Ecological Investigation, Genetic Evolution For Parts Of Enterovirus And Isolation, Identification Of CPV For Giant Panda

Intestinal infectious diseases, as one of the important diseases of giant panda (Ailuropoda melanoleuca), is more harmful than virus infectious diseases in the giant panda population. Due to the species limits, intestinal susceptible to viral disease of systemic research for the giant panda is still blank, This study selected a greater threat on the five kinds of pandas susceptible to enterovirus that canine distemper, canine parvovirus, canine coronavirus, rotavirus and canine adenovirus as the research object, the establishment of the molecular epidemiology detection method, and to investigate the methods used in different regions, different feeding methods and different years pandas carry enteroviruses; A positive part of survey were cloned and sequenced as the virus mutated gene, using bioinformatics software for analysis, study their genetic variation trends; some of the positive samples for virus isolation using cell culture, get pandas source virus strains. This study includes the following aspects:1. Establish the molecular detection method of giant panda enterovirusBased on the published sequence of the nucleocapsid protein gene of enterovirus in GenBank,4 pairs of specific primers was designed. As a template with the standard vaccine strain, and after optimization of the reaction system, a rapid PCR method for detection of enterovirus was established. Those method had good reproducibility and stability, There were no cross reaction with other virus. This method can be used for clinical diagnosis and laboratory research.2. The ecology investigation of giant panda intestinal virusesApplications established four kinds of testing methods and laboratories already established a rotavirus detection methods for 188 giant pandas materials to epidemiological investigation, these samples collected in 2011 to 2013, respectively from the Sichuan captivity and wild giant panda from Minshan, Qionglai, Xiangling of the three big mountains. The results showed that there are 8 CDV positive,19 CPV positive,1 CCV positive,2 CAV positive,13 positive RV. Prevalence of intestinal diseases in the wild type giant pandas are below the captive giant panda,2011 and 2012 is lower than 2013, pathogen species and intestinal diseases prevalence of giant pandas are differences in five different regions, The experimental data in this study enriched the content of the giant panda enterovirus ecology.3. Phylogenetic analysis of the canine distemper virus and canine parvovirus strains detected from giant pandaPositive panda source samples of canine distemper and parvovirus was used to carry out genetic evolution analysis respectively, the mutant gene:H gene and VP2 gene, and cloning sequencing, genetic evolutionary tree construction were analyzed, and the results show that the giant panda source canine distemper belongs to Asia-1 gene type, the giant panda source parvovirus 370th amino acid in VP2 gene locus mutation, still belongs to the New CPV-2a type.4. The identification of giant panda source separation of intestinal virusPair virus positive samples for virus isolation and identification adapt to extend the cell, the virus will be able to grow in MDCK cells and cause obvious mesh and cell loss of the diseased cells, with specific primers of CPV gene amplification, get about 340 bp fragment, sequencing and comparing with corresponding sequence of canine parvovirus, homology of 98% above, identify the virus is canine parvovirus.