Natural Bovine Coronavirus Infection in a Calf Persistently Infected with Bovine Viral Diarrhea Virus: Viral Shedding, Immunological Features and S Gene Variations

The evolution of a bovine coronavirus (BCoV) natural infection in a calf persistently infected with bovine viral diarrhea virus (BVDV) was described. The infected calf developed intermittent nasal discharge, diarrhea and hyperthermia. The total number of leukocytes/mL and the absolute differential number of neutrophils and lymphocytes resulted within the normal range, but monocytes increased at T28 (time 28 post-infection). Flow-cytometry analysis evidenced that the CD8+ subpopulation increased at T7 and between T28 and T35. BCoV shedding in nasal discharges and feces was detected up to three weeks post infection and high antibody titers persisted up to T56. The RNA BCoV load increased until T14, contrary to what was observed in a previous study where the fecal excretion of BCoV was significantly lower in the co-infected (BCoV/BVDV) calves than in the calves infected with BCoV only. We can suppose that BVDV may have modulated the BCoV infection exacerbating the long viral excretion, as well as favoring the onset of mutations in the genome of BCoV detected in fecal samples at T21. An extensive study was performed to verify if the selective pressure in the S gene could be a natural mode of variation of BCoV, providing data for the identification of new epidemic strains, genotypes or recombinant betacoronaviruses.

Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

COVID-19 patients shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. In our benchmarking study, we recommend a standardized protocol for the preservation, extraction and detection of viral RNA from stool. This protocol includes a preservative, viral RNA extraction steps, and PCR-based quantification methods to maximize yield and detection of SARS-CoV-2 RNA. Our protocol takes advantage of commercially available reagents and equipment to maximize ease of access and consistency across studies. Additionally, we apply an attenuated bovine coronavirus vaccine as a spike-in control, and synthetic RNA standards to improve standardization and reliability of the assay. While we recommend both ddPCR and RT-qPCR-based assays, we acknowledge that ddPCR may be prohibitively expensive due to the necessity of specialized equipment and reagents. This protocol was developed with a focus on SARS-CoV-2 RNA, but may apply to other coronaviruses as well. We estimate that this protocol takes between 6 to 8 hours total to quantify the viral RNA load in a fecal sample.

Next-Generation Sequencing Reveals Four Novel Viruses Associated with Calf Diarrhea

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus; the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Novel Preparation Technique of Hyperimmune Globulins against Bovine Coronavirus as Surrogate of Beta Coronavirus

Consuming time and effort to prepare hyperimmune globulins using Freund’s adjuvant is a sophisticated and harsh technique. In this work, a novel, safe, and rabid method was proposed using monolaurin as an immune-stimulating agent in hyperimmune globulins production against Bovine coronavirus (BCoV). The mentioned virus was used as a surrogate to family Betacoronavirus. Bovine coronavirus (Mabus strain) with titer log10 5.8 tissue culture infective dose infectivity (TCID50)/ml was used in this experiment. The inactivation of the virus was done using 1% ascorbic acid for 24h. Monolaurin emulsion (10% w/v) of was prepared by sonication using tween 20 and water. The inactivated bovine coronavirus was added to the emulsion by 20% of the final volume. The immunoglobulins were prepared by inoculating the inactivated virus with the adjuvant in rabbits and evaluated on the Madin-Darby bovine kidney (MDBK) cell line by virus neutralization test (VNT). The effect of the adjuvant was assessed by histopathological examination of vital organs such as the kidney and liver. The antibody titer against the BCoV was reached its peak, log2 1024 TCID50/ml, at the 3rd-week post-inoculation in the rabbits. The level of the globulin reached a high level and its peak (14.3g/dL) at the end of the experiment. No abnormalities were seen in the livers and kidneys of the negative control group of rabbits. Monolaurin showed a new level of safety and efficacy when used as an adjuvant during the preparation of the immunoglobulins against BCoV.

Heat efficiently inactivates coronaviruses inside vehicles

Heat is an established method to inactivate coronaviruses, and there is utility in using heat to reduce viral load on common touch points in vehicles exposed to a person shedding SARS-CoV-2. As SARS-CoV-2 is a Biosafety level (BSL)-3 pathogen, real world testing of heat as a sanitation method for public and private vehicles becomes a challenge, requiring a surrogate coronavirus that can be handled safely outside of a BSL-3 facility. In this study, we used Bovine Coronavirus (BCoV) as a surrogate for SARS-CoV-2 to test the efficacy of heat-based betacoronavirus inactivation. In vitro, a 30-minute exposure to 56°C completely inactivated BCoV in solution, and a 15-minute exposure reduced recovery of BCoV >1000-fold. When heated to 56°C for 15 minutes, the infectivity of BCoV spotted and dried on typical porous and non-porous automobile interior materials was reduced by 99 - 99.99%. When BCoV was spotted and dried on hard plastic (seat) material placed inside an out of service transit bus, 56°C heat for 30 minutes reduced BCoV infectivity 85 - 99.5%. Thus, 56°C is an accessible, rapid, and effective method to inactivate coronaviruses inside motor vehicles.

Isolation of bovine coronavirus (BCov) in cell cultures

One of the most common viruses in the world that causes disease in cattle is the bovine coronavirus (BCoV). This virus is the causative agent of respiratory and gastrointestinal infections in newborn calves, resulting in significant economic losses in both dairy and meat farming. Considering the complex epizootic situation with the coronaviruses in the world and partial antigenic affinity of BCoV with coronaviruses of other species of animals and humans, the isolation of new strains of coronaviruses, their identification and optimization of cultivation conditions becomes extremely important and relevant. The aim of our research was to determine the features of methods of isolation of bovine coronavirus and to select methods for its cultivation in cell culture in order to obtain the virus with the highest titers of infectious activity. Isolation of BCoV was performed in monolayers of MDBK and the primary-trypsinized calf kidney culture cells, using 20 samples collected from calves with clinical signs of respiratory or/and gastrointestinal disease. 16 samples were positive for BCoV by means of Real-Time PCR test. Up to fifth serial passage, only 4 of these isolates presented typical syncytial cytopathic effect. It has been experimentally established that the continious calf kidney cell culture line (MDBK) and the primary-trypsinized calf kidney culture (CK) are suitable for BCoV isolation and accumulation. The infectious titer of bovine coronavirus at the level of the fifth passage in the cultures of MDBK and CK cells reached 5.54 ± 0.16 lg TCD50/ml and 5.59 ± 0.14 lg TCD50/ml, respectively. However, due to the high cost of obtaining primary-trypsinized cell cultures, this isolation method may be unacceptable to most pharmaceutical companies and laboratories. Also after 5 serial passages, the viral material was again examined in Real-Time PCR to confirm the isolation of BCoV - the study of 4 samples with a characteristic syncytial CPE had a positive result in Real-Time PCR. However, of the Real-Time PCR-positive 12 samples, the virus could not be isolated in continuous cell cultures of MDBK and Vero, as well as in primary-trypsinized cattle lung and kidney cell cultures. This fact may indicate the presence of different strains of BCoV circulation in farms in our country. Further research is planned to be focused on optimizing the methods and modes of BCoV strains isolation, as well as to identify and study the cultural properties of new strains of BCoV circulating in Ukraine. We will also continue the study of the obtained viral isolate for the subsequent development of tools for the diagnosis and immunoprophylaxis of coronavirus infection in veterinary medicine.

Seroprevalence of bovine coronavirus and factors associated with the serological status in dairy cattle in the western region of Thailand

Background and Aim: Bovine coronavirus (BCoV) is a pathogen affecting the productivities of dairy cattle worldwide. The present study aimed to determine the seroprevalence and factors associated with BCoV serological status using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Materials and Methods: A cross-sectional study was conducted in the western region of Thailand. Blood samples were collected from 30 dairy herds. In total, 617 blood serum samples were tested using a commercial indirect ELISA for BCoV-specific immunoglobulin G antibodies. A questionnaire was used to collect data on the factors which have been identified as risk factors for BCoV antibody detection. The age and history of diarrhea of each animal were recorded. Fisher's exact test was performed to univariately assess the association between BCoV serological status and possible risk factors. Variables with Fisher's exact test p<0.10 were then evaluated using multivariate logistic regression to identify factors associated with BCoV serological status. The Bonferroni adjustment was used for multiple comparisons of significant variables in the final multivariate logistic regression model. Results: No herd was free from antibodies to BCoV. The individual seroprevalence of BCoV was 97.89% (604/617). The prevalence within herds was in the range of 45.45-100%. Cattle >3 years of age were more likely to be seropositive to BCoV compared to cattle <1 year of age (p=0.003), with the odds ratio being 81.96. Disinfecting diarrhea stools were a protective factor for being BCoV seropositive, with odds ratios of 0.08 and 0.06 compared to doing nothing (p=0.008) and to clean with water (p=0.002), respectively. Conclusion: BCoV seropositive dairy cattle were distributed throughout the western region of Thailand. The probability of being seropositive for BCoV increased with increasing animal age. Cleaning the contaminated stool with appropriate disinfectants should be recommended to farmers to minimize the spread of the virus.