Studies on Diagnosis and Pathogenesis of Equine Herpesviruses-1 and -4 by Polymerase Chain Reaction

Sharma, Prakash Chand (1994) Studies on Diagnosis and Pathogenesis of Equine Herpesviruses-1 and -4 by Polymerase Chain Reaction. PhD thesis, University of Glasgow.

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Abstract

The work presented in this thesis focuses on the development of polymerase chain reaction (PCR) assays for diagnosis and differentiation of equid herpes viruses-1 and -4 (EHV-1 and EHV-4) infections in suspected field cases and contact animals and in experimentally infected specific-pathogen-free (SPF) foals. The technique has been applied to study the pathogenesis of EHV-1 and EHV-4 infections in these foals. Further, the effect of homologous (EHV-1:EHV-1) challenge on nasal virus excretion and duration of viremia has been studied. Studies on heterologous (EHV-4:EHV-1) challenge have also been conducted with one objective being to determine whether challenge virus could reactivate virus inoculated to produce primary infection (reactivation of EHV-4 by EHV-1 in this instance). Following secondary challenge (homologous or heterologous), dexamethasone administration was performed in order to reactivate latent virus(es). The chapterwise details of the thesis are as follows: Chapter 1 describes the general features the family Herpesviridae structure of herpesvirion, genomes of herpesviruses, classification of herpesviruses, their replicative cycles and latency. A brief account of various equid herpesviruses (equid herpesviruses-1 to -8) is given and the literature on various aspects of EHV-1 and EHV-4 has been reviewed comprehensively. The general materials and methods used throughout the course of study are described in Chapter 2. Chapter 3 gives an account of polymerase chain reaction (PCR) and its application in various fields of science. The EHV-1/EHV-4 PCR assay is detailed. Type common primers derived from the conserved regions of homologues of HSV genes encoding glycoproteins gH and gC and a nested set comprising a primer derived from thymidine kinase (tk) gene and three from the gH gene have been used to amplify specific regions of EHV-1 and EHV-4. The targets for amplification in the PCR assays were; tissue culture supernatants from EHV-1 and EHV-4 infected cultures and recombinant plasmic DNAs containing EHV-1 and EHV-4 target. The amplified products could be distinguished by type-specific probes selected from the divergent regions of the genes and internal to the primers. Reaction conditions were optimised and assay sensitivity assessed. Chapter 4 describes the use of PCR techniques to the detection of EHV-1 or EHV-4- specific DNA in nasopharyngeal swab samples from suspected field cases and their in-contacts. The primers used in the PCR assay were: inner gH, nested set and gC. The assays were conducted blind and later decoded and compared with virus isolation data. Of 98 samples, 91 of which were EHV-1 and EHV-4-negative and 7 positive, all 7 positives were confirmed by PCR and 5 virus-negative were PCR-positive. The results indicated that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infections. The studies on the experimental infections of specific-pathogen-free (SPF) foals are described in Chapter 5. The experiment was conducted in three phases: primary infection, secondary infection/challenge and reactivation. Two groups of two SPF foals were infected by inoculating 107 pfu of EHV-1 strain Ab4 or EHV-4 strain MD intranasally. Nasal secretions and peripheral blood leucocytes (PBMCs) were collected at days -2, 1, 3, 5, 8, 11 and 18 post-infection (p.i.) and analysed by PCR. Results were compared with the virus isolation and co-cultivation data. The latter work was carried out by research group at Cambridge led by Dr Hugh Field. Rectal temperatures as high as 106F were recorded in EHV-1 infected foals and classical signs of EHV-1 induced disease were observed. The EHV-4 infected foals exhibited a mild disease. EHV-1 specific DNA was detected in the nasal secretions of the EHV-1 infected foals til day 18 post-infection, the day of last sampling. In the PBMCs of EHV-1 infected foals, EHV-1 specific DNA was detectable till day 11 p.i. At the peak of infection at day 5 in one foal and at day 8 in another foal. 5 x 10+ and 5 x 104 PBMCs respectively gave a positive signal in PCR assay. Nasal virus excretion by virus isolation was demonstrable till day 11 p.i. in both the EHV-1 infected foals. EHV-1 viraemia was observed by infectious centre assay at days 3, 5 and 11 in one foal and at day 11 post infection in another. The EHV-4-specific DNA in EHV-4 infected group was detectable till day 18 and 15 respectively in the nasal secretions of the two foals. EHV-4 specific DNA was detectable till day 15 p.i.; the infection was mild throughout as 5 x 105 cells gave a positive signal in PCR assay. In another foal, EHV-4 specific DNA was detectable till day 11 p.i. in the PBMCs, the peak of infection observed at day 11 when 5 x 104 cells gave a positive signal. No infectious centre was demonstrated in co-cultivation studies of leucocytes of EHV-4 infected foals indicating thereby that the assay is less sensitive as compared to PCR. Our studies provide definitive evidence of EHV-1 and EHV-4 specific DNA sequences in PBMCs of foals infected under specific pathogen-free conditions, following primary infection with EHV-1 and EHV-4. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David E Onions
Keywords: Veterinary science, Animal diseases, Virology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75373
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:23
Last Modified: 19 Nov 2019 20:23
URI: http://theses.gla.ac.uk/id/eprint/75373

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