Isolation,Identification And Pathogenicity Of Muscovy Duck Reovirus From Goose Origin

Muscovy duck reovirus disease is a viral infectious disease caused by Muscovy duck reovirus(MDRV).The clinical signs of the disease are primarily paralysis,and the typical lesion on autopsy is a grayish-white spot on the surface of the liver.Since 2021,there has been an outbreak of infectious disease with paralysis and a large number of white necrotic spots on all tissues and organs of the body as the main characteristics in major goose breeding areas in Shandong,Henan and Jiangsu in China.The disease has severely hindered the healthy and rapid development of China's goose industry.In this study,12 strains of goose of MDRV were isolated from spleen tissue samples of naturally infected geese,and strain HN21was selected from the isolates for pathogenicity studies.A Taq Man probe RT-q PCR detection method was established according to the pathogenesis of the disease,and the method was used to detect the distribution of the virus in different tissues and organs after infection with the virus in goslings of different ages,the rule of detoxification and the situation of viremia,in order to prevent and control the disease.In this study,12 strains of the virus were isolated from samples collected from geese suspected to be infected with Muscovy duck virus in Henan,Jiangsu and Liaoning.The?C gene of the isolates was determined and 12 isolates were identified as goose-origin Muscovy duck reovirus.Genetic evolutionary analysis with the avian reovirus?C gene from NCBI showed high homology among the 12 isolates,with nucleotide homology ranging from 97.9%to 100%and deduced amino acid homology ranging from 98.1%to 100%.There were 16bases difference between the 12 strains,with large variations in bases 337-340 and 791-798.There were 8 differences in amino acid sequences between the 12 strains.The 12 isolates had the highest homology with the classic strain ZJ2000M,with nucleotide homology ranging from 95.4%to 96.7%and amino acid homology ranging from 95.2%to 96.7%.Compared with the nucleotide sequence of the classical strain ZJ2000M,there were 45 differences.12strains had 18 amino acid changes compared to the classical strain ZJ2000M sequence.The genetic evolutionary tree showed that the 12 isolates remained in the same evolutionary lineage as the waterfowl-derived ARV and were most closely related to the previously reported ZJ2000M strain.Specific primers and probes with an amplification length of 154 bp were designed based on the goose-origin MDRV S4 gene sequence.The recombinant plasmid containing the target fragment was constructed using the p MD18-T vector,and after successful identification,the standard quality plasmid was prepared by 10-fold dilution.The standard plasmid was used as a template to optimize the reaction conditions and construct a standard curve to establish a Taq Man probe for the detection of goose-derived MDRV.Fluorescence quantitative RT-PCR method.The method has high sensitivity,strong specificity and good repeatability,and can be used for the early qualitative and quantitative diagnosis of goose-derived MDRV infectionIn this study,viruses of HN21 strain were selected from the isolated strains to study the pathogenicity of goslings at different days of age.and the goslings in the experimental group were infected by oral and intramuscular injection routes,each inoculated with 0.5 mL(TCID₅₀of 10^-6.51/0.1mL)of virus solution each,and the goslings in the control group were inoculated with the same dose of saline.Clinical signs of infected goslings were observed and recorded.At 1 d,2 d,4 d,7 d,11 d,16 d and 22 d after artificially infecting the goslings,three goslings from each group were randomly selected to be weighed and their weight recorded.Three samples of cloacal swabs and blood were collected randomly from each group to detect the detoxification in the cloaca and blood of the goslings.The lesion tissues were collected and fixed for pathological histological study,while each tissue organ was collected for viral load determination.The results of the study showed that the clinical symptoms were similar in goslings of different infection days.The necropsy changes showed that white necrotic spots appeared in various tissues and organs of the 10-day-old attack group goslings,mainly including liver,spleen,lung,kidney,pancreas and intestine.In addition,the affected goslings also showed necropsy changes such as joint swelling and intra-articular hemorrhage.The necropsy changes of goslings in the 20-day-old attack group were generally the same as those in the10-day-old attack group,but to a lesser extent.The 30-day-old attack group only showed white necrotic spots in the liver and spleen,but not in other tissues and organs.The above results show that the infectivity of the virus is closely related to the age of infection,and the younger age of the infected goslings,the more serious the degree of disease.The histopathological results showed that the goslings in each challenge group showed severe damage to the whole body tissues and organs,among which the liver,spleen and kidney were the most severely necrotic,accompanied by inflammatory cell infiltration.Body weight gain was influenced in healthy goslings infected with goose-origin MDRV at different days of age.The results of viral load in blood and cotton swab detoxification pattern showed that viraemia and cloacal detoxification were observed in geese of different days of age in the early stage of infection.The viral load of different tissues showed that goose-origin MDRV could be detected in all tissues and organs of goslings in different day-old experimental groups 1 d after the attack,among which the viral content was higher in spleen and liver,and the viral load of geese in the small day-old attack group was higher than that in the large day-old attack group.The above results indicate that there is a difference in the infectivity of MDRV for different day-old goslings,and the younger the age of infection,the more pathogenic it is for geese.The dynamic distribution of MDRV in gosling can provide a reliable theoretical basis for the prevention and control of goose-origin Muscovy duck reovirus disease.