at low levels;contacting with poultry(pigeon)at work and suffering from chronic respiratory diseases are risk factors for H9N2 and H5N1 infections;poultry occupational exposure population is more susceptible to H9N2 and H5N1 viruses than the general population,and is a key population for AIVs prevention and control.6.Significance and innovationThe SPF sentinel chickens infected new flu virus through contact with wild birds,proving that the avian influenza virus can be transmitted from wild birds to poultry under natural conditions.We have found that the silent infections of H7N9,H9N2,and H5N1,but no silent infection of H5N6 may exist in occupationally exposed poultry by means of hemagglutination inhibition test and microneutralization test.Individual silent infections of H7N9 and H9N2 exist in swine occupational exposure population and general population.Poultry exposed populations are more susceptible to H9N2 and H5N1 viruses than the general population.There are at least 10 different subtypes of avian flu virus in local poultry and their environment,and reassortment is very common. |
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