Molecular Tools to Identify and Characterize Malignant Catarrhal Fever Viruses (MCFV) of Ruminants and Captive Artiodactyla

Abstract

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

Keywords: Herpesviridae; consensus Panherpes PCR; gamma(γ)-herpesvirus; long-distance PCR; malignant catarrhal fever virus (MCFV).

Figure 1

Schematic representation of the herpes virion. A linear, double-strand deoxyribonucleic acid (DNA) molecule (red and orange) is surrounded by an icosahedral capsid (light blue), which in turn is covered by tegument (white). The outer surface of the tegument (grey) is associated with a lipid bilayer envelope (red and grey), which contains integral glycoproteins. Glycoprotein B trimer (green), glycoprotein C monomer (brown), glycoprotein D homodimer (light blue), glycoprotein H (purple) and glycoprotein L (orange) heterodimer allow the entry of the virion into the host cell.

Figure 2

Classes of viruses belonging to the family Hespesviridae based on the arrangement of repeated sequences in the genome. Human herpesvirus-6A and Human herpesvirus-6B (subfamily β-herpesvirinae) and Equine herpesvirus-2 (subfamily γ-herpesvirinae) genomes belong to class 1, while many members of the subfamily γ-herpesvirinae belong to class 2; members of the genus Varicellavirus (subfamily α-herpesvirinae) belong to class 3 genome, whereas Herpes simplex virus (subfamily α-herpesvirinae) and Cytomegalovirus (subfamily α-herpesvirinae) belong to class 4. The pol gene, which contains three conserved regions (A, B and C), is present in the architecture of all genome classes (illustrations not at scale). These regions encode highly conserved amino acid domains (DFA, ILK, TGV, IYG and KG1). PCR primers (dashed arrows) according to VanDevanter et al. are shown under the different class architectures. Primer’s directions are specified by dashed arrows. The product obtained by the Panherpes nested PCR is between region B and region C. The nomenclature used in all classes is U (unique), U_L (unique long), U_S (unique short), TR (terminal repeat), TR_L (terminal long repeat), IR_L (internal long repeat), TR_S (terminal short repeat), IR_S (internal short repeat); TR_L and TR_S include a (terminal direct repeat) and IR_L and IR_S a^I (internal inverse repeat). The orientations of repeated sequences are specified by block arrows.

Figure 3

Representation of the deoxyinosine-substituted primers generated by Ehlers et al. modifying the original degenerate primers and introducing deoxyinosine in all primer positions with 3- and 4-fold degeneration. For example, in the DFA degenerate primer, the degenerate N base was substituted with I^a.