A Rapid Nucleic Acid Visualization Assay for Infectious Bovine Rhinotracheitis Virus That Targets the TK Gene.

Infectious bovine rhinotracheitis virus (IBRV) can cause various degrees of symptoms in the respiratory system, reproductive system, and whole body of cattle. It also can lead to persistent and latent infection in cattle, posing a challenge to timely control of infectious bovine rhinotracheitis (IBR) in farms and causing large financial losses in the global cattle industry. Therefore, the goal of this study was to establish a rapid, simple, and accurate method that can detect IBRV in order to facilitate the control and eradication of IBR in cattle. We combined recombinant polymerase amplification (RPA) with a closed vertical flow visualization strip (VF) and established an RPA-VF assay that targets the thymidine kinase (TK) gene to rapidly detect IBRV. This method (reaction at 42°C for 25 min) was able to detect a minimum of 3.8 × 101 copies/µL of positive plasmid and 1.09 × 101 50% tissue culture infective dose (TCID50) of the IBRV. This assay has high specificity for IBRV and does not cross-react with other respiratory pathogens in cattle. The concordance between the RPA-VF assay and the gold standard was 100%. In addition, this assay was also suitable for the detection of DNA from clinical samples extracted by a simple method (heating at 95°C for 5 min), which can achieve the rapid detection of clinical samples in the field. Overall, the present sensitivity, specificity, and clinical applicability assessments indicated that the RPA-VF assay we developed can be utilized as a quick and accurate on-site test for IBRV detection in farms. IMPORTANCE IBRV causes different degrees of clinical symptoms in cattle and poses a great threat to the cattle industry. The infection is persistent and latent, and the elimination of IBRV in infected herds is difficult. A rapid, simple, and accurate method to detect IBRV is therefore vital to control and eradicate IBR. Combining RPA with an VF, we established an RPA-VF assay for the rapid detection of IBRV, which can complete the test of clinical samples in 35 min. The assay shows good sensitivity, specificity, and clinical applicability and can be used as an on-site test for IBRV in farms.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation.

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-alpha mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1beta antibody or human soluble TNF-alpha receptor (sTNF-alphaR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1beta antibody, sTNF-alphaR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.

The zinc RING finger of bovine herpesvirus 1-encoded bICP0 protein is crucial for viral replication and virulence.

Bovine herpesvirus 1 (BHV-1) infected cell protein 0 (bICP0) stimulates productive infection, in part by activating viral gene expression. The C(3)HC(4) zinc RING finger of bICP0 is crucial for activating viral transcription and productive infection. In this study, we used a bacterial artificial chromosome containing a wild-type (wt) virulent BHV-1 strain to generate a single amino acid mutation in the C(3)HC(4) zinc RING finger of bICP0. This virus (the 51g mutant) contains a cysteine-to-glycine mutation (51st amino acid) in the C(3)HC(4) zinc RING finger of bICP0. A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. The 51g mutant virus expressed higher levels of the bICP0 protein than did the 51g rescued virus (51gR) but yielded reduced virus titers following infection of permissive bovine cells. The 51g mutant virus, but not the 51gR virus, grew poorly in bovine cells pretreated with imiquimod to stimulate interferon production. During acute infection of calves, levels of infectious virus were 2 to 3 logs lower in ocular or nasal swabs with 51g than with 51gR. Calves latently infected with the 51g mutant did not reactivate from latency because virus shedding did not occur in ocular or nasal cavities. As expected, calves latently infected with 51gR reactivated from latency following dexamethasone treatment. These studies demonstrate that mutation of a single well-conserved cysteine residue in the C(3)HC(4) zinc RING finger of bICP0 has dramatic effects on the growth properties of BHV-1.

Ectodysplasin-1 deficiency in a German Holstein bull associated with loss of respiratory mucous glands and chronic rhinotracheitis.

A 2-year-old German Holstein bull was identified as a carrier of a mutation within the X-chromosomal ED1 gene, which encodes a TNF-related signalling molecule mainly involved in ectodermal development. The clinicopathological appearance was associated with hypotrichosis, hypodontia, and a reduced number of eccrine glands, in addition to chronic rhinotracheitis and partial squamous metaplasia. Furthermore, for the first time in an ED1-deficient animal, a complete lack of respiratory mucous glands was observed. This suggests that the ED1 gene plays a role in the development of mucous glands, the absence of which resembles a feature of X-linked anhidrotic ectodermal dysplasia (ED1) in human patients.

Type-I interferon genotypes and severity of clinical disease in cattle inoculated with bovine herpesvirus 1.

Genomic DNA samples and health records from 98 unrelated, mixed-breed cattle inoculated with bovine herpesvirus 1 (BHV-1) were examined to determine the relationship between interferon (IFN) genotype and severity of clinical disease. Cattle were retrospectively classified as moderately or severely affected on the basis of rectal temperature, feed intake, and weight gain after intranasal inoculation of BHV-1. Southern blot analysis of 16 type-I IFN genes identified alleles at 3 IFN loci (IFNB1, IFNW4, and IFNW8) that were significantly associated with the more severe clinical phenotype (odds ratios = 4.1 [P = 0.01], 2.3 [P < 0.05] and 2.4 [P = 0.06], respectively). A second allele at the IFNB1 locus was associated with the milder disease phenotype (odds ratio = 2.9, P < 0.05). These results indicate that selective breeding programs aimed at altering the frequency of these alleles in cattle populations may potentially improve animal health and lessen the economic impact of BHV-1 infection on cattle producers.

Immediate-early RNA 2.9 and early RNA 2.6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein.

Bovine herpesvirus 1 (BHV-1) contains three major immediate-early (IE) genes involved in regulation of the productive cycle of replication. Two spliced IE RNAs, IER4.2 (4.2 kb) and IER2.9 (2.9 kb), are under the control of a single promoter; IER1.7 (1.7 kb) is transcribed from a different promoter in the opposite direction. Examining the kinetics of transcription, we found that the IER4.2/2.9 promoter was turned off at the end of the IE period. An alternative promoter became active, directing synthesis of an unspliced early RNA, ER2.6 (2.6 kb), which was colinear with the second exon of IER2.9 except for its 5' end in the intron about 10 bases upstream of the splice site. Sequence analysis revealed a single open reading frame common to IER2.9 and ER2.6 with a coding potential of 676 amino acids. The putative protein, named p135, contained a cysteine-rich zinc finger domain near the N terminus with homology to ICP0 of herpes simplex virus type 1, to protein 61 of varicella-zoster virus, to early protein 0 of pseudorabies virus, and to other viral and cellular proteins. The remaining parts of p135 exhibited only limited homology, mainly with pseudorabies virus protein 0, but the entire sequence was highly conserved between two strains of BHV-1 (K22 and Jura). The latency-related antisense transcript covered a large portion of ER2.6 excluding the zinc finger coding region. In transient expression assays, p135 activated a variety of promoters, including that for ER2.6, but repressed the IER1.7 promoter. Thus, p135 combines functional characteristics of ICP0, a strong transactivator, and of protein 61, a repressor. BHV-1 seems to have evolved a subtle mechanism to ensure the continued synthesis of p135 while turning off IER4.2, which encodes p180, the herpes simplex virus type 1 ICP4 homolog.