Biological Characteristics Of The Isolates Of Avian Leukosis Virus Subgroup A And K Associated With Myeloid Avian Leukosis

Avian leukosis viruses（ALVs）,a group of important tumorigenic retroviruses,can induce chicken various tumors and seriously threaten the poultry industry development.ALVs from chicken are divided into seven different subgroups,A^E,J and K.Viruses of different ALV subgroups have different pathogenicity and tumorigenic spectrum,for example,subgroup A and B primarily induce chicken lymphoblastoma,while viruses of subgroup J usually induce chicken myelocytoma,though they can induce other typical tumors sometimes.In the past two decades,myelocytoma and hemangioma induced by ALV-J have been widely reported and studied in China.However,the related reports about the other ALV subgroups are relatively rare.Especially,there have no report about the neoplastic lesions induced by ALV-K and the myelocytoma induced by ALV-A in natural case.In this study,pathological observation,isolation and identification of pathogen and analysis of viral genomic characteristics were carried out on two different neoplastic cases from a layer chicken flock and an indigenous chicken flock in Hubei province.Furthermore,two recombinant viruses were rescued by reverse genetics and were inoculated into different SPF chickens to study the viral pathogenicity and tumorigenic characteristics,respectively.Moreover,the transcription profiling of liver tissue from SPF chicken inoculated with the rescued ALV-A to analyze and understand the mechanism of myelocytoma induced by ALV-A.The results are as follows:1.Pathological observation and pathogen detectionIn order to investigate the tumor type and etiology of diseased chickens,pathological observation and pathogen detection were performed.Histological examination showed lymphoblastoma and myelocytoma in the sick chickens.ELISA of cloacal swab showed that all the sick chickens displayed positive using ALV group-specific antigen P27.However,Multi-PCR detection using the specific primer for ALV-A,ALV-B and ALV-J showed that only one fragment corresponding to ALV-A was been amplified from the liver DNA of sick commercial layers but no fragment was amplified from the sick indigenous chicken.PCR detection using common primers for all ALV env genes indicated that a specific DNA fragment was amplified in sick indigenous chicken and sequence comparison showed that the env region of the PCR product shared highly homology with the ALV-K isolate JS11C1.2.Isolation and identification of the virusesThe cancerous livers from two sick chicken flocks were picked out and filtered liver tissue suspensions were made to co-culture with DF-1cells respectively.The bright green fluorescence was detected in the DF-1 cells inoculated with the filtered liver tissue suspension of sick layers by IFA using the monoclonal antibody AF3.Similarly,the bright green fluorescence was also detected in the DF-1 cells inoculated with the filtered liver tissue suspension of sick indigenous chicken by IFA using the anti-ALV-K SU mono-specific antiserum.The results indicate that the isolate from layer belongs to ALV-A while the isolate from indigenous chicken belongs to ALV-K,named them as HB2015012 and HB2015032,respectively.Culture test in vitro showed that HB2015012 and HB2015032 have similar replication capacity with the reference strain ALV-J HB2010001 and HB2015029.The infectivity test showed that HB2015032 is able to infect the JingJiang duck embryonic fibroblast（DEF）.3.Genomic characteristics of HB2015012 and HB2015032Sequencing showed that the full-length provirus genomes of HB2015012 and HB2015032 are 7735bp and 7703bp in length,respectively,both have the typical genetic structure of type C retrovirus.Sequence comparison showed that gag gene and pol gene of HB2015012 and HB2015032 are relatively conserved at the nucleotide level.However,the pol gene of HB2015032 contains a mutation C to T,which generates a stop codon at 5345-5347?nt in genome,leading to production of a truncated Pol protein with 22 amino acid（aa）shorter than the other subgroups.The result suggested that the pol gene of HB2015032may be derived from ALV-J.Both isolates share similar genome structure with ALV-J in the 3′UTR+LTR region,including entire DR1,E element,U3,R and U5.Among them,the U3 of either HB2015012 or HB2015032 contains almost all the regulation elements of ALV-J prototype HPRS-103,including two CAAT,Y,CArG and PRE boxes,one NFAP-1 and TATA box.In particular,E element of HB2015032 has a 1-bp deletion,corresponding to7388 bp in HPRS-103,resulting in the production of a c-Ets-1 binding site associated with the vascular endothelial cell differentiation.This deletion was previously observed in some ALV-J strains associated with hemangioma of China layer chickens,and was believed to be responsible for the new pathogenicity of ALV-J.In conclusion,these results indicate that HB2015012 and HB2015032 are natural recombinant viruses with ALV-J 3’UTR+LTR,belonging to two different subgroups ALV-A and ALV-K.Moreover,HB2015032 is an ALV-K with the backbone of ALV-J,and its E element maybe come from the hemangioma type ALV-J.4.Establishment of reverse genetic system and the rescue of virusGenomic DNA of either HB2015012 or HB2015032 was cloned into pTopo-Blunt simple.The recombinant plasmid was transfected into DF-1 to rescue the viruses.The results showed that the infectious strains of rHB2015012 and rHB2015032 were rescued successfully.Culture tests in vitro showed that the replication capacity of the rescue virus rHB2015012 and rHB2015032 were similar to the maternal viruses.5.Pathogenicity of rHB2015012 and rHB2015032The pathogenicity of the viruses was test by inoculating rHB2015012 or rHB2015032 into 1 day old SPF chicks with 2×10³TCID₅₀.The incidence of ALV-induced tumors in chickens inoculated with rHB2015012 was 65%while the tumor incidence in control chickens inoculated with DMEM was 35%.The incidence of tumors in chickens inoculated with rHB2015032 was 75%while the tumor incidence in chickens inoculated with DMEM was 55%.On the basis microscopic pathology,40%tumors induced by rHB2015012 and 55%of tumors induced by rHB2015032 were characterized as myelocytome,suggesting both rHB2015012 and rHB2015032 has the ability to induce myelocytome,but the horizontal transmission and tumorigenic capacity of rHB2015032 is stronger than rHB2015012.6.Establishment of Multi-PCR systems for ALV-A/J/K and recombinant ALV-A,ALV-K with ALV-J-like UTR+LTRALV-K is a novel ALV subgroup virus isolated from local chicken flocks in China recently.It has been neglected for a long time,because it only causes subclinical symptom or not.So far,there has no report about specificity of PCR for ALV-K.In this study,Multi-PCR systems for ALV-A/J/K and recombinant ALV-A,ALV-K with ALV-J-like UTR+LTR were established respectively.The results showed that the established Multi-PCR systems have higher specificity and sensitivity（up to 10² DNA copies/μL）,suggesting they can be used in the detection of these viruses in clinical samples.7.Liver transcription analysis of the chicken infection with rHB2015012The liver transcription profiling of the chicken infected with rHB2015012 were analyzed through the RNA-Seq high throughput sequencing.As a result,1825differential expression genes（DEG）were screened,including 1288 up-regulation genes and 537 down-regulation genes.Gene Ontology and KEGG pathway enrichment showed that part of the DEGs were associated with many important functions,mainly including cellular process,biological regulation,metabolic process,response to stimulus and immune system process.Further analysis showed those genes were majorly involved in some notable signaling pathways,such as pathways in cancer,osteoclast differentiation pathway,JAK-STAT signaling pathway,PI3K-Akt signaling pathway.In detail,85 DEGs were enriched in pathways in cancer,76 DEGs were enriched in PI3K-Akt signaling pathway,72 DEGs were enriched in osteoclast differentiation pathway,and 30 DEGs were related to hematopoietic cell lineage,36 DEGs were related to cell adhesion molecules.72DEGs enriched in osteoclast differentiation pathway can activate the JAK-STAT pathway associated with myelocytoma generation.In addition,17 DEGs were enriched in acute myeloid leukemia pathway.P53 signaling pathway in MAPK signaling pathway were suppressed and PI3K-Akt signaling pathway were activated,which may promote the proliferation of c-myc.