Genetic characterisation of Indian bovine herpesvirus-1 with other ruminant herpesviruses in relation to glycoproteins C

Introduction

Respiratory diseases in bovines, such as Infectious Bovine Rhinotracheitis (IBR) and Bovine Respiratory Disease Complex (BRC), are associated with Bovine Herpesvirus 1 (BoHV-1), a significant pathogen found worldwide (Jefferson et al., 2023) [8] . The virus was first identified in Germany as the causative agent of reproductive disorders like Infectious Vulvovaginitis (IPV). Other conditions it can cause includes Infectious Balanoposthitis (IBP), vision impairment, systemic newborn disease and latent infections in ruminants (Hostnik et al., 2021) [6] . In India, after first isolation of BoHV-1 organism from keratoconjunctivitis cases, both subtypes BoHV-1.1 and BoHV-1.2, have been recovered from IBR, IPV, IBP cases, whose homology was identical to the cooper strain. Serological evidence of IBR has been reported in various species of animals in different parts of the world for example cattle, buffalo, sheep, goats, dromedaries, deer, elephants, hippopotami. Five related herpesviruses, which cause serological cross-reactions with BoHV-1, may interfere with IBR eradication and control programs. These viruses include bovine herpesvirus 5 (BoHV-5), bubaline herpesvirus-1 (BuHV 1), caprine herpesvirus 1 (CaHV-1), cervine herpesvirus 1 (CvHV-1) and rangiferine herpesvirus 1 (RanHV-1) (Ros et al., 1999; Yadav et al., 2024) [21,26] . Related ruminant herpesviruses are known to cause species-specific diseases. Outbreaks of fatal meningoencephalitis caused by BoHV-5 in calves have been reported in several countries, including Australia, Hungary, the United States, Argentina and Italy. In India, the first case of BoHV-5 was detected in a cattle abortion case in Rajasthan (Kumar et al., 2020) [10] . Initially, BuHV-1 was associated with subclinical disease in water buffalo and the virus has been isolated from the prepuce of buffaloes in Australia, vulvovaginitis cases in India (Yadav et al., 2024) [26] , after pharmacological reactivation in Italy, and from abortion cases in the Mediterranean region (Amoroso et al., 2013) [1] . CapHV-1, which is globally distributed, causes generalised disease in young kids, primarily affecting the digestive tract, while in adult goats, the infection often remains unnoticed or may cause abortion, vulvovaginitis, or balanoposthitis. It is suspected that sheep and goats might be involved in the transmission of BoHV-1, as the presence of the virus has been detected in Iran, Egypt, and Pakistan. CerHV-1 was first isolated in 1982 from an outbreak of ocular disease on a red deer farm in Scotland and is now widespread in both freeliving and farmed red deer. RanHV-1 was isolated from reindeer in Finland. Species differentiation during seroepidemiology is important in proper diagnosis and control strategies. Glycoprotein C (gC) one among many types I transmembrane glycoprotein, is a non-essential protein, N-terminal forms a spike that interacting with heparan-sulphate on the cell surface contributing to the host cell type tropism (Rashmi et al., 2024) [19] . It consists of 521 (BoHV-1) amino acid residues that belong to the immunoglobulin superfamily. (Fitzpatrick et al., 1989) [4] . Both N-terminal and C-terminal regions are highly variable to distinguish the subtypes and amplification of residues from 321 to 450 by PCR region, has often been envisaged as a target for differentiating the subtypes by few authors (Rijsewijk et al., 1999; Esteve et al., 2008) [20,3] . Similarly in the present study the total of 192 amino acid residues of gC, towards carboxy terminal region were subjected for genome alignment and phylogenetic analysis in relation to other ruminant herpes viruses

Materials and Methods Collection and Processing of Samples

Five isolates of Bovine alpha-herpesvirus type 1 (BoAHV-1), archived at the ICAR-NIVEDI laboratory from 2010 to 2023 (Table 1), were selected for revival in this study. The virus was propagated using MDBK cell lines on EMEM medium supplemented with 10% Foetal Bovine serum and antibiotic. The cells were regularly observed under an inverted microscope at 6, 24, and 48 hours to monitor the development of cytopathic effects (CPE). Once 90% cell growth was achieved or rounding of cells -characteristic CPE was observed (Fig 1), the virus-inoculated cells were subjected to three cycles of freezing and thawing. Approximately 200 microliters of the cultured virus were then aliquoted and stored at -20 °C until further use Polymerase chain reaction: Viral DNA was extracted from the cell culture supernatant using the DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the manufacturer's protocol. The eluted DNA was then stored at -20 °C. For molecular characterization of the isolates, PCR amplification was performed on the partial gC genes. The primers used for this amplification, gC F (560 -GAA GGA GCG CAA GTG GAT GCT CT-583) and gC R (1138-CGG AGT CGT CGA CCG TAA AGA CGT-1115), target the carboxy-terminal region of the gC gene and were previously designed from the same lab (Ranganath et al., 2013) [18] . The polymerase chain reaction (PCR) was conducted using a Bio-Rad S1000 Touch PCR Thermal Cycler in a 25 μL reaction mixture within a 200 μL thin-wall PCR tube. Each reaction contained 12.5 μL of Green Taq PCR Master Mix (Thermo Scientific), 0.5 μL of forward primer (10 pmol/μL), 0.5 μL of reverse primer (10 pmol/μL), 5 μL of template DNA, and nuclease-free water to reach a final volume of 25 μL. After centrifugation, the mixture was subjected to a thermal cycling program: initial denaturation at 94 °C for 15 minutes, followed by 34 cycles of denaturation at 94 °C for 50sec, annealing at 65 °C for 50 sec, and extension at 72 °C for 50 sec, with a final extension at 72 °C for 10 minutes. The expected PCR product bands were visualised by running 15 μL of the PCR product on a 1.7% agarose gel and analysing it with a Bio-Rad Gel Doc 2000. All PCR bands of expected size were gel-purified using the NucleoSpin Gel & PCR Clean-up Mini kit (MACHEREY-NAGEL) and subsequently submitted for Sanger sequencing at Indus BioSolutions Ltd, Bangalore, India. Using forward and reverse primers mentioned above both sense and antisense strands of amplicons were sequenced. The resulting nucleotide sequences were analysed using BLAST, aligned, and annotated with GeneTool, then submitted to GenBank. Accession numbers received for all five sequences, along with details such as origin, year, sex, and sample type from which the virus was isolated, are provided in Table 1. In addition to the sequences from the five isolates in this study (Table 1), sequences of representative bovine herpesvirus isolates from different genotypes (BoAHV-5), and related herpesviruses such as Bubaline herpesvirus, caprine herpesvirus 1 (CaHV-1), cervine herpesvirus 1 (CvHV-1) and rangiferine herpesvirus 1 (RanHV-1) were retrieved from GenBank (Table 2). Multiple sequence alignments were performed using CLUSTALW, followed by construction of the phylogenetic trees based on the maximum likelihood approach according to the Tamura-3-parameter model using mega11 software (Tamura et al., 2021) [9] .

Results

Virus Isolation and Cell Culture: Five BoAHV-1 isolates collected and stored between 2010 to 2023 were utilised in this study. Monolayers of MDBK cells were inoculated with glycerol stock virus samples for revival following standard protocols (OIE Manual, 2023) [12] . Microscopic examination of cell monolayers revealed the presence of grape-like clustering within 30 to 48 hours post inoculation (fig 1).

PCR Amplification:

The expected product sizes 572 bp for the glycoprotein C genes was amplified and visualised with ethidium bromide staining after agarose gel electrophoresis. The desired bands eluted and sequenced were submitted to NCBI and

Genome alignment and Phylogenetic analysis: Broadly

BoHV-1, BuHV-1, BoHV-5, CeHV-1 formed one cluster and CaHV-1 and RanHV-1 formed another. All five partial gC sequences from the present study exhibited 99.7% to 100% similarity among them and close relation to BoAHV 1.1. Genome with a homology of 98.3 to 100% to vaccine strains from USA-Bovishield Gold MLV, Titanium IBR MLV, Switzerland Cooper strain, ATCC Los Angeles strain, previously reported BoAHV 1.1 strains of India and Egypt. Genome identity of 91.6 to 92.0% observed for Brazil and Indian BoHV-5 strains, 92.2% for Bubaline herpes virus, 89.6 to 89.85 for cervid herpes virus of China, Italy, Switzerland., 67.8 to 68% for rangiferin herpes Finland and 66.9 to 67.1% for Caprine herpes virus of Switzerland, Italy, China.

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