Identification Of Co-infection Of Marek’s Disease Virus And Avian Hepatitis E Virus And Pathogenic Investigation Of Hepatitis E Virus

Hepatitis E virus(HEV)is the main pathogen of Big liver and spleen disease(BLS)or Hepatitis-Splenomegaly(HS).Decreased laying rate and increased mortality rate in layers and broilers breeders were usually obsereved during HEV infection at 30 to 72 weeks old.Marek’s disease virus(MDV)is one of the most common tumor virus in poultry,which induces Marek’s Disease(MD)along with highly contagious malignant lymphoma to cause immunosuppression,and finally resultes in significant economic losses to poultry industry.At present,sdudies related to avian HEV infection in poultry are rarely reported in China.The multiple mixed infection of MDV,REV and ALV revealed that the co-infection of MDV with other viruses is more harmful than the MDV infection individually.Up to now,there are no reports of avian HEV and MDV co-infection.In the present study,we collected 25 sick chickens and 5 apparently healthy chickens from egg-type bredders.The results showed hepatosplenomegaly with tumors.Histopathological observation showed that hepatocellular vesicular degeneration,and lymphocyte infiltration in some small blood vessels and liver sinus.Anticoagulants from 30 chickens were collected to inoculate with CEF for 7 to 9 days.The cell supernatant was used to detect ALV-P27 antigen by ELISA,and the cells were testd for H19 by indirect immunofluorescence(IFA).ELISA results showed that the samples were all negative for ALV-P27 antigen.IFA results showed that the cells were positive for MDV.There were not positive cells were detected with ALV and REV monoclonal antibody JE9 and 11B118.DNA samples were isolated from CEF and the liver samples of 30 chickens mentioned above to detect MDV,ALV,REV.The results showed that the positive rate of MDV was46.7%(14/30),while ALV and REV were all detected negative.MDV positive samples were cloned and sequenced.Meq gene sequencing showed that the homology of the isolated MDVstrain was 98.7% ~ 100% compared with the classical MDV reference strain.Analysis of the base sequence showed that the isolated MDV was a virulent strain,and the base located at575-577 was CAC.There is no deletion of proline(P)in the EELCAQLCSTPPPPI repeats of the multi-proline functional region of the meq gene.We conjected that the isolated strain was derived from wild virus according to the comprehensive analysis.RNA were extracted from liver samples,then,reverse transcription for cDNA.The chicken HEV ORF2 gene was amplified by nested PCR,and the positive samples were cloned and sequenced.According to the avian HEV ORF2 partial gene sequence,we compared the isolated samples with reference strain to identify the homology.RT-PCR results showed that the detection rate of avian HEV was 56%(14/25)in the liver samples of sick chickens,and the detection rate of avian HEV was 80%(4/5)in the apparently healthy chickens.The sequencing results showed that the homology of detected HEV ORF2 was77.3% ~ 98.3% with the classical reference strain,which belonged to avian HEV gene type 3.The liver samples of the HEV-positive chickens was grind with PBS buffer and filtered.We injected the virus intravenously into four two-week-old SPF chickens by intravenous injection.The control chickens were injected with normal saline.We collected livers and extracted RNA 14 days post injection.HEV RNA was identified and the positive results were cloned and sequenced to determine the source of the virus.The liver samples were collected and the target bands with 250 bp size were detected in the poisoning group by nested PCR.No avian HEV RNA was detected in the control group.The test results showed that the avian HEV used in the toxic suspension can be retransmitted to the chicken.In order to understand the infection rate of avian HEV in the breeding grounds with tumor disease,we investigated seven poultry farms with tumor disease for avian HEV.40 feces and 20 anticoagulant were collected from the HY-LINE variety brown which was suspected to be suffering from large hepatopathic spleen.In addition,a total of 165 samples were collected from six other breeding grounds with tumor disease.RNA was extracted and reverse transcription was performed for nested PCR detection of avian HEV.The results showed that the detection rate of avian HEV was 67.5%(27/40)in the fecal samples from the HY-LINE variety brown,and the detection rate of avian HEV in plasma samples was 20%(4/20).However,no avian HEV was detected in the samples collected from the other 6breeder farms.We selected two samples randomly for cloning sequencing.The results of sequence alignment showed that the homology of HEV sequences was 98.3% ~ 100% with the three birds isolated from the HY-LINE variety brown,and the phylogenetic tree analysis showed that they belonged to genotype 3 and were in the same branch.To investigate the status of avian HEV infection of live avian and wild bird in the region,and provide data for the prevention and control of avian HEV and wild bird disease surveillance.East Asia-Australia migration route goes through the north and south of China:the Yellow River Delta Nature Reserve and the Jiangsu Yancheng Wetland Rare Bird Nature Reserve.From 2014 to 2016,2691 samples of wild birds and live poultry were collected.RNA was extracted and quantitified by qPCR.The results showed that no positive samples were detected and 7 samples were suspected suffered.The suspicious samples were verified by nested PCR and we found that all of the target bands were not amplified.In this survey,no avian HEV was found in wild birds and marketed poultry samples.