A Metagenomics and Case-Control Study To Identify Viruses Associated with Bovine Respiratory Disease

Abstract

Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirtyeight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P < 0.0001), bovine rhinitis A virus (P ‫؍‬ 0.005), and the recently described bovine influenza D virus (P ‫؍‬ 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and realtime PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies. IMPORTANCEBovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact. Bovine respiratory disease (BRD) is the most common and costly problem in the cattle industry, accounting for 70 to 80% of morbidity and 40 to 50% of mortality in U.S. feedlots (1, 2). The cattle industry is one of the largest agricultural sectors of the United States economy, with approximately three-quarters of a million farms raising cattle. The annual costs of BRD have been estimated at over 1 billion dollars per year (3, 4).In BRD, bacterial infections are thought to be opportunistic infections precipitated by viral infections causing damage to t...

Pathogenesis of Influenza D Virus in Cattle

“…It is well known that BRD is a multifactorial respiratory infection marked by primary viral pathogens followed by bacterial colonization in the upper and lower respiratory tract. Similarly, a recent metagenomic study showed that bovine adenovirus 3, bovine rhinitis A virus, and IDV were the top three viruses associated with BRD, either alone or in coinfections, in dairy calves in California, which would be consistent with a hypothesis of IDV predisposing to or exacerbating disease (5). Of course, it is also likely that other conditions, such as inclement weather, transportation, and close housing, might contribute to the more severe IDV-induced disease in the field.…”

“…A metagenomic study in dairy calves with bovine respiratory diseases (BRD) found that IDV was present in 62% of 50 calves that were coinfected with bovine adenovirus 3, bovine rhinitis A virus, or another BRD-associated pathogen (5). Our virologic surveillance found that IDV was present in 23.6% of 55 calves affected with BRD but in only 2.4% of 82 healthy calves upon arrival at the same facility (4).…”

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses

“…In the absence of a definitive diagnosis for the skin lesions, metagenomic sequencing was performed on seven lip lesions and one nonlesioned lip, according to previously described protocols, to further investigate a potential underlying viral etiology (21)(22)(23)(24). In brief, a tissue homogenate was centrifuged through a 0.22-m filter to enrich viral particles by size and then treated with nucleases to deplete host nucleic acids.…”

“…Nucleic acids from nuclease-resistant viral particles were extracted using the QIAquick viral-RNA column purification system, followed by sequence-independent amplification using random priming. First-strand synthesis (for both DNA and RNA) was performed with a 28-base oligonucleotide whose 3= end consisted of eight random nucleotides (primer N1_8N, CCTTGAAGGCGGACTGTGAGNNNNNNNN), using Superscript III reverse transcriptase (Invitrogen) (21)(22)(23)(24). A second strand was synthesized using Klenow fragment DNA polymerase (New England BioLabs).…”

Prevalence of infections with a new type of influenza virus – influenza D virus in humans and animals

“…Z puli przebadanych próbek jedynie 6 surowic (2,8%) reagowało w teście HI, w niskim mianie (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), ze szczepami ludzkimi ICV.…”