A serological survey of pathogens associated with the respiratory and digestive system in the Polish European bison (Bison bonasus) population in 2017-2022.

BACKGROUND: The European bison (Bison bonasus) is a near threatened species and requires health monitoring. The aim of the present study was to determine the prevalence of antibodies to pathogens known to cause respiratory and digestive illness in ruminants. RESULTS: In the studied 328 European bison, the highest seroprevalence was observed for Bovine herpesvirus-1 (BoHV-1) (50.27%), Bovine Coronavirus (BCoV) (26.36%), and Bluetongue Virus (BTV) (12.83%). For Mycoplasma bovis strains and Bovine Viral Diarrhea Virus (BVDV), positive results were rare. Interestingly, a higher prevalence of BTV antibodies was noted in the northeastern populations and older animals. CONCLUSIONS: Our findings indicate that the Polish European bison population appears to have considerable contact with BoHV-1; however, this does not appear to be of great significance, as clinical symptoms and post-mortem lesions are rarely noted in Polish European bison population. The high seroprevalence of BTV in the north-east of Poland is an ongoing trend, also noted in previous studies. It is possible that European bison may perpetuate the virus in this region. This is the first report of antibodies for BCoV in European bison.

Establishment and applications of a duplex quantitative real-time RT-PCR assay for simultaneous detection of bovine rhinitis A virus and bovine rhinitis B virus

Bovine rhinitis virus (BRV) is an important pathogen responsible for the bovine respiratory disease complex (BRDC) and can be divided into two genotypes (BRAV and BRBV). To establish a duplex quantitative real-time RT-PCR assay for simultaneous detection of BRAV and BRBV, specific primers and TaqMan probes targeting the 5'NTR of BRAV and 3'NTR of BRBV were designed. A duplex quantitative real- time RT- PCR assay for simultaneous detecting BRAV and BRBV was preliminarily established by optimizing reaction conditions for each step. The assay specifically detects BRAV and BRBV, and no crossreaction with other common bovine respiratory pathogens, including IDV, BCoV, BVDV-1, BRSV, BPIV-3, BAdV-3, mycoplasma bovis, Pasteurella multocida, Mannheimia haemolytica, Escherichia coli, and Salmonella, was observed. In addition, the sensitivity test showed that the detection limits of this assay were 3.2x101 copies/L for both BRAV and BRBV plasmid standards. Besides, the repeatability test showed that the variation coefficients of this assay were less than 0.05 from both lot-to-lot and intra-lot. These results showed that the assay has high specificity, extreme sensitivity, and good repeatability. Moreover, a total of 43 nasal swabs of BRDC cattle were tested by our assay and four other quantitative real-time RT-PCR assays, including 3 BRAV assays and 4 BRBV assays. The results showed that the detection rates of our assay were 32.56%(14/43) for BRAV and 30.23%(13/43) for BRBV, and the detection rates of other quantitative real-time RT-PCR assays were 0(0/43), 2.33%(1/43), 23.26%(10/43) for BRAV and 27.91% (12/43), 27.91%(12/43), 27.91%(12/43), 27.91%(12/43) for BRBV, indicating that our assay has a more substantial detection capability than other assays. This study firstly established a duplex quantitative real-time RT-PCR assay for simultaneous detection of BRAV and BRBV, and the assay exhibited high specificity, sensitivity, and stability. Moreover, the study firstly confirmed the existence of BRAV in China, contributing to the prevention and control of BRDC.

Template Entrance Channel as Possible Allosteric Inhibition and Resistance Site for Quinolines Tricyclic Derivatives in RNA Dependent RNA Polymerase of Bovine Viral Diarrhea Virus.

The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds' most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals.

Epidemiological investigation of bovine viral diarrhea virus infection in swinery

From 2017 to 2020, 1 078 piglet diarrhea samples were collected from 6 pig farms in different districts of Shanghai. Multiple RT-PCR method was used for detection and analysis to study the infection status of bovine viral diarrhea virus (BVDV) in swinery in Shanghai. The results showed that the overall detection rate of BVDV in swinery in Shanghai was 7.14% (77/1 078), and showed an increasing trend year by year. The mixed infection rate of BVDV and other diarrhea pathogens was high, with the highest dual infection rate (65%, 26/40), mainly BVDV/PASTV (61.54%, 16/26). On this basis, the triple infection rate was 25% (10/40), mainly BVDV/PAStV/PKoV (40%, 4/10) infection mode;The quadruple infection rate was 10% (4/40), which was dominated by BVDV/PAStV/PEDV/PSV (50%, 2/4) infection. The BVDV prevalence in swinery was seasonal, and the prevalence in spring (10.36%) and autumn (13.59%) was higher than that in summer (6.8%) and winter (2.66%). The positive rate of BVDV in different pig farms was significantly different by 0-24.07%. In view of the detection rate of diarrhea virus dominated by PEDV in pig farm 2 had been high in recent years, this study further monitored the infection of BVDV in this pig farm, and found that the detection rate of BVDV in this pig farm was increasing year by year from 2017 to 2019, with the highest detection rate in 2019 (8.61%, 42/488);The mixed infection of BVDV and other diarrhea pathogens was also serious, with the dual infection rate of 57.58% (19/32), triple infection rate of 21.21% (7/32), quadruple infection rate of 21.21% (7/32), respectively. This study enriched the epidemic data of BVDV in swinery in Shanghai, and could provide reference for the prevention and control of pig epidemics.

Antiviral Potential of the Genus Panax: An updated review on their effects and underlying mechanism of action.

Viral infections are known as one of the major factors causing death. Ginseng is a medicinal plant that demonstrated a wide range of antiviral potential, and saponins are the major bioactive ingredients in the genus Panax with vast therapeutic potential. Studies focusing on the antiviral activity of the genus Panax plant-derived agents (extracts and saponins) and their mechanisms were identified and summarized, including contributions mainly from January 2016 until January 2022. P. ginseng, P. notoginseng, and P. quinquefolius were included in the review as valuable medicinal herbs against infections with 14 types of viruses. Reports from 9 extracts and 12 bioactive saponins were included, with 6 types of protopanaxadiol (PPD) ginsenosides and 6 types of protopanaxatriol (PPT) ginsenosides. The mechanisms mainly involved the inhibition of viral attachment and replication, the modulation of immune response by regulating signaling pathways, including the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway, phosphoinositide-dependent kinase-1 (PDK1)/ protein kinase B (Akt) signaling pathway, c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. This review includes detailed information about the mentioned antiviral effects of the genus Panax extracts and saponins in vitro and in vivo, and in human clinical trials, which provides a scientific basis for ginseng as an adjunctive therapeutic drug or nutraceutical.

Viral RNA extraction using an automatic nucleic acid extractor with magnetic particles and genetic characterization of bovine viral diarrhea virus in Tokachi Province, Japan, in 2016-2017.

In this study, the viral genome extraction performance of automatic nucleic acid extractors and manual nucleic acid extraction kits was compared. We showed that compared with manual kits, the automatic extractors showed superior genome extraction performance using bovine viral diarrhea virus (BVDV) genome-positive cattle sera and bovine coronavirus/infectious bovine rhinotracheitis virus-spiked cattle nasal swabs. In addition, the subgenotyping of BVDV strains detected in Tokachi Province in Japan during 2016-2017 was performed. Results showed that most of these BVDV strains belonged to subgenotype 1b, while few strains belonged to subgenotypes 1a and 2a. This study showed the high applicability of automatic nucleic acid extractors in extracting multiple viral genomes and the dominant subgenotype of BVDV in Tokachi.

A phylogenetic perspective of antiviral species of the genus Artemisia (Asteraceae-Anthemideae): A proposal of anti SARS-CoV-2 (COVID-19) candidate taxa.

Introduction: Different classes of disease-causing viruses are widely distributed universally. Plant-based medicines are anticipated to be effective cures for viral diseases including the COVID-19, instigated by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). This study displays the phylogenetic perspective of Artemisia and proposes some candidate taxa against different viral diseases, including SARS-CoV-2. Methods: Data of Artemisia with antiviral activity were obtained from different published sources and electronic searches. A phylogenetic analysis of the nrDNA ITS sequences of reported antiviral Artemisia species, along with the reference species retrieved from the NCBI GenBank database, was performed using the maximum likelihood (ML) approach. Results: In total, 23 Artemisia species have been documented so far with antiviral activity for 17 different types of viral diseases. 17 out of 23 antiviral Artemisia species were included in the ITS phylogeny, which presented the distribution of these antiviral Artemisia species in clades corresponding to different subgenera of the genus Artemisia. In the resultant ML tree, 10 antiviral Artemisia species appeared within the subgenus Artemisia clade, 2 species appeared within the subgenus Absinthium clade, 3 species appeared within the subgenus Dracunculus clade, and 2 species appeared within the subgenus Seriphidium clade. Discussion: Artemisia species from different subgenera with antiviral activity are prevalent in the genus, with most antiviral species belonging to the subgenus Artemisia. A detailed analysis of taxa from all subgenera, particularly the subgenus Artemisia, is therefore proposed in order to discover compounds with potential anti-SARS-CoV-2 activity.

Anti diarrheal effect of a commercialized mixed herbal medicine and in conjunction with probiotics in diarrheic water buffalo (Bubalus bubalis) calves

The study assessed the efficacy of a commercialized mixed herbal medicine in alleviating diarrhea in water buffalo (Bubalus bubalis) calves. The study involved 15 diarrheic water buffalo calves regardless of sex and with less than a year old from one farm divided into three treatments using randomized block design. Treatment 1 was served as control given with antibiotics and intestinal protectants.;Treatment 2 was mixed herbal medicine and probiotics and lastly, Treatment 3 was mixed herbal medicine only. The calves were treated three times a day for seven days for Treatments 2 and 3 while Treatment 1 (control) were treated once a day for 7 days. The animals were ob served and scoring of diarrhea were done and recorded daily for the next 7 days. Results of the study showed significant decrease in diarrhea scores on Day 6 and 7 post-treatment in Treatments 1 and 2 compared to the control. At Day 8 post-treatment, all calves showed soft to apparently normal stool. Genetic analysis of the possible causative agent of diarrhea revealed infection caused by rotavirus A, bovine coronavirus, BVDV, and ETEC. Results revealed that diarrhea caused by these pathogens can be alleviated by the herbal medicine and herbal medicine in addition of probiotics parallel to antibiotic treatment.

Efficacy of oleandrin and PBI-05204 against bovine viruses of importance to commercial cattle health.

BACKGROUND: Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV). and bovine coronavirus (BCV) threaten the productivity of cattle worldwide. Development of therapeutics that can control the spread of these viruses is an unmet need. The present research was designed to explore the in vitro antiviral activity of the Nerium oleander derived cardiac glycoside oleandrin and a defined N. oleander plant extract (PBI-05204) containing oleandrin. METHODS: Madin Darby Bovine Kidney (MDBK) cells, Bovine Turbinate (BT) cells, and Human Rectal Tumor-18 (HRT-18) cells were used as in vitro culture systems for BVDV, BRSV and BCV, respectively. Cytotoxicity was established using serial dilutions of oleandrin or PBI-05204. Noncytotoxic concentrations of each drug were used either prior to or at 12 h and 24 h following virus exposure to corresponding viruses. Infectious virus titers were determined following each treatment. RESULTS: Both oleandrin as well as PBI-05204 demonstrated strong antiviral activity against BVDV, BRSV, and BCV, in a dose-dependent manner, when added prior to or following infection of host cells. Determination of viral loads by PCR demonstrated a concentration dependent decline in virus replication. Importantly, the relative ability of virus produced from treated cultures to infect new host cells was reduced by as much as 10,000-fold at noncytotoxic concentrations of oleandrin or PBI-05204. CONCLUSIONS: The research demonstrates the potency of oleandrin and PBI-05204 to inhibit infectivity of three important enveloped bovine viruses in vitro. These data showing non-toxic concentrations of oleandrin inhibiting infectivity of three bovine viruses support further investigation of in vivo antiviral efficacy.

Development and Application of an Insulated Isothermal RT-PCR for On-site Detecting Nebovirus

Nebovirus (NeV) is an emerge diarrhea-causing virus in calves, the aim of this study is to establish an insulated isothermal RT-PCR(iiRT-PCR) for detecting NeV on field. Based on the RdRp sequences of NeV in GenBank database, a pair of primers and a fluorescent TaqMan probe were designed and synthesized. After optimizing the react system and condition, the iiRT-PCR method for detection of NeV was established. The iiRT-PCR assay could amplify specific fragment of NeV, without amplification of irrelevant pathogens, including bovine coronavirus, bovine norovirus, bovine rotavirus, bovine viral diarrhea virus, bovine torovirus, bovine Cryptosporidium parvum, bovine Eimeria. The intra- and inter-coefficients of variation were 3.07%-3.12% and 2.45%-3.01%, respectively, and the detection limit of viral nucleic acid of the assay was 5.38 copies•μL-1. One hundred and one calf diarrhea samples, collected from Hongyuan County, Ruoergai Prefecture, Xichang City, Liangshan Yi Autonomous Prefecture and Langzhong City in Sichuan Province during 2020-2021, were used to detect NeV, and 64.36% samples were detected as NeV positive. The study established an iiRT-PCR method for NeV detection with good specificity and reproducible as well as high sensitivity. Moreover, combined with the premixed detection reagent and PetNAD nucleic acid extraction kit, this assay could be used to NeV detect on-site, and only 1 hour from nucleic acid extraction to result report, which contribute to the fast detection for NeV.

ANTIVIRAL EFFECT OF CERIUM DIOXIDE NANOPARTICLES ON THE MODEL OF THE CAUSATIVE AGENT OF BOVINE VIRAL DIARRHEA

Cerium dioxide nanoparticles (CeO2) have a wide range of biological properties, including antiviral activity. Preparations based on cerium oxide nanoparticles can be effective against animal RNA viruses, which have the greatest epizootic significance for Ukraine and potentially against human viruses, including SARS-CoV-2. In our studies, we determined the effect of cerium dioxide nanoparticles on bovine viral diarrhea virus (BVDV), a pestivirus that is the etiological agent of bovine viral diarrhea. To determine the antiviral efficacy of CeO2, a cytopathogenic strain of BVDV “BK-1” was used as an etiological agent of bovine viral diarrhea, which induces a cytopathic effect on cow embryo lungs (CEL) cell culture. When evaluating the antiviral efficacy of CeO2, it was determined that the MPC for CEL cell culture is 0.057 μg/cm3, and the CC50 is 0.077 μg/cm3. The antiviral activity of CeO2 was evaluated by the IC50 index, which was 0.036 μg/cm3 and the selectivity index, which after co-administration of CeO2 with BVDV was 2.14. There was a decrease in the titer of infectious activity of the virus during treatment with CeO2 by 2.09 lg TCD50/cm3. © 2021, Zabolotny Institute of Microbiology and Virology, NAS of Ukraine. All rights reserved.

Inactivation of Enveloped Bovine Viral Diarrhea Virus and Non-Enveloped Porcine Parvovirus Using Low-Pressure Non-Thermal Plasma.

As the worldwide population has been experiencing since 2020, viruses represent a serious threat to global well-being. To avoid viral transmission through surgery or medical examination, sterilization of medical material is needed. From emerging sterilization processes, the use of non-thermal plasma (NTP) arises as a promising technique to efficiently reduce microbial burden on medical devices, including new complex polymers as thermosensitive ones. Thus, we evaluated the antiviral efficacy of a low-pressure NTP process taking place in a sealed bag. For this purpose, two different plasmas, O2 100% plasma and Ar 80%-O2 20% plasma, were tested against two viruses: the bovine viral diarrhea virus and the porcine parvovirus, surrogates of human hepatitis C virus and human parvovirus B19, respectively. The efficacy of both NTP treatments on viral load can be detected after only five minutes. Moreover, the longer the NTP treatments last, the more the load decreases. The most effective load reduction was obtained with a 120-min O2 plasma treatment inducing a minimum of four-log viral load reduction. So, this process demonstrated strong virucidal capacity inside a sealed bag and represents a very interesting opportunity in the field of fragile medical devices sterilization or disinfection.

Surfactants - Compounds for inactivation of SARS-CoV-2 and other enveloped viruses.

We provide here a general view on the interactions of surfactants with viruses, with a particular emphasis on how such interactions can be controlled and employed for inhibiting the infectivity of enveloped viruses, including coronaviruses. The aim is to provide to interested scientists from different fields, including chemistry, physics, biochemistry, and medicine, an overview of the basic properties of surfactants and (corona)viruses, which are relevant to understanding the interactions between the two. Various types of interactions between surfactant and virus are important, and they act on different components of a virus such as the lipid envelope, membrane (envelope) proteins and nucleocapsid proteins. Accordingly, this cannot be a detailed account of all relevant aspects but instead a summary that bridges between the different disciplines. We describe concepts and cover a selection of the relevant literature as an incentive for diving deeper into the relevant material. Our focus is on more recent developments around the COVID-19 pandemic caused by SARS-CoV-2, applications of surfactants against the virus, and on the potential future use of surfactants for pandemic relief. We also cover the most important aspects of the historical development of using surfactants in combatting virus infections. We conclude that surfactants are already playing very important roles in various directions of defence against viruses, either directly, as in disinfection, or as carrier components of drug delivery systems for prophylaxis or treatment. By designing tailor-made surfactants, and consequently, advanced formulations, one can expect more and more effective use of surfactants, either directly as antiviral compounds or as part of more complex formulations.

Ivermectin also inhibits the replication of bovine respiratory viruses (BRSV, BPIV-3, BoHV-1, BCoV and BVDV) in vitro.

Bovine respiratory disease (BRD) complex is an important viral infection that causes huge economic losses in cattle herds worldwide. However, there is no directly effective antiviral drug application against respiratory viral pathogens; generally, the metaphylactic antibacterial drug applications are used for BRD. Ivermectin (IVM) is currently used as a broad-spectrum anti-parasitic agent both for veterinary and human medicine on some occasions. Moreover, since it is identified as an inhibitor for importin α/ß-mediated nuclear localization signal (NLS), IVM is also reported to have antiviral potential against several RNA and DNA viruses. Since therapeutic use of IVM in COVID-19 cases has recently been postulated, the potential antiviral activity of IVM against bovine respiratory viruses including BRSV, BPIV-3, BoHV-1, BCoV and BVDV are evaluated in this study. For these purposes, virus titration assay was used to evaluate titers in viral harvest from infected cells treated with non-cytotoxic IVM concentrations (1, 2.5 and 5 µM) and compared to titers from non-treated infected cells. This study indicated that IVM inhibits the replication of BCoV, BVDV, BRSV, BPIV-3 and BoHV-1 in a dose-dependent manner in vitro as well as number of extracellular infectious virions. In addition, it was demonstrated that IVM has no clear effect on the attachment and penetration steps of the replication of the studied viruses. Finally, this study shows for the first time that IVM can inhibit infection of BRD-related viral agents namely BCoV, BPIV-3, BVDV, BRSV and BoHV-1 at the concentrations of 2.5 and 5 µM. Consequently, IVM, which is licensed for antiparasitic indications, also deserves to be evaluated as a broad-spectrum antiviral in BRD cases caused by viral pathogens.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

Characterization of bovine ileal epithelial cell line for lectin binding, susceptibility to enteric pathogens, and TLR mediated immune responses.

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Sequential exposure to bovine viral diarrhea virus and bovine coronavirus results in increased respiratory disease lesions: clinical, immunologic, pathologic, and immunohistochemical findings.

Bovine coronaviruses (BoCVs) have been found in respiratory tissues in cattle and frequently associated with bovine respiratory disease (BRD); however, pathogenesis studies in calves are limited. To characterize the pathogenesis and pathogenicity of BoCV isolates, we used 5 different BoCV strains to inoculate colostrum-deprived calves, ~ 2-5 wk of age. Later, to determine if dual viral infection would potentiate pathogenicity of BoCV, calves were inoculated with BoCV alone, bovine viral diarrhea virus (BVDV) alone, or a series of dual-infection (BVDV-BoCV) schemes. A negative control group was included in all studies. Clinical signs and body temperature were monitored during the study and samples collected for lymphocyte counts, virus isolation, and serology. During autopsy, gross lesions were recorded and fixed tissues collected for histopathology and immunohistochemistry; fresh tissues were collected for virus isolation. Results suggest increased pathogenicity for isolate BoCV OK 1776. Increased body temperature was found in all virus-inoculated groups. Lung lesions were present in calves in all dual-infection groups; however, lesions were most pronounced in calves inoculated with BVDV followed by BoCV inoculation 6 d later. Lung lesions were consistent with mild-to-moderate interstitial pneumonia, and immunohistochemistry confirmed the presence of BoCV antigen. Our studies demonstrated that BVDV-BoCV dual infection may play an important role in BRD pathogenesis, and timing between infections seems critical to the severity of lesions.

Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay.

BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.