Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1.

BACKGROUND: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes. RESULTS: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/µL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. CONCLUSIONS: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Serological Responses of Guinea Pigs and Heifers to Eight Different BoAHV-1 Vaccine Formulations.

Bovine alphaherpesvirus 1 (BoAHV-1) infection affects the production and reproductive performance of dairy and beef livestock, resulting in considerable economic losses. In addition to biosecurity measures, vaccination programs are effective strategies for controlling and preventing BoAHV-1 infection and transmission. We evaluated the serological immune response against BoAHV-1 induced by eight different formulations of commercial vaccines: three modified live vaccines and five killed vaccines containing BoAHV type 1 or types 1 and 5. In the first experiment, 50 BoAHV-1-seronegative guinea pigs were assigned to eight groups; each individual in the treatment groups received two doses (one-fifth of the bovine dose). The second experiment was conducted using 29 crossbred Holstein × Gir heifers in four groups of six to nine animals each. The serological immune response against BoAHV-1 was measured using virus neutralization and enzyme-linked immunosorbent assays to measure the total IgG against BoAHV. We evaluated the effects of the vaccine, time, and interaction of the vaccine and time on neutralizing antibodies against BoAHV-1. Killed vaccines produced low levels of antibodies against BoAHV-1, whereas modified live vaccines produced high levels of antibodies capable of providing neutralizing titers in the vaccinated animals, with the thermosensitive modified live vaccine showing the highest levels of antibodies.

Seroepidemiology of bovine alphaherpesvirus 1 (BoAHV-1) in commercial and smallholder dairy herds in north Shewa, central highlands of Ethiopia.

Bovine alphaherpesvirus 1 (BoAHV-1) is the most important respiratory and reproductive disease-causing pathogen in dairy cattle. Despite BoAHV-1 has become widespread and a major challenge to the dairy industry, little is known about its epidemiology in dairy herds in Ethiopia. A cross-sectional study was conducted from November 2022 to May 2023 to determine the seroprevalence and potential risk factors associated with BoAHV-1 seropositivity in dairy herds in North Shewa, the central highlands of Ethiopia. A total of 511 blood samples were collected from randomly selected cattle herds (n = 142) and examined antibodies against BoAHV-1 using ELISA test. A retrospective survey was also done to gather information related to reproductive disorders. The overall seroprevalence of BoAHV-1 was 61.84% (95% CI: 57.53-65.97) at the animal level and 85.21% (95% CI: 78.28-90.21) at the herd level. Multivariable logistic analysis revealed that the risk of being BoAHV-1 seropositive was nine times higher in cows older than six years (OR = 9.16; 95% CI: 3.09-27.16; P = 0.000), five times higher (OR = 4.51; 95% CI: 1.23-16.53; P = 0.019) in cows with a history of abortion, three times higher (OR = 2.75; 95% CI: 1.72-4.22; P = 0.029) in cows with a history of retained fetal membrane, and three times higher (OR = 2.83; 1.86-9.31; P = 0.03) in animals with clinical signs of ocular and/or nasal discharge. This study demonstrates a significant circulating of BoAHV-1 in the dairy cattle population in study districts. Thus, a comprehensive approach that includes strict farm biosecurity and vaccination should be practiced for effective BoAHV-1 control and prevention and to promote the growing dairy industry in the central highlands of Ethiopia.

Isolation of the Initial Bovine Alphaherpesvirus 1 Isolate from Yanbian, China.

Bovine infectious rhinotracheitis (IBR), caused by bovine alphaherpesvirus 1 (BoAHV1), poses significant challenges to the global cattle industry due to its high contagiousness and economic impact. In our study, we successfully isolated a BoAHV1 strain from suspected infected bovine nasal mucus samples in Yanji city, revealing genetic similarities with strains from Sichuan, Egypt, and the USA, while strains from Xinjiang, Beijing, Hebei, and Inner Mongolia showed more distant associations, indicating potential cross-border transmission. Additionally, our investigation of BoAHV1 infection dynamics within host cells revealed early upregulation of gB, which is critical for sustained infection, while the expression of gC and gD showed variations compared to previous studies. These findings enhance our understanding of BoAHV1 diversity and infection kinetics, underscoring the importance of international collaboration for effective surveillance and control strategies. Furthermore, they lay the groundwork for the development of targeted therapeutics and vaccines to mitigate the impact of IBR on the cattle industry.

Antiviral activity of Taurisolo® during bovine alphaherpesvirus 1 infection.

Bovine alphaherpesvirus 1 (BoAHV-1), the pathogen causing Infectious Bovine Rhinotracheitis (IBR) and predisposing to polymicrobial infections in cattle, provokes farm economic losses and trading restrictions in the world. However, nontoxic antiviral agents for BoAHV-1 infection are still unavailable, but plant extracts, such as flavonoid derivatives possess activity against BoAHV-1. Taurisolo®, a nutraceutical produced by Aglianico grape pomace, has recently shown promising antiviral activity. Herein, the potential activity of Taurisolo® during BoAHV-1 infection in Madin Darby bovine kidney (MDBK) cells was tested. Taurisolo® enhanced cell viability and reduced morphological death signs in BoAHV-1-infected cells. Moreover, Taurisolo® influenced the expression of bICP0, the key regulatory protein of BoAHV-1, and it strongly diminished virus yield. These effects were associated with an up-regulation of aryl hydrocarbon receptor (AhR), a transcription factor involved in microbial metabolism and immune response. In conclusion, our findings indicate that Taurisolo® may represent a potential antiviral agent against BoAHV-1 infection. Noteworthy, AhR could be involved in the observed effects and become a new target in antiviral therapy.

Infectious Bovine Rhinotracheitis Post-Eradication Program in the Autonomous Province of Bolzano, Italy: A Retrospective Study on Potential Bovine Herpesvirus Type 2 Cross-Reactivity.

Bovine alphaherpesviruses, BoAHV, can cause respiratory, genital and neurological disorders. In particular, bovine alphaherpesvirus type 1 (BoAHV1) is one of the most significant ruminant pathogens worldwide and it can heavily damage the livestock industry. BoAHV1 can cause infectious bovine rhinotracheitis (IBR) along with fertility disorders. Bovine alphaherpesvirus type 2 (BoAHV2) can cause two different conditions as well: pseudo-lumpy skin disease (PSLD) and bovine herpetic mammillitis (BHM). The autonomous province of Bolzano (Italy) has adopted several strategies to control and eradicate IBR, and it was declared in 2000 to be IBR-free by the European Commission. Since 2001, a post-eradication monitoring program has overseen the serological analysis of bulk milk and, in the presence of a positive result, a follow-up examination is performed on the individual blood serum of all bovines older than 24 months that belong to bulk milk-positive herds. Despite the detection of positives in both bulk milk and serum samples, South Tyrol has been declared IBR-free, as these positives have never been confirmed through seroneutralization. Between 2014 and 2022, approximately 41,000 bulk milk (averaging 4300 samples/year) and 3229 serum samples were tested for BoAHV1. The aim of this study was to evaluate the post-eradication program for IBR with a particular focus on the potential cross-reactivity with BoAHV2; for this reason, serum samples were also tested for BoAHV2 antibodies. This study could be of great importance for those countries that submit herds to an IBR monitoring and eradication program; performing further analyses to confirm and explain false positive outcomes would increase the reliability of the obtained results.

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.