Genetic Diversity Of Subgroup J Avian Leukosis Virus Under The Selection In Different Tissues,Cells And Drugs

Avian leukosis virus(ALV)can cause benign,malignant and subclinical infections in chickens,resulting in growth retardation,egg drop and immunosuppression.Since AL was discovered,the disease spread around the world quickly,especially in recent years,Avian leukosis virus subgroup J(ALV-J)has been widely spread in Chinese chickens,causing a serious threat to poultry industry in China.The quasispecies is a special form of the virus in the mutation selection balance,which is the important cause for genetic diversity.ALV-J,as a typical retrovirus,also has a quasispecies phenomenon.In this study,conventional sequencing and High-throughput sequencing were carried out on the research of ALV-J in flocks or individual chicken in China;explored the evolution rules of ALV-J in different host;we also established the animal model for exploring the viral variation from vertical transmission of ALV-J;drug selection pressure model from zidovudine was established in vitro,and high-throughput sequencing was used to reveal the mechanism of drug-resistance,through which,we improved the understanding of genetic diversity on ALV-J in different host,tissues and cells.1.Genetic diversity of subgroup J Avian leukosis virus in different individuals of the same chicken flockIn order to assess the epidemic state of ALV in indigenous chickens in China,10 novel strains of ALV-J,named JS16JH01 to JS16JH10,were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors.To further understand their virological characteristics,the proviral genome of ENV-LTR was sequenced and compared with the reference strains.The homology of the gp85 gene between the 10 ALV-J strains and NX0101 was in the range from89.7–94.8% at the nuclear acid level.In addition,their gp85 genes were quite varied,with identities of 92–98% with themselves at the nuclear acid level.There were several mutational sites and deletions in the amino acid sequence of gp85 genes after comparison with other reference strains of ALV.Interestingly,a novel insertion in the gp85 region was found in two strains,JS16JH01 and JS16JH07,compared with NX0101 and HPRS-103.At present,owing to the large-scale purification of ALV in China,laying hens and broiler chickens with ALV infection are rarely detected,but ALVs are still frequently detected in the local chickens,which suggests that more efforts should be applied to the purification of ALV from indigenous chickens.2.Analysis of quasispecies of ALV-J using sanger and highthroughput sequencingALV-J exists as a complex mixture of different,but closely related genomes named quasispecies subjected to continuous change according to the Principles of Darwinian evolution.To verify the applicability of highthroughput sequencing in the study of quasispecies.The present study seeks to compare conventional Sanger sequencing with deep sequencing using MiSeq platform to study quasispecies dynamics of ALV-J.The accuracy and reproducibility of Mi Seq sequencing was determined better than Sanger sequencing by running each experiment in duplicate.According to the mutational rate of single position and the ability to distinguish dominant quasispecies with two sequencing methods,conventional Sanger sequencing technique displayed high randomness due to few sequencing samples,while deep sequencing could reflect the composition of the quasispecies more accurately.In the mean time,the research of quasispecies via Sanger sequencing was simulated and analyzed with the aid of re-sampling strategy with replacement for 1000 times repeat from high-throughput sequencing data,which indicated that the higher antibody titer,the higher sequence entropy,the harder analyzing with the conventional Sanger sequencing,resulted in lower ratios of dominant variants.In sum,deep sequencing is better suited for detecting rare variants comprehensively.The simulation of Sanger sequencing that we propose here will also help to standardize quasispecies researching under different selection pressure based on next-generation sequencing data.3.Genetic Diversity of Avian Leukosis Virus Subgroup J in a SPF chicken using deep sequencingTo study the genomic diversity of ALV-J in different organs from one chicken more comprehensively,viral RNA was sequenced using conventional Sanger sequencing and high-throughput sequencing,respectively.Compared with the primary inoculation virus,the dominant variants in follicle evolved the most than other organs which can distinguish it from other organs at gp85-A,gp85-B and LTR-U3 regions.Combining with conventional sequencing,we proved that it is a co-evolution process in these 3 regions.Dominant variants with LSD insert mutation were only found in gp85-B from liver,spleen and plasma,but there is no similar mutation in other organs.Global selection pressure values showed that the virus appears to be under the greater selective pressure in the ovary and follicle than other organs.The genomic diversity of ALV-J existed not only in the population level,but also in different organs from an ALV-J infected chicken,which indicated that more attention should be givento the purification process of infected chickens.But whether the ovary sample plays an important role for increasing the diversity of the gene in the process of ALV vertical transmission,the vertical transmission model will be developed in the future study.4.The evolution of ALV-J in the process of vertical transmission by HiSeq sequencingPrevious studies found that the ALV-J gp85 gene sequences from different tissues in the same infected chicken were significantly different,and the virus in follicle vary farly with viruses in the other organs,but whether the virus in follicle could be transmitted to offspring by vertical transmission,which organs have the highest homology with the offspring? To solve this problem,the vertical transmission model of ALV-J was constructed,and 3 hens with persistent viremia but negative antibodies and laying were obtained.After collecting eggs and hatching,we got 3 offspringss with positive viremia corresponding to the 3 hens.The virus RNA was extracted directly from the plasma,liver,spleen,kidney,follicle from the hens,and plasma from the corresponding chicks.RT-PCR was applied to the 2 hypervariable regions of gp85 and LTR-U3 regions for high-throughput sequencing using HiSeq2500 platform.The top3 dominant mutant from each organ were obtained and analyed,the results revealed that the gp85-B segment appeared same evolutional trend between chicks and hens.The first dominant mutant formed a new branch between the virus from the plasma in chicks and follicle in hens in the phylogenetic tree.Homology analysis showed that the virus in the plasma of the chick had the highest homology with the virus in the follicle in the hen.The comparison of amino acid sequence also found that the virus in the plasma in the chick had the same amino acid mutations with the virus in the follicle.SNP analysis showed that the virus in the chicks had different mutation sites with virus in the hen.All the above results confirmed that the virus in the plasma of the chicks is more likely to be from the virus in the follicle of the hen.The evolution rule of ALV-J in vertical trasmission has increased its genetic diversity and accelerated the evolution,making ALV-J easier to survive in different host environments.5.A deep sequencing reveals significant diversity among dominant variants and evolutionary dynamics of avian leukosis viruses in two infectious ecosystemsAs a typical retrovirus,the evolution of ALV-J in different infectious ecosystems is not characterized,what we know is there are a cloud of diverse variants,namely quasispecies with considerable genetic diversity.This study is to explore the selection of infectious ecosystems on dominant variants and their evolutionary dynamics of ALV-J between DF-1cells and specific-pathogen-free(SPF)chickens.High-throughput sequencing platforms provide an approach for detecting quasispecies diversity more fully.An average of about20,000 valid reads were obtained from two variable regions of gp85 gene and LTR-U3 region from each sample in different infectious ecosystems.The top 10 dominant variants among ALV-J from chicken plasmas,DF-1 cells and liver tumor were completely different from each other.Also there was a difference of Shannon entropy and global selection pressure values(ω)in different infectious ecosystems.In the plasmas of two chickens,a large portion of quasispecies contained a 3-peptides “LSD” repeat insertion that was only less than0.01% in DF-1 cell culture supernatants.In parallel studies,the LTR-U3 region of ALV-J from the chicken plasmas demonstrated more variants with mutations in their transcription regulatory elements than those from DF-1 cells.Our data taken together suggest that the molecular epidemiology based on isolated ALV-J in cell culture may not represent the true evolution of virus in chicken flocks in the field.6.The variation of ALV-J under drug selection pressurre and the mechanism of drug resistance from Zidovudine(AZT)AZT is an anti HIV drug with anti-retrotranscriptase activity,and retrovirus ALV also has a reverse transcriptase gene.Previous studies have found that ALV-J produced resistance under the AZT selection,in order to study the mutation of ALV-J under drug selection and the mechanism of generation of drug resistance.An ALV-J infectious clone rSD1005 had developed drug resistance to azidothymidine(AZT)after fifty generations in DF-1 cells.High throughput sequencing of two hypervariable regions was carried out to observe the difference of evolution.The analysis of boxplots demonstrated that residue change patterns in the pol-A segment differed significantly(p < 0.05)in the group treated with AZT.Additionally,the point mutation T196 A that may be related to drug resistance was determined.rSD1005–T196A was constructed based on rSD1005 by point mutation,and rSD1005–T196A showed high drug-resistance to AZT compared to rSD1005 by verification,which suggested the mutation T196 A is closely related to the drug-resistance.In this study,we found the drug resistance of ALV-J under drug selection pressure and identified the key nucleic acid sites that determined the drug resistance.It established a reliable model for studying the molecular mechanism of retroviral drug-resistance in vivo.