Molecular Characterisation of US6 (gD) and US8 (gE) Glycoprotein Genes Located on a Unique Short Region of Bovine Alphaherpesvirus-1 (BoAHV-1) Genome

Bovine alphaherpesvirus type 1 (BoAHV-1) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae and class Varicellovirus. Its core comprises a linear 135 kbp, double-stranded DNA genome safeguarded by 162 capsomers in an icosahedral symmetry (Jefferson et al., 2023). Viral glycoproteins encoded by BoAHV genes are organised into a single unique long unit (UL) and a single unique short unit (US), flanked by two inverted repeat sequences known as internal repeat and terminal repeat. These viral glycoproteins, located in the envelope on the virion surface, play pivotal roles in pathogenesis and immunity. Serologically related viruses causing either Infectious Bovine Rhinotracheitis (IBR) or Infectious Pustular Vulvovaginitis (IPV) are collectively referred to as Bovine alphaherpesvirus-1 (Gibbs and Rweyemamu, 1977), which are further classified into two genotypes: BoAHV-1.1 and BoAHV-1.2. BoAHV-1.2 is subdivided into BoAHV-1.2a and BoAHV-1.2b subtypes (Metzler et al., 1985; Ranganatha et al., 2013; Zhou et al., 2020). Another closely related virus, BoAHV-5, causes sporadic infections and higher mortality in calves due to encephalitis rarely reproductive infections. In India, BoAHV-5 has been isolated from reproductive tracts but not from cases of encephalitis (Kumar et al., 2020). Among the various glycoproteins found on the envelope, Gb (UL27), gD (US6) and gC (UL44) are essential, while gE (US8) non-essential for virus replication (Rashmi et al., 2024). However, all of these glycoproteins are highly immunogenic in nature. PCR assay or ELISA developed targeting these glycoproteins are used in diagnostics (OIE, 2023).
       
Although rarely a very deadly illness, IBR can cause significant economic losses through abortion, loss of body condition, decreased milk output, death of newborn calves, temporary failure of conception, poor feed conversion, subsequent bacterial pneumonia and expensive treatment. Losses in large milking herds have been estimated to range from $25 to $55 per cow (Townley, 1971). Mortality rates in dairy herds are around 3%, whereas in feedlots, they can range from 20-30% and sometimes reach as high as 100%, as reported in Hungary (Barenfus et al., 1963). The reproductive form of the illness has lowered the conception rate from 80% to 45-50% in artificially inseminated cows (Laveso et al., 1984). Townley (1971) documented an acute outbreak of the respiratory type of IBR in a dairy herd, which had a 30% morbidity rate and resulted in a $500 loss. During an epidemic of the disease¢s nerve form, Australia experienced a 30% death rate and a 90% morbidity rate (Gardiner et al., 1964). Additionally, an annual economic loss of €62 million is attributed to IBR, affecting 80% of the dairy industry in Ireland (Hanrahan et al., 2020). In India such economic loss estimates has not yet made but the five year cumulative seroprevalence of 31.37% from twenty five states and three union territories depicts the substantial economic loss (Patil et al., 2022).
       
Glycoprotein D (US6), in conjunction with gB and the gH-gL complex, facilitates the viral fusion process. It is noteworthy that gD is the primary inducer of high neutralizing antibody titres against BoAHV, making it a pivotal vaccine candidate (Dubuisson et al., 1992). With a molecular weight of 71 kDa, glycoprotein D exhibits a 79.9% amino acid identity between BoAHV-1 and BoAHV-5 (Delhon et al., 2003). ELISA based on gD are used to identify the infection. On the other hand, the association of gE with gI and US 9 sequence protein facilitates the axonal sorting and reverse axonal transport of viruses in neurons (Ning et al., 2022). Deletion of the US8 do affect the cell-to-cell movement of virus but not the viral copy number. Further gE deleted vaccines and ELISA based on gE are commonly used to differentiate infected from vaccinated animals in disease control programmes (Petrini et al., 2020).

Collection and processing of samples
 
Ten isolates of Bovine alpha-herpesvirus type 1 (BoAHV-1), archived at ICAR-NIVEDI laboratory over a 12-year period (Table 1), were chosen for revival in this study. The MDBK cell lines to propagate the virus were obtained from NCCS Pune. Cells were regularly observed under an inverted microscope at 6, 24 and 48 hours to monitor the cytopathic effects (CPE) development. Once 90% growth was achieved or characteristic CPE was observed (Fig 1), the cells inoculated with virus samples were subjected for three cycles of freezing and thawing. Around 200 microliter of cultured virus were subsequently aliquoted and stored at -20°C until use.
 

 

 
Polymerase chain reaction
 
Viral DNA extraction from cell culture supernatant was performed using the DNeasy Blood and Tissue Kit (QIAGEN, Germany) following the manufacturer's instructions. The eluted DNA was stored at -20°C. Molecular characterization of the isolates involved PCR amplification of the partial gD and gE genes. Details of the primers used and their respective product sizes are provided in Table 2. Polymerase chain reaction (PCR) was carried out using a Bio-Rad S1000 Touch PCR Thermal Cycler in a 25 ml total volume reaction mixture in a 200 mL capacity thin-wall PCR tube. Each reaction consisted of 12.5 mL of Green Taq PCR Master Mix (Thermo Scientific), 0.5 mL of forward primer (10 pmol/mL), 0.5 mL of reverse primer (10 pmol/mL), 5 mL of template DNA and nuclease-free water to 25.0 mL. The resulting mixture, after centrifugation, underwent a precise thermal profile as follows: initial denaturation at 95°C for 10 minutes, followed by 29 cycles at 95°C for 1-minute denaturation, specific annealing temperatures of 60°C for gE and 61°C for gD and extension at 72°C for 1.5 minutes, concluding with one cycle at 72°C for 10 minutes. The expected band sizes were visualised using a Bio-Rad Gel Doc 2000 after running 15 mL of PCR product on a 1.7% agarose gel. All PCR amplicons were gel-purified (QIAquick® Gel Extraction Kit, Qiagen, USA) and submitted for sequencing.  
 

 
Phylogenetic analysis
 
Following gel purification, gE and gD gene fragments were subjected for Sanger sequencing at Indus BioSolutions Ltd, Bangalore, India, using the forward and reverse primers listed in Table 2. The nucleotide sequences were BLAST analysed, aligned using GeneTool and submitted to GenBank. Generated Accession numbers of all twenty sequence were listed in Table 1. In addition to the sequences of the twenty isolates from the present study (Table 1), sequences of representative bovine herpesvirus isolate of different genotypes, BoAHV-5 and related herpesviruses such as Bubaline herpesvirus, were retrieved from GenBank (Table 3). Multiple sequence alignments were performed using CLUSTALW followed by Neighbor-Joining tree to assess phylogenetic relationships with various lineages using Mega 11.
 

Virus isolation and cell culture
 
BoAHV-1 isolates collected and stored between 2009 to 2023 were utilised in this study. Monolayers of MDBK cells were inoculated with glycerol stock virus samples for revival following standard protocols (OIE, 2023). Microscopic examination of cell monolayers revealed the presence of grape-like clustering within 30 to 48 hours post infection (Fig 1).
 
PCR amplification
 
The expected product sizes of 343 bp (Fig 4) and 652 bp (Fig 5) for the US6 (glycoprotein D) and US8 (glycoprotein E) genes respectively were amplified and visualised with ethidium bromide staining after agarose gel electrophoresis.
 
Genome alignment and phylogenetic analysis of US6 gene
 
All partial US 6 (gD) sequences from the present study exhibited 99.7% to 100% similarity among them and close relation to BoAHV 1.1. Seven isolates showed 100% nucleotide identity with vaccine strains from the USA, including Bovishield Gold, Pyramid IBR MLV, Switzerland cooper strain, ATCC Los Angeles strain, previously reported BoAHV 1.1 strains of India and Egypt.  The remaining three virus isolates (688, 742 and 744) displayed identity ranging from 99.3% to 99.7% forming a sub branch in the phylogenetic tree, however no difference in open reading frame was observed. Variable levels of identity were observed with BHV-1.2 strains (ranging from 96.8% to 98.7%), BoAHV-5 (89.9% to 90.5%) and BuHV-1 (90.5% to 91.2%). Overall, the least genome identity was observed against the BoAHV5 strains (Fig 2). 
 

 
Genome alignment and phylogenetic analysis of US8 gene
 
All isolates of present study displayed close relation to BoAHV 1.1subtype, similar to the US6 gD gene.  The nucleotide sequence alignment of the partial US8 (gE) gene of the seven virus isolates and published sequences, including Bovishield Gold, Pyramid IBR MLV, Switzerland cooper strain, ATCC Los Angeles strain, previously reported BoAHV 1.1 strains of India, Italy, China, Egypt, revealed 100% homology. Three virus isolates (685, 746 and 11705) exhibited variable percentages of homology (ranging from 99.3% to 99.5%). In case of 11012 virus, at 578 nucleotide position nucleotide C was present instead of T, leading to change in the amino acid to P (Proline) instead L (Leucine). However, isolate 685 and 746 though exhibited nucleotide change at 591 positions, no amino acid change was found. The levels of genomic similarity ranged from 99.2% to 100% for BoAHV-1 isolates (n=10), 99.00% to 99.7% between BoAHV-1.2 reference isolates, 89.00% to 89.7% for BoAHV-5 and 90.08% to 91.03% between BuAHV 1. Bubaline herpesviruses were found to be closer to BoAHV-1 than to BoAHV-5 (Fig 3).
 

       
In this study, Bovine alphaherpesvirus samples obtained from the repository of the ICAR- NIVEDI Virology lab, isolated from various sources such as nasal swabs, vaginal swabs, blood and semen samples from cattle, were successfully revived on MDBK cell lines following established protocols. Among the ten virus isolates studied, four originated from Orissa, three from Pune, two from Karnataka and one from Kolkata. Confirmation of BoAHV-1 presence was achieved through the display of characteristic cytopathic effects and amplification of the gD and gE genes, with expected product sizes of 343 bp and 652 bp, respectively (Fig 4 and 5).
 

 

       
All twenty PCR-amplified partial gene sequences (ten gE and ten gD) underwent direct sequencing and phylogenetic analysis using the Neighbour joining method. Partial nucleotide sequence analysis of the essential US6 (gD) gene and non-essential US8 (gE) gene allowed for the determination of genetic relatedness of the isolates with other herpesvirus strains from worldwide. The analysis revealed two clusters of the virus, with all ten isolates grouped as BoAHV-1.1, along with various reference BoAHV-1 and BoAHV-1.2 strains. Related herpes viruses such as BoAHV 5 and BuAHV formed separate cluster. This finding is consistent with previous studies by other authors, albeit based on different glycoprotein genes gB (Patil et al., 2012; Chandranaik et al., 2014; Surendra et al., 2015; Patil et al., 2016; Kumar et al., 2020; Guo et al., 2022), gC (Fitzpatrick et al., 1989; Sivarama et al., 1999; Esteves et al., 2008; Surendra et al., 2015; Kumar et al., 2020; Guo et al., 2022) and gD (Surendra et al., 2015; Noor et al., 2019; Guo et al., 2022). Clustering of all BoAHV 1.1 Indian isolates with Cooper strain observed in the present study was supported by other previous studies (Patil et al., 2006; Patil et al., 2012; Patil et al., 2016; Chandranaik et al., 2014). In contrast to the tree based on the US8 (gE) gene, the phylogenetic tree based on the US6 (gD) gene showed a clearer branching of BoAHV 1.2 from BoAHV 1.1. This variation is explained by the increased subtype polymorphism, especially in the US6 gene. The observation contradicted a comparative amino acid analysis of the US 6 (gD) and UL36 genes of Indonesian BoAHV-1.2 field isolates, which found that subtype polymorphism was higher in the UL36 area than in the gD region (Noor et al., 2019). Further, higher genome identity among subtypes in the US8 gene could indeed contribute to amino acid identity and development of more sensitive immunoassays for sero-epidemiology.
       
In India, numerous serological and molecular epidemiological studies conducted since 1976 have confirmed the endemic nature of BoAHV infection across almost all states and union territories (Patil et al., 2017; Rashmi et al., 2024). While testing and culling of infected animals have been implemented in bull mother farms, widespread virus control through vaccination and differentiation of vaccinated from infected animals remains limited. Understanding the prevalent strains and mutations within these strains is imperative for effective disease control programs. The implementation of the Differentiating Infected from Vaccinated Animals (DIVA) vaccination strategy, necessitates the use of glycoprotein- E based immune assays to differentiate between infection and vaccination status. In this aspect the findings of current studies suggest that, the higher genome identity observed among BoAHV 1 subtypes in the US8 gene could facilitate the development of more sensitive immunoassays for sero-epidemiology, which might cross detect BoAHV 5 and also BuAHV.

IBR caused by BoAHV-1 is endemic India and is one of the important diseases that needs immediate attention in bull mother farms/semen collection centres. Isolation of the virus is very difficult as the virus remains latent in infected animals. The isolates used in this study were characterised by nucleotide sequencing and all of them belonged to BoAHV-1.1. Seven isolates from each phylogenetic analysis showed 100% genome identity to vaccine strains from USA-Bovishield Gold MLV, Pyramid IBR MLV, Switzerland Cooper strain, ATCC Los Angeles strain, previously reported BoAHV 1.1 strains of India and Egypt. The analysis highlights the higher genome identity among BoAHV-1 subtypes in the gE gene, suggesting its potential for developing more sensitive immunoassays for seroepidemiology.

The authors declare that they have no conflict of interest.