| Avian leukosis(AL)is a variety of neoplastic infectious diseases caused by avian leukosis virus(ALV).There are currently 7 subgroups of ALV that can naturally infect chickens,namely A,B,C,D,E(endogenous),J and K,of which the most common subgroups in the clinic are the A,B and J.Subgroups A and B mainly cause lymphocytic leukosis,while subgroup J mainly induces myeloid leukosis.ALV infection can lead to the incidence of various clinical tumors and subclinical infections.It is one of the most harmful/threat diseases of the qualitychickens at present.Quality-chickens are the birds of local excellent breeds in China that are independently bred and produced by many breeding companies of all kinds of scale.The annual production is about 5 billion birds,accounting for about half of the broilers produced in China,of which Guangxi and Guangdong accounted for about 40%.In order to track the genetic diversity and the latest changes of ALV epidemic strains in the quality-chickens,clinical samples of the suspected ALV infected quality-chickens in Guangxi and Guangdong were collected in the study during 2017-2020 and used for the isolation and identification of ALV by the DF-1 cell culture.A total of 43 ALV strains were isolated;The env gene sequences of the isolates were identified and all belonged to the J subgroup(ALV-J),of which19 strains belonged to Clade 1.1,1 strain belonged to Clade 1.2,and 23 strains belonged to Clade 1.3;Through the analysis of the selection pressure and amino acid(aa)entropy value of the env gene,it was found that the Clade 1.3 isolates had the highest genetic diversity;According to the sequence alignment analysis of the viral envelope protein ENV,it was found that 10 isolates had obvious aamutation sites and they all belonged to the Clade 1.3,with 2 isolates containing a potential N-glycosylation site(NLS)at the aa residues 192-194.And the site is located in the functional domain of an α-helix(190-200 aa residues)in the secondary structure of the protein,which was found by homology modeling with Alpha Flod2.The co-infection cases of different ALV subgroups were found recently.In order to explore the influence between different subgroups,isolate GX20YL16 J of the predominating Clade 1.3 and the isolate GX18PX02 of ALV-B were used to infect and co-infect the chicken embryo fibroblast(CEF)cells.Then,the cell cultures were collected respectively at 1,3,5,7 and 9 days post-infection(dpi)for the real-time quantitative PCR(q PCR)detection,and the growth curves of viruses were established.The results showed that during 1-3 dpi,the ALV-J viral copy numbers in the co-infection were significantly higher than that in the ALV-J mono-infection(P<0.001),while those of ALV-B in the co-infection were not significantly different from those in the ALV-B mono-infection(P>0.05).It indicates that ALV-B can increase the growth of ALV-J during 1-3 dpi in the coinfection.In order to further analyze the differential expression of host genes in the process of the ALV-B’s enhancive effect on the growth of ALV-J in the coinfection,the CEF models of ALV-B and ALV-J infection were set up in the study.The samples of the infected cell cultures were collected from the groups of the mono-infection and co-infection of ALV-B and/or ALV-J for the transcriptome analysis at 1 dpi and 3 dpi,respectively.According to the sequencing results,it was found that the differential genes enriched at 3 dpi in each infection group were more than those at 1 dpi;Differential enrichment analysis of the differential express genes shared in the mono-infection and the co-infection groups,and the GO analysis found that the differential express genes were mainly enriched in the activating chemokines,cytokines and the chemokine-mediated signaling pathways,producing inflammatory responses,and participating in the positive regulation of immune system processes,etc.;The KEGG pathway is mainly enriched in cellular processes,metabolism,signal transduction,and immune response pathways,etc.The study found that Toll-like receptors(TLRs)signaling pathway was significantly enriched in each infection group on 1 and 3 dpi.Through differential enrichment analysis,the significantly different cytokines were found co-enriched at 1 dpi and 3 dpi between the co-infection and ALV-B mono-infection groups,and the relevant key factors,including TLR3,TLR7,IRF7,IL-6,IκBα,IL-12β,IL-1β,CASP8,IKK? and IL-8-L2,were screened out that activated the TLRs signaling pathway.And the results were verified by the RT-q PCR detection of these 10 cytokines.The results showed that the expression levels of these 10 cytokines were up-regulated in each infection group and assured the transcriptome sequencing results.The results of the study demonstrated that the 43 ALV isolates obtained from the quality-chickens in Guangxi and Guangdong during 2017-2020 all belonged to the J subgroup,mainly Clade 1.3 and 1.1.The Clade 1.3 isolate has the highest genetic diversity,and some of the changes were found in the functional domain structure of gp85 of the ENV protein;Based on the phenomenon that ALV-B could increase the growth of ALV-J in the co-infection,the transcriptome data and the verification results of the mono-infection and co-infection of ALV-B and/or ALVJ in CEF at 1 dpi and 3 dpi were analyzed.It was found that ALV-B enhanced the growth of ALV-J in the co-infection mainly through the joint action of the upregulation of multiple cytokines in the TLRs signaling pathway,revealing that the TLRs signaling pathway may play a key role in the process.And it will provide useful data for the subsequent studies on the pathogenic and tumorigenic mechanisms of the co-infection of different subgroups in chickens. |
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