Optimisation of rapid untargeted nanopore DNA virus metagenomics using cell cultures and calves experimentally infected with bovine herpes virus-1

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle in Ireland, and internationally. This disease is caused by many well-known, and an ever-increasing number of newly associated viruses and bacteria. Consequently, diagnosis of BRD pathogens by targeted real-time polymerase chain reaction (qPCR) diagnostics is too expensive and slow to enable a same-day response that is targeted at the causative pathogen(s). To address this, we developed a same-day, sample to result, untargeted metagenomic MinION sequencing protocol for the identification of DNA viruses associated with BRD from nasal swabs. The procedure comprises non-viral nucleic acid depletion, nucleic acid extraction, rapid transposase-based tagmentation with barcoded adapters, non-biased PCR amplification of tagmented nucleic acid, sequencing on a MinION device, then rapid analysis of resulting sequences on cloud-based software EPI2ME WIMP. The protocol was developed using BoHV1-infected foetal lung cell cultures where we achieved 96% enrichment of the BoHV-1 sequence. Subsequently, the protocol was successfully applied to untargeted detection of BoHV-1 in nasal swabs from calves experimentally challenged with BoHV-1.

Serological and molecular detection of Bovine Herpes Virus 1 infection in cattle by enzyme linked immunosorbent assay and polymerase chain reaction

The present study was undertaken to assess the seroprevalence of Bovine Herpes Virus 1 (BoHV 1) in cattle in and around Thrissur district employing ELISA and to detect the presence of the virus from suspected cases of abortion in cattle using PCR assay. A total of 182 serum samples were screened using commercially available ELISA kit of which 22 were found positive for BoHV1 antibody with a seropositivity of 12.09 per cent. For the detection of BoHV1 virus in aborted cases, a PCR assay was standardised targeting glycoprotein C gene of BoHV1. Out of a total of 13 aborted foetal tissue samples, six were found to be positive with an amplicon size of 179 bp. Further, the amplicons were confirmed by nucleotide sequencing. Phylogenetic analysis of the sequences showed that our sample from Kerala grouped with isolates from India (Gujarat and UP), Brazil, Switzerland, and USA.