Optimization of the in situ detection of viral protein and nucleic acid in tissue: mouse hepatitis virus and canine distemper virus
Date Issued
2011
Date
2011
Author(s)
Liang, Chung-Tiang
Abstract
Mouse hepatitis virus (MHV) is the leading viral pathogen of laboratory mice in Taiwan. This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method for detection of MHV in tissues. Mouse hepatitis virus antigen was clearly detected in samples of liver, stomach, caecal and colonic mucosa, and spleen. This method may prove useful in diagnosis when commercial antisera are unavailable or when immunosuppression prevents serological diagnosis. (Chapter II). Canine distemper virus (CDV) causes a highly contagious disease, which has been reported in Taiwan for many years; however the causative and its genes have never been identified and pathogenesis are poorly understood. The objectives of the dissertation were to set up a fast and easy diagnostic method of CDV infection, to isolate the field virus and do phylogenetic analysis of the viral H gene, to characterize the pathology of CD in Taiwan, and to assess the frequency of CNS demyelination and other pathological lesions in cases of CDV infection confirmed by immunohistochemistry (IHC) and/or reverse transcription polymerase chain reaction (RT-PCR). This study describes a modified non-biotin polymerized horseradish peroxidase (HRP) immunohistochemical method for the diagnosis of CDV infection from formalin-fixed, paraffin-embedded tissues. This method confirmed seven out of eight (87.5%) suspected cases. Labeled CDV antigen was observed in cerebrum, cerebellum, meninges, glial cells, neurons, vascular endothelium, periventricular areas and pericytes, and choroid plexus; grey and white matter and central canal of the spinal cord; renal pelvis and tubular epithelium, and urinary bladder epithelium; macrophages and lymphocytes in splenic white pulp and lymph nodes; skin epidermis; bronchiolar epithelium and alveolar macrophages; hepatic Kupffer cells, and gastric and intestinal mucosal epithelium; stratified squamous epithelium of the tongue and oesophagus.With the non-biotin HRP detection system, pretreatment by autoclaving followed by microwave heating gave better labeling results than did microwave pretreatment alone. No obvious difference was noted between the labeling results produced by the non-biotin HRP detection system and the Super Sensitive TM Link-Label IHC detection system (Chapter III, VI). For the purpose of confirming the IHC labeling and improving the detection of CDV nucleoprotein RNA, in situ hybridization (ISH) was applied in paraffin-embedded tissues from selected dogs with spontaneous CDV infections. In addition to proteinase K, autoclaving in various solutions (Trilogy, TBS S3006, H-3301, and S1700) for pre-treatments were compared. The intensity was assessed by using the integrated optical density (IOD) and the integrity of the tissue morphology. The combination of proteinase K digestion and autoclaving in a Trilogy solution resulted in a 5- to 100-fold ISH signal enhancement of CDV RNA. This modified technique can be useful in the retrospective viral studies across a broad range in the future (Chapter IV). For the purpose of CDV genotyping, during the period from 2003 to 2005, two CDV strains were isolated from 17 non-vaccinated puppies with suspected canine distemper by co-culture with peripheral blood mononuclear leucocytes and B95a cells. In addition, four cloned hemagglutinin (H) genes were obtained from 166 dogs infected with CDV. Indirect immunofluorescence assays and antigen tests confirmed that they were CDV. Analysis of the H genes of the six identified strains revealed that the deduced amino acid sequences contained nine potential sites for N-linked glycosylation, as had been found for the H proteins of Japanese isolates. The seventh site is characteristic to the Taiwan strains described in this report and to the recently reported Japanese strains. Furthermore, phylogenetic analysis of the H gene showed that the six isolates belong to the Asia-1 group and are closely related to the recently reported Japanese and Chinese strains (Chapter V). To realize the histopathological lesions of CDV in Taiwan, fifty two (IHC or RT-PCR positive) affected dogs were obtained from either animal clinics or dog shelters from 2000 to 2009, within which 32 were from clinics and 20 were from shelters. Postmortem and laboratory examination included, gross findings, histopathology, Luxol-fast blue cresyl echt violet (LFB-CEV) histochemistry, non-biotin HRP anti-CDV immunohistochemistry, and phosphoprotein gene RT-PCR. Clinic cases had histories of treatment and or vaccination. Twenty four clinic cases (75%) were puppies less than 6 months old. Seventeen shelter cases (85%) were identified as ‘adults’ greater than 6 months old. There were 27 males and 25 females. Eleven dog breeds were represented, but most dogs (35/52, 67%) were crossbred. Totally, 79% (41/52) showed lymphoid depletion, 71% (37/52) had interstitial pneumonia, 65% (34/52) had CNS demyelination, 32% (17/52) had catarrhal enteritis. Younger dogs from clinic group frequently had lymphoid depletion (31/32, 96%), inclusion bodies (28/32, 87%), pneumonia 81% (26/32, 81%), and CNS demyelination (26/32, 81%), which were all statistically significantly different from those from shelter. Enteritis was identified in about one third of the animals in both groups. The distribution of inclusion bodies also showed significant difference in urinary bladder, lymphoid tissues, lung, and alimentary tract between the two groups. Twenty nine co-infections and other associated lesions were identified. However, no significant difference was seen in the frequency of occurrence between two groups with the exception of interstitial nephritis. In conclusion, lymphoid depletion, pneumonia, and CNS demyelination were the most common CDV-infected principal lesions, and the inclusion bodies had a high occurrence in lymph nodes, spleen as well as mucosa epithelium of lung, stomach, urinary bladder and kidney (Chapter III, VI). In the present study, it has been demonstrated that CDV in Taiwan has at least one clade of the phylogenetic tree showing more than 95% amino acid similarity (< 5% amino acid variation) in the H gene. The modified non-biotin polymerlized horseradish peroxidase (HRP) IHC labeling and ISH method are easy, optimal and accurate for retrospective diagnosis of CDV and MHV. The occurrence of CDV-associated pathological lesions depend on the source of the animals, treatment history, age and tissue distribution.
Subjects
mouse hepatitis virus
canine distemper virus
immunohistochemistry
reverse transcription polymerase chain reaction
in situ hybridization
SDGs
Type
thesis
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