Molecular Epidemiology Of ALV-J In Pakistan And ALV-K In China

Avian leukemia(AL)is a general name of tumor disease which caused by Avian Leukemia Virus(ALV),mainly including hemangioma leukosis,lymphoid leukosis,and myeloid leukosis.At present,although AL has been effectively controlled in some developed countries(such as the United Kingdom and the United States)after years of continuous purification,AL epidemic situation is still severe in many developing countries.For the latter,it is particularly important to conduct an epidemiological investigation of ALV to assist in formulating reasonable purification and detection measures.In this study,2 strains of subgroup J avian leukemia virus(ALV-J)were isolated from Pakistan for the first time,and 3 strains of subgroup K avian leukemia virus(ALV-K)were isolated from local chickens in China,and the molecular epidemiology and pathogenicity of ALV-J and ALV-K were systematically studied.Although ALV infection was reported in Pakistan in 2001,ALV has never been isolated.In order to master the molecular epidemiological characteristics and possible sources of the ALV epidemiological strain in Pakistan,two ALV-J strains(PK19FA01 and PK19SA01)were isolated and identified from 43 samples of local chicken in Pakistan,and their genome-wide sequences were amplified and analyzed.The results showed that the mutations of the two isolates were mainly concentrated in the gp85,3'UTR and 3'LTR regions.Gp85 gene sequence analysis showed that PK19FA01 had higher homology with the ALV-J prototype strain HPRS-103(UK),while PK19SA01 was more similar to the ALV-J strains in the USA.The sequence analysis results of 3 'UTR and 3' LTR showed that 135 bp was missing in the rTM region of 3 'UTR of the two isolates,and both of them contained an intact E element,and the homology of the E element of the two isolates was 96%.The U3 region,which located in 3'LTR,homology of both was 98.7%.The 3'UTR and 3'LTR sequences of the two isolates were more similar to those of the American ALV-J strains.Therefore,based on the above sequence characteristics,it is speculated that the ALV epidemic strain in Pakistan may originate from the United States or the United Kingdom,but may recombine in the chicken population of Pakistan.Since the first discovery of the ALV-K strain in China in 2012,it has been widely prevalent in China.In order to further understand the epidemiological characteristics and pathogenicity of ALV-K strains in China,three ALV-K strains(JS15SG01,JS14LH01 and JS13LH14)were isolated from the collected clinical samples in this study,and sequence analysis was conducted after amplification and acquisition of the whole genome sequence.The results showed that :(1)gp85 amino acid sequence analysis indicated that JS15SG01 had a high homology with ALV-K strains(JS13LH01,JS13-LH1,etc.)in mainland China and ALV-K strains(km-5844,sp-40,etc.)in Japan.The other two isolates(JS14LH01 and JS13LH14)have higher homology with the first ALV-K strain JS11C1 in China.The homology between the 3'UTR region of the three isolates was as high as 99%,and more than 98% with this of ALVE.These sequence characteristics indicated that the Chinese ALV-K strains had different sources and recombined with ALV-E and ALV-J,simultaneous.As a mixed strain of ALV-K,ALV-E and ALV-J,JS15SG01 has not been studied on its pathogenicity before.Therefore,in this study,JS15SG01 was infected with 1-day-old SPF chickens and studied on its pathogenicity.The results showed that: the virus shedding began in the second week after infection,but the positive rate was not high(<40%)and presented with intermittent virus shedding.The positive rate of viremia reached 100% in the second week after infection and was intermittent in the later period.The specific antibody began to turn positive at 12 weeks,with a positive rate of 38% at 19 weeks.Pathogenicity studies have shown that after JS15SG01 infection,chickens exhibited intermittent viral shedding and viremia and were less likely to produce specific antibodies.During the entire animal experiment period,SPF chickens in the infected group showed no clinical symptoms.Histopathological results showed that macrophages proliferated in the brain and spleen tissue sections of SPF chickens in the infected group.In this study,two ALV-J strains were isolated from Pakistani flocks for the first time,and the whole genome sequence of the first Pakistan ALV-J isolate was obtained.The possible source was speculated.These data enrich the epidemiological data on ALV in Pakistan and help Pakistan to take more effective measures to prevent and control the prevalence of avian leukemia.In addition,three ALV-K strains were isolated.It was first discovered that JS15SG01 is a recombinant strain of ALV-K and ALV-E and ALVJ subgroups,and its pathogenicity was evaluated.These results laid the foundation for further understanding of the genetic evolution and pathogenic characteristics of ALV-K in China.