IDENTIFICATION AND CHARACTERISATION OF POTENTIALLY NOVEL HERPESVIRUSES IN VERTEBRATE SPECIES

The Herpesviridae family includes a diverse group of viruses that can infect a broad spectrum of vertebrate hosts, ranging from fish to mammals. The structural unit of these viruses, known as the virion, contains a linear, double-stranded DNA genome that is encapsulated within an icosahedral capsid, which is further surrounded by a protein layer called the tegument. The outer surface of the tegument is associated with a lipid envelope, which incorporates glycoproteins that facilitate viral attachment and entry into host cells. Herpesviruses have co-evolved with specific hosts and are extremely adapted to them; indeed, in these adapted hosts they establish a life-long latent and sub-clinical infection. However, various pathological manifestations can still arise in fetuses, newborns, and immunocompromised individuals as a result of primary infection or reactivation. Furthermore, herpesviruses can cross species barriers and infect non-adapted hosts, which can lead to severe consequences. Non-adapted hosts typically lack the evolutionary adaptations necessary to effectively control the virus, resulting in increased viral pathogenicity and significant clinical disease. Based on biological and phylogenetic characteristics this virus family is divided into three sub-families: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. Each sub-family includes distinct genera and species. However, many herpesviruses that infect non-human species remain unclassified due to their limited genetic data available in existing databases. This insufficiency complicates both the classification and the identification of these viruses. This issue is particularly evident in studies involving Malignant Catarrhal Fever Viruses (MCFVs) and alphaherpesviruses found in reptiles and pinnipeds, where there is considerable viral diversity but insufficient genomic resources. Consequently, the classification of these viruses often lacks clarity and precision, making species and strain-level identification especially challenging. A significant factor contributing to this problem is the widespread reliance on the Pan-herpes PCR assay as the primary detection and identification method. While this approach is effective for broadly detecting herpesviruses, it typically generates short DNA polymerase gene sequence fragments, which complicate identification and characterisation of these viruses. To address these limitations, my research aimed to develop more effective molecular techniques that could generate longer, more comprehensive genetic sequences for improved herpesvirus identification and classification. The key innovations included the use of phylogenetic techniques to verify virus identification and the application and validation of Long-PCR to extend the sequences available, by linking two adjacent, conserved gammaherpesviral genes (DNA polymerase and glycoprotein B). Initially, Pan-herpes PCR approach was applied to detect herpesviruses in one hundred and thirty-eight wild animals belonging to several species of Artiodactyla. Viral sequences from positive samples were subsequently identified and analysed with phylogenetic techniques to investigate genetic diversity and evolutionary relationships. Gammaherpesvirus-positive samples were then used to perform Long-PCR for further in-depth analysis. As a result, and combining several molecular identification methods, two new unpublished strains of ovine gammaherpesvirus 2 (OvGHV-2), belonging to the MCFV group, were identified, along with two potential new members of the Macavirus genus. The study also demonstrated that the Long-PCR technique is a reliable method for precise identification and accurate phylogenetic analysis, particularly in cases where more advanced techniques, such as Next Generation Sequencing (NGS), are not feasible. Unfortunately, the Long-PCR approach cannot be applied to the study of alphaherpesviruses due to the genomic arrangement of their DNA polymerase and gB genes, which are not located in close proximity within their genomes. Therefore, only the Pan-herpes PCR followed by phylogenetic analysis was employed to investigate herpesviruses present in clinical samples obtained from UK farmed crocodiles. This study identified four alphaherpesviruses from these samples. Of these, one was represented by a strain of crocodyline herpesvirus 1 (CrHV-1), while the other three sequences corresponded to distinct strains of crocodyline herpesvirus 2 (CrHV-2) species. Additionally, phylogenetic analysis, which included database-retrieved sequences of all known reptile viruses, confirmed that chelonid herpesviruses could be classified as a distinct genus, proposed to be named Chelonivirus. The investigation into pinniped herpesviruses involved analysing both classified and unclassified herpesviruses identified in Odobenidae, Otariidae and Phocidae via phylogenetic analysis. Sub-family classification of herpesviruses detected in this animal superfamily remains challenging due to limited sequence data and the absence of an officially approved taxonomy. My approach led to the identification of genetic similarities and differences, which facilitated a more accurate taxonomy and ultimately enhanced our understanding of the diversity and evolution of pinniped herpesviruses. In conclusion, through the development, testing, and integration of several molecular methods for identifying and characterising herpesviruses, I have provided valuable new insights and validated approaches. These findings contribute to a deeper understanding of the evolutionary history of these viruses, leading to improved classification and identification of potentially new adapted and non-adapted hosts. Additionally, in the near future, these results could be used to study the co-evolution between viruses and their adapted hosts.