| Avian leukosis virus(ALV)belongs to the Alpharetrovirus genus of the Retrovirus family.According to the antigenicity of viral envelope glycoprotein,viral interference test,host range and genomic molecular biology characteristics,ALVs are divided into 10 subgroups,named as ALV-A,B,C,D,E-I and J,respectively.Among which ALV-A,-B,-C,-D and ALV-J are exogenous ALVs,and ALV E-I are endogenous ALVs.ALUs can lead to decreased performance of chickens,immunosuppression and a variety of tumor performances,thus seriously influencing the healthy development of poultry industry.So ALV is one of the important pathogens attracting global attentions to control it.In recent years,the situations of prevention and control for ALVs in China is very severe.Much more reports have showed that ALV-J is the main epidemic strain in broiler,laying hens and local flocks,while studies on ALV-A/B subgroup relatively remain few,simultaneously evidences reveal that the infection rate and viral variability of ALV-1/B is increasing in flocks.In order to study the infection status and pathogenicity of exogenous ALV in Hetian chicken breeds of Fujian Province,in this study,clinical samples from suspected cases of ALV infection were collected for ALV isolation with cell culture.The isolates were first identified by indirect immunofluorescence assay and PCR amplification,and then subjected to complete genomic sequencing and analysis through segmented PCR to determine the classification status of this virus.Next,6-day-old white Leghorn chick embryos were inoculated with this isolate to simulate vertical transmission,in addition,1-day-old and 42-day-old SPF white Leghorn chicks were also infected by cohabitation infection to simulate horizontal transmission,all above groups were set to detect the effects of different transmission patterns of this virus on the growth performance,immune performance,infection status and tumor phenotype in vivo.The main contents and results are as follows:1 Isolation and identification of ALV-BThe anilomian swabs and venous blood were collected from suspected cases of ALV infection,and the ALV group specific antigen p27 in the anorectal cotton swab samples was detected by ELISA.Then the positive swabs-corresponding venous blood samples were pretreated and inoculated into the single-layer DF-1 cells to isolate the virus,simultaneously,the blank control group was set up.After 3 passages in cultured DF-1 cells,the p27-positive supernatant was used for immunofluorescence assay(IFA)unsing p27-specific monoclonal antibody(McAb)and ALV-J-specific McAb as primary antibody.The results showed that the strain expressed p27 antigen but had no ALV-J-specific gp85 protein,suggesting that it was a non-J subgroup of exogenous ALV.gp85-specific PCR and further sequencing and analysis showed that the ALV-B-specific gp85 gene was identified,and the strain was named as FJ15HT0.2 Complete genomic sequencing and analysis of FJ15HT0In order to further understand the genomic characteristics of the FJ15HT0 strain,according to gene sequence of ALV reference strain,eight pairs of overlapping primers were designed to cover the complete genomic sequence of the ALV-type provirus.The target bands were amplified by segmented PCR,cloned and identified,and then three positive clones were selected for sequencing.The complete genomic sequence of the ALV-type provirus was obtained by software splicing.The full length of proviral genome was 7785 bp,and the genomic structure was 5'-LTR-gag-pol-env-LTR-3' with no proto-oncogenes,which was consistent with the typical genomic structure of complete-copy retroviruses.The FJ15HT0 strain was found to be a recombinant strain through homology comparison with the sequence of classical reference ALV strains.The main results were as follows:(1)The analysis of gag and pol gene sequenceSequence analysis showed that gag and pol gene of FJ15HT0 strain were conserved.Compared with other ALV reference strains,the homology of gag-and pol-deduced amino acids was more than 96%and 97%,respectively.(2)The analysis of env gene sequenceThe env gene encodes the gp85 and gp37 proteins,in which the deduced amino acid sequence of gp85 was more than 91%homologous to the ALV-B reference strains,while less than 90%homologous to the other subgroups.The deduced amino acid sequence of gp37 had 90.6%homology with that of ALV-E reference strain,and only 55.6%homology with that of J subgroup reference strain HPRS103,and average homology of 88.7%with ALV-B,ALV-C and ALV-D.(3)The analyses of LTR sequence and regulatory elementsCompared with other reference strains,the nucleotide homology of FJ15HT0 strain was 71-90.5%.The highest homology with the J subgroup reference strain HPRS103 was 90.5%.The U3 region of 3'-terminal contained the same Cis-regulatory elements with the ALV-J HPRS103 strain,such as two Y Boxes and two CArGs sequences,which meet the characteristics of exogenous ALV with enhanced replication capacity.(4)UTR sequence analysisCompared with other subgroups strains,the nucleotide homology of 5'UTR of FJ15HT0 strain ranged from 83%to 94.5%,with 94.5%homology with ALV-C Prague C strain.The nucleotide homology of the 3'UTR sequence of the FJ15HT0 strain with ALV reference strains was 60.3-82.4%,with the highest homology(82.4%)with the ALV-A reference.Based on the above results of sequence analysis,it was found that the FJ15HT0 strain was a recombinant ALV strain with ALV-B-specific gp85 gene sequence,ALV-E-specific gp37 sequence and ALV-J-specific LTR sequence.3 The characteristics of the FJ15HT0 strain48 SPF chick embryos were randomly divided into 4 groups,A,B,C,and D group,each consisting of 12 embryos.As for group A,6-day-old embryos were inoculated with DMEM as blank control;as for group B,6-day-old embryos were infected with FJ15HT0 strain through yolk sac inoculation;as for group C and D,6-day-old embryos were inoculated with DMEM,and then cohabited with group B on 1-day age and 42-day age,respectively;The group A and other groups were separately reared in different cages till to 11-week(w)age.Each quarantine until 11 weeks old.(1)The effect of different infection models on the viral sheddingMeconium or cloaca cotton swab were collected from each group at 1-day,1-week,2-week,3-week,4-week,5-week,6-week,7-week,8-week,9-week,10-week and 11-week age,respectively,for determination of p27 antigen with the ALV antigen detection kit.The p27 antigen was positive in the inoculation group from 1-day to 11-week age.The p27 antigen was negative in both 1-day-old and 42-day-old cohabitation infection group,which indicated that FJ15HT0 strain could vertically infected chicks with continuous viral shedding,while chicks infected with FJ15HT0 strain through early-and late-stage horizontal transmission had no shedding of detectable viruses.(2)The effect of different infection models on the production of virus-specific antibodiesThe blood samples were respectively collected from chicks in each group at 2-week age,3-week,5-week,7-week,9-week and 11-week age for detection of anti-ALV-A/B antibodies.The results showed that the anti-ALV-A/B antibodies in beth blank control group and inoculation group were absent from 2-week to 11-week age.One case of ALV-A/B antibodies-positive chick appeared at 3-week,5-week,11-week age in the 1-day-old cohabitation infection group,with positive rate of 9.1%,11.1%and 14.3%,respectively,and two positive cases appeared at 7-week age,with positive rate of 25%.One case of ALV-A/B antibodies-positive chick appeared at 9-week age in the 42-day-old cohabitation infection group,with positive rate of 28.6%,and three positive cases appeared at 11-week age,with positive rate of 42.9%It was suggested that the absence of anti-ALV-A/B antibodies met the characteristics of immune tolerance induced by embryo infection,and positive antibodies were detectable in the co-infected chickens at 3-week-age.(3)The effect of different infection models on the viremiaThe chickens were sacrificed at 2-week,3-week,5-week,7-week,9-week,11-week,and the plasma was isolated and inoculated into the DF-1 cells,after 7 days,the p27 antigen was detected by ELISA.The viremia was detectable in the inoculation group from 1-day to 11-week.Partial positive cases were found in the 1-day-old cohabitation infection group at 2-week,3-week,and 5-week age,respectively,while all samples were negative in 42-day-old cohabitation infection group.It was suggested that chicken embryo vaccination and early cohabitation infection could cause viremia in chickens,but late cohabitation infection failed to induce viremia.(4)The effect of different infection models on the growth performanceChickens in each group were weighed from 1-week to 11-week age and the data were analyzed statistically.The results showed that the infection of FJ15HT0 strain through the embryo inoculation had obviously inhibitory effect on chickens growth.The average weights of inoculation group during 2-11th week were significantly decreased(P<0.05)in comparison with those in blank control group,with only 61.8%of the weight in the blank group at 11-week age.The FJ15HT0 strain also had an inhibitory effect on the weights of chicks in 1-day-old cohabitation infection group,the average weights of this group during 3-11th week were significantly reduced(P<0.05)in comparison with those in blank control,with only 67.8%of the weight in the blank group at 11-week age.The impact of FJ15HT0 strain on the chicken growth performance in the 42-day-old cohabitation infection group was not obvious when compared with that in the blank group.There was no significant differences in the average weights during 1-11th week between inoculation group and 1-day-old cohabitation infection group(P>0.05).The average weights of inoculation group during 6-11th week were significantly decreased(P<0.05)in comparison with those in 42-day-old cohabitation infection group.Additionally,the average weights of the 1-day-old cohabitation infection group was significantly lower(P<0.05)than those of the 42-day-old cohabitation infection group during 2-4th week and 6-11 week.(5)The effect of different infection models on the immune organ indexesThe chickens were sacrificed at 11-week age,and the thymus,spleen and bursa of Fabricius were weighed for calculation of the immune organ indexes.The results showed that the immune organ indexes of spleen,thymus and bursa of the inoculation group was not significantly different from those of the blank group(P>0.05).The spleen immune organ index of the 1-day-old cohabitation infection group was significantly lower than that of the blank group(P<0.05),while no significant differences was found in thymus index as well as bursa index.In addition,there was no significant difference in the three immune organ indexes between the 42-day-old cohabitation infection group and the blank group(P>0.05).(6)The effect of different infection models on the NDV,AIV-H5 antibody levelsAt 7-day age,all chicks were vaccinated with chicken Newcastle disease LaSota strain through eye-and nose-dropping ways,and subcutaneously immunized with recombinant avian influenza virus H5 subtype divalent inactivated vaccine.Secondary immunization was carried out in the same way at 21-day age.The blood plasma samples were collected from each group at 2-week,3-week,5-week,7-week,9-week and 11-week age for determination of antibody titers of Newcastle disease virus(NDV)using hemagglutination inhibition(HI)test.The results showed that embryo inoculation of FJ15HT0 strain surpressed the immune responses derived from NDV live vaccine in the infected chickens.The level of anti-NDV antibody in inoculation group at 3-week,9-week and 11-week age was significantly lower than that in blank group(P<0.05).Additionally,the levels of anti-NDV antibody in 1-day-old and 42-day-old cohabitation infection group at 9-week and 11-week age significantly decreased(P<0.05)in comparison with that in blank group.The above results suggested that embryo inoculation of FJ15HT0 strain and cohabitation infection had a certain inhibitory effect on the production of anti-NDV antibody in the infected chickens.In addition,the blood plasma samples were collected from each group at indicated time points and then subjected to detection of the anti-AIV-H5 antibody titer with HI test.Results showed that there was no significant difference(P>0.05)in the antibody titers among the inoculation group,1-day-old cohabitation infection group,as well as 42-day-old cohabitation infection group.(7)The effect of different infection models on the induced tumor phenotypeThe heart,liver,spleen,kidney,bursa of fabricius,thymus tissue were collected from each group at 11-week age for preparation of paraffin sections,followed by HE staining and observation of the pathological changes with optical microscope.The results showed that embryo inoculation of FJ15HT0 strain resulted in the presence of tumor lesions in the spleen and liver of the infected chickens.The lesions were solid tumors with clear boundaries and different sizes,in which the deeply stained immature lymphocytes were aggregated.It was indicated that lymphoma occurred in infected chickens.No tumors was found in the blank control group and the cohabitation infection groups.Therefore,it was confirmed that embryo inoculation of FJ15HT0 strain had strong tumorigenic ability to SPF chickens.In conclusion,we isolated an exogenous ALV FJ15HT0 strain,analyzed its complete genome and finally confirmed that FJ15HT0 strain was a naturally recombinant exogenous ALV.For further study,the effects of different infection models on the infection status,growth performance,immune performance and tumor phenotype of the flocks were investigated through animal experiments.Our results systematically reveal the molecular characteristics and pathogenic characteristics of FJ15HT0 strain,which will provide a theoretical basis for understanding the prevalence and genetic evolution of ALV in Hetian chicken breeds,and provide important references for the effective control of avian leukemia in local flocks. |
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