| Avian encephalomyelitis(avian encephalomyelitis, AE), caused by avian encephalomyelitis virus(avian encephalomyelitis virus, AEV), is a contagious infectious disease that is characterized with the neurological signs, such as rapid tremors of head and neck, ataxia in young chicks. Only a transient egg production and hatching rate decline occurred in laying hens. AEV is a positive single-stranded RNA virus, which is a member of the family Picornaviridae within the genus Tremovirus. Since 1932, AE was first reported in America, this contagious disease has now spread worldwide. Large economic losses in the poultry industry were encountered globally because of the prevalence of AE. Lacking of the corresponding therapeutic methods of AE, vaccine administration approach is an efficient measurement to prevent and control the prevalence of AE. Therefore, it is of great importance to study the tissue distribution of AEV in infected chicks for the pathogenicity mechanisms of AEV. In this study, an AEV XianYang strain was isolated and identified from the suspected AE chick brain tissues, then quantitative PCR(qPCR) and immunohistochemistry(IHC) assays were performed for the detection of virus load and viral antigens for the tissue distribution of AEV in infected chicks. The following results were obtained:The clinical samples were collected from the suspected clinical AE chicks in a broiler farm in Xianyang, Shannxi Province, then the pathogen was isolated and identified from the suspected brain tissue. The samples were propagated on 6 day-old specific pathogen-free embryonated eggs for three passages. Subsequently, IHC and animal regression assays were performed to identify the pathogen. At last, the pathogen, named avian encephalomyelitis virus XY13, was isolated and identified. A pair of specific primers based on the highly conserved domain of VP1 gene of AEV 1143 strain published in GenBank were designed, the VP1 fragment was amplified by RT-PCR, cloned into pMD19-T vectors, transformed into E. Coli DH5? competent cells and sequenced, then the genetic phylogenetic analysis between XY13 and reference strains was performed. The results indicated that the target fragment of VP1 gene encoding 96 amino acid residues is 288 bp in length; the nucleotides and deducted amino acids similarity between XY13 and reference strains based on the sequence analysis is 74.6% ~ 99.7%, 76.0% ~ 97.9%, respectively. The results of genetic phylogenetic analysis showed that AEV XY13 strain placed on the same branch with SX strain was mostly related to SX strain, meanwhile placed on a bigger branch with 1143, L2 Z, SX, and YL strain; whereas distantly related to NH-937, Van Roekel and 204 C strains. In addition, according to the evolutionary distances between AEV XY13 and reference strains, the evolutionary distances between XY13 and 1143, L2 Z, SX, YL was less than 0.011, whereas, the evolutionary distances between XY13 and NH-937, Van Roekel, 204 C was more than 0.060.The virus load in different tissues were detected by qPCR assay. The brain materials in broth was diluted in a 10-fold series ranging from 10-1 to 10-7, the diluent was inoculated into yolk sac of 6 day-old SPF embryonated eggs, then the embryo lesions were observed and recorded daily until 12 days post inoculation(dpi). The 50% embryo infectious dose(EID50) was calculated according to the algorithm of Reed-Muench method. Fifty-five one day-old White Leghorn SPF chicks were randomly separated into two groups. Group I was the experimental group with 33 chicks, and group II was the control group with 22 chicks. Each chick in group I was orally inoculated with a dose of 105EID50 /0.2 ml of XY13 and each chick in group II was orally administered a dose of 0.2 ml PBS. After the treatments, three chicks selected from group I and two chicks selected from group II were euthanized by cervical dislocation at 2, 4, 6, 7, 8, 10, 12, 15, 18, 22, and 24 dpi for samples collection. The brain, liver, intestine, proventriculi, bursa of Fabricius, spleen, and kidney tissues were aseptically collected at each interval for virus load detection by qPCR. At last, the clinical symptoms of the chicks in experimental group were observed at 6 dpi, five chicks were depressed and paralysis. One chick with paralysis was died at 7 dpi. The symptoms gradually disappeared after 10 dpi. The results of virus load detection by qPCR showed that the viral RNAs were detectable as early as 2 dpi in the examined samples including brain, liver, intestine, proventriculi, bursa of Fabricius, spleen and kidney, and the virus load in the brain was lower than other tissues. The virus load in the brain increased continuously and peaked at 6 dpi when the typical AE neurological symptoms were most obvious in the experimental group. The virus load in the digestive system including intestine and proventriculi maintained a constant level until the end of the experiment. Whereas, the virus load in the liver gradually decreased in the early infection, then gradually increased during the later infection stage, which indicated that AEV has stronger tissue tropism in the digestive system such as liver, intestine, and proventriculi. Meanwhile, the virus load in the bursa of Fabricius and spleen fluctuated during the whole inoculation period. A gradually continuous decline in the kidney followed from 2 dpi to 24 dpi.IHC assay was performed to detect AEV antigens in different tissues including the brain, liver, intestine, proventriculi, bursa of Fabricius, spleen, and kidney at 2 dpi, 7 dpi, and 12 dpi. Relative expression of viral antigens was estimated, and the correlation analysis between virus load and viral antigens in different tissues at 2 dpi, 7 dpi, and 12 dpi was performed. The results showed that positive brown staining in different tissues were observed at 2 dpi, 7 dpi, and 12 dpi. A correlation between the virus load and virus antigens was analysed, the results showed that viral antigens distribution in different tissues tested by IHC presented a significantly positive correlation with virus load in different tissues tested by qPCR at 2 dpi, 7 dpi, and 12 dpi, respectively.Above all, AEV XY13 strain was closely related to SX strain, whereas, distinctly related to NH-937, Van Roekel and 204 C strains, which indicated that the isolate XY13 had a certain genetic evolutionary diversity with the known reference strains. Tissues distribution of AEV based on qPCR indicated that AEV is characterized with stronger tissue tropism in the digestive system such as liver, intestine and proventriculi, whereas, with weak tropism in the nervous, immune, and urinary systems. A good correlation between viral antigens and virus load was observed, this correlation could exactly reflect the tissue distribution of AEV in chick, which could service on the pathogenicity mechanism of AEV in the future. |
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