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.

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: https://theses.gla.ac.uk/id/eprint/75373