Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein.

Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 µg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Development of a 1-step TaqMan real-time PCR method for detection of the Bovine Group A Rotavirus.

BACKGROUND: The purpose of this study was to develop a 1-step real-time quantitative fluorescence polymerase chain reaction (QF-PCR) method for detecting Bovine Group A Rotavirus (BRVA). The primers and probe were designed targeting the VP6 gene of BRVA. The standard substance was obtained through in vitro transcription. The primers, probe concentration, and annealing temperatures were optimized to determine the optimal system and conditions for the reaction. The specificity, sensitivity, and repeatability of the method were assessed and compared with a reported real-time QF-PCR method for clinical samples. RESULTS: The results indicated that the detection method can achieve a sensitivity of 3.47 copies/µL and exhibit good specificity by exclusively detecting BRVA without cross-reactivity to other common pathogens in cattle and sheep. The standard curve exhibited a robust linear correlation, and the amplification efficiency was calculated to be 105%. The intra-group and inter-group coefficients of variation were less than 2%. A total of 96 clinical samples were tested and compared with the real-time QF-PCR method that was reported. The coincidence rate was 90.63% (87/96). Furthermore, the clinical samples revealed that the prevalence of BRV in cattle from Fujian Province was 85.42% (82/96). CONCLUSION: This study has successfully developed a 1-step real-time QF-PCR method for BRVA, which offers an efficient and sensitive technical support for the rapid diagnosis and epidemiological investigation of BRVA.

Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus.

Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/µL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/µL, 1 copy/µL and 2.4 copies/µL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections.

Alterations of microbiota and metabolites in the feces of calves with diarrhea associated with rotavirus and coronavirus infections.

The changes in the composition of intestinal microbiota and metabolites have been linked to digestive disorders in calves, especially neonatal calf diarrhea. Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are known to be the primary culprits behind neonatal calf diarrhea. In this study, we analyzed changes in the fecal microbiota and metabolites of calves with neonatal diarrhea associated with BRV and BCoV infection using high-throughput 16S rRNA sequencing and metabolomics technology. The microbial diversity in the feces of calves infected with BRV and BCoV with diarrhea decreased significantly, and the composition changed significantly. The significant increase of Fusobacterium and the reductions of some bacteria genera, including Faecalibacterium, Bifidobacterium, Ruminococcus, Subdoligranulum, Parabacteroides, Collinsella, and Olsenella, etc., were closely related to diarrhea associated with BRV and BCoV infection. Metabolites in the feces of BRV and BCoV-infected calves with diarrhea were significantly changed. Phosphatidylcholine [PC; 16:1(9 Z)/16:1(9 Z)], lysophosphatidylethanolamine (LysoPE; 0:0/22:0), lysophosphatidylcholine (LysoPC; P-16:0) and LysoPE (0:0/18:0) were significantly higher in the feces of BRV-infected calves with diarrhea. In contrast, some others, such as desthiobiotin, were significantly lower. BRV infection affects glycerophospholipid metabolism and biotin metabolism in calves. Two differential metabolites were significantly increased, and 67 differential metabolites were significantly reduced in the feces of BCoV-infected calves with diarrhea. Seven significantly reduced metabolites, including deoxythymidylic acid (DTMP), dihydrobiopterin, dihydroneopterin triphosphate, cortexolone, cortisol, pantetheine, and pregnenolone sulfate, were enriched in the folate biosynthesis, pantothenate and CoA biosynthesis, pyrimidine metabolism, and steroid hormone biosynthesis pathway. The decrease in these metabolites was closely associated with increased harmful bacteria and reduced commensal bacteria. The content of short-chain fatty acids (SCFAs) such as acetic acid and propionic acid in the feces of BRV and BCoV-infected calves with diarrhea was lower than that of healthy calves, which was associated with the depletion of SCFAs-producing bacteria such as Parabacteroides, Fournierella, and Collinsella. The present study showed that BRV and BCoV infections changed the composition of the calf fecal microbiota and were associated with changes in fecal metabolites. This study lays the foundation for further revealing the roles of intestinal microbiota in neonatal calf diarrhea associated with BRV and BCoV infection.

Analysis of Fecal Microbial Changes in Young Calves Following Bovine Rotavirus Infection.

The objective of the present study was to identify changes in fecal microbiota and predict the functional features of healthy calves and those infected with rotavirus over time. Six Holstein calves (average body weight 43.63 ± 1.19 kg, age-matched within 5-7 d) were randomly selected and distributed into two groups which contained three calves each. Fecal samples were taken 3 days before inoculation and on days 1 and 7 post-inoculation. The 16S rRNA gene amplicon sequencing was performed. Bacterial diversity tended to decrease in the rota group, as indicated by the alpha (evenness, p = 0.074 and Shannon, p = 0.055) and beta (Bray-Curtis dissimilarity, p = 0.099) diversity at 1 day post-inoculation. Differences in the bacterial taxa between healthy and rota-infected calves were detected using a linear discriminant analysis effect size (LDA > 2.0, p < 0.05). Rota calves had a higher abundance of certain bacterial taxa, such as Enterococcus, Streptococcus, and Escherichia-Shigella, and a lower abundance of bacteria that contribute to the production of short-chain fatty acids, such as Alistipes, Faecalibacterium, Pseudoflavonifractor, Subdoligranulum, Alloprevotella, Butyricicoccus, and Ruminococcus, compared to the healthy calves. The observed changes in the fecal microbiota of the rota-infected group compared to the healthy group indicated potential dysbiosis. This was further supported by significant differences in the predicted functional metagenomic profiles of these microbial communities. We suggest that calves infected with bovine rotavirus had bacterial dysbiosis, which was characterized by lower diversity and fewer observed genera than the fecal microbiota of healthy calves.

Complete genome constellations of two bovine rotavirus A strains isolated in Japan reveal a unique T9 NSP3 genotype.

Full genome sequencing of two bovine rotavirus A (RVA) strains isolated in Japan in 2019 revealed two genotype constellations; one had a constellation of G8-P[1]-I2-R2-C2-M2-A3-N2-T9-E2-H3. Thereupon, genotype T9 carried by RVA/Bovine-tc/JPN/AH1041/2022/G8P[1], constitutes a rare NSP3 genotype, and only two unusual Japanese bovine RVA strains have thus far been reported to carry this genotype. The other RVA/Bovine-tc/JPN/AH1207/2022/G6P[5] strain possessed a constellation of G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Phylogenetic analyses indicate that the majority of gene segments were most closely related to Japanese bovine RVAs, suggesting that both strains might have derived through multiple reassortment events from RVA strains circulating within Japanese cattle. The emergence of RVA strains in Japan and their reassortment with locally circulating atypical RVAs could have implications for current vaccination strategies.

An end-point multiplex PCR/reverse transcription-PCR for detection of five agents of bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is frequently associated with single or mixed viral, bacterial and/or protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each potential agent; a time-consuming, laborious and expensive process. Herein, we describe an end-point multiplex PCR/reverse transcription-PCR (RT-PCR) for detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we optimized multiplex PCR/RT-PCR conditions. Next, we evaluated the analytical sensitivity of the assay and assessed the performance of the reaction by testing 95 samples of diarrheic calf feces. The analytical specificity was evaluated against bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our assay was about 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, about 5 × 10-4 CFU for S. enterica, 5 × 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No non-specific amplification of other bovine diarrhea agents was detected. Out of 95 samples analyzed, 50 were positive for at least one target, being 35 single and 15 mixed infections. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was the most frequent in mixed infections (11/15). Positive and negative multiplex results were confirmed in individual reactions. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster and easier NCD diagnosis, which may be useful for routine diagnosis and surveillance studies.

Genome characterization of a Turkish bovine rotavirus field isolate by shotgun metagenomics.

A bovine rotavirus (BRV) isolate from Kirsehir was isolated from feces of a neonatal calf with diarrhea, identified, and sequenced by shotgun sequencing. Its genotype constellation is G10-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The structural genes and the non-structural genes NSP1, NSP3, and NSP4 of the Kirsehir isolate were similar in sequence to those of BRVs identified in Turkey. However, VP2, NSP2, NSP4, and NSP5/6 showed similarity to those of rotaviruses from different animal hosts. These findings not only expand our current understanding of the diversity of rotaviruses but also contribute to our understanding of the evolution of rotaviruses at both the national and global levels and reinforce the significance of conducting further research on rotaviruses in Turkey.

Ammi-visnaga extract; a novel phyto-antiviral agent against bovine rotavirus.

The spread of bovine rotavirus has a great impact on animal productivity, milk products, and human public health. Thus, this study aimed to develop a novel, effective and accessible Phyto-antiviral treatment made from methanolic Ammi-visnaga seed extract against rotavirus infection. Rotaviruses were isolated from raw milk and cottage cheese samples randomly collected from Cairo and Qalubia governorates. They were all identified serologically, however, only three of them were both biologically and molecularly confirmed. The methanolic extract derived from Khella seeds (MKSE) was chemically analyzed with mass chromatography. The cellular toxicity of MKSE was tested on Caco-2 cells and its antiviral activity against one of the isolated bovine rotaviruses (BRVM1) was tested by both the cytopathic inhibition assay and the plaque reduction assay. Our results showed that 17.3% of the total collected 150 dairy samples were bovine rotavirus antigen positive. Three representatives of them were phylogenetically identified to be included in group A based on a 379 bp coat protein gene. Visnagin, Benzopyran, Khellin, and Benzenepropanoic acid were the major active components found in the MKSE. The maximum non-toxic concentration of MKSE was 5 µg/mL and the CC50 value was 417 µg/mL. The MKSE exhibited in-vitro antiviral activity against BRVM1 indicated by inhibition of the viral cytopathic effect (SI = 204.5, IP = 98%), causing a 1.5 log decrease in BVRM1 TCID50 and reducing the viral plaques count by the percentage of 93.14% at MNTC (5 ug/ml). In conclusion, our study showed that bovine rotavirus represents a severe health problem that needs attention in Egypt, and it supports using MKSE as a potential natural anti-rotavirus agent.

Comparison of antigenicity between recently isolated bovine rotaviruses A and vaccine strains by cross-neutralizing antibody tests and their significance for immunization.

Three bovine rotaviruses A (RVAs) isolated from a cattle farm in Japan were serotyped by serum neutralization assay, as compared with the RVA strains contained in a vaccine used on the same farm. Antisera were prepared against the three isolates and the vaccine strains of bovine RVA. The results of cross-neutralization tests revealed that the RVA isolates from this farm differed somewhat in serotype. Collected plasma from calves for 6 weeks after colostrum ingestion showed that maternal antibodies acquired against all isolates gradually decreased, but antibodies toward one isolate increased by 6 weeks after the mentioned decreasing. These results suggest that rotavirus vaccines administered to cows should include all serotypes commonly found in calves with diarrhea.

Characterization of bovine rotavirus isolates from diarrheic calves in Türkiye.

BACKGROUND: Neonatal calf diarrhea, which is the most common cause in calf deaths, leads to significant economic losses in dairy farming around the world. Diarrhea develops due to infectious and non-infectious reasons. Group A Rotaviruses (RVA) are the leading and predisposing factor for acute neonatal gastroenteritis. METHODS AND RESULTS: In this study, 20 diarrheic fecal samples were collected from one farm in Balikesir province of Turkey. During virus isolation, a total of 2 stool samples were detected to produce cytopathogenic effects in MA-104 cell line. The two samples (RV-36, RV-38) were tested positive with antigen ELISA kits detecting RVA antigens. In order to detect the presence of rotavirus viral nucleic acid in cell supernatants, VP6 gene region-specific RT-PCR test was performed and the samples RV-36 and RV-38 were positive for RVA viral nucleic acid. By RT-PCR using genotype specific primers, both the isolates RV-36 and RV-38 formed amplicons compatible with G10 and P[11] genotypes of RVA. RVA nucleic acids segments were also visualized by poliacrilamide gel electrophoresis (PAGE) method. The phylogenetic tree constructed according to the VP6 gene region showed that these isolates were in the Rotavirus A group and in the I2 cluster same as other bovine and some human RVA isolates. CONCLUSION: Succesful isolation of RVA G10P[11] was echieved in the cattle farm. As rotaviruses play the most important role in the etiology of diarrhea in newborn calves respected genotype G10P[11] should be considered in selection of the vaccines applied to the dams. Those isolates can be further evaluated as vaccine candidate.

A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses.

Introduction: Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high. Methods: To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes. Results: This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/µL, 96.0 copies/µL, 12.8 copies/µL, 16.4 copies/µL, 18.2 copies/µL, and 65.3 copies/µL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence. Discussion: Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

Efficacy of prepartum vaccination against neonatal calf diarrhea in Nelore dams as a prevention measure.

BACKGROUND: Neonatal calf diarrhea (NCD) is the leading cause of calf morbidity and mortality in beef cattle. Cow's vaccination in last stage of pregnancy is one of the most important measures to mitigate the risk of NCD outbreaks. The aim of this study was to evaluate the efficacy of prepartum single dose vaccination against NCD, especially Bovine Rotavirus type A (BoRVA) and Bovine Coronavirus (BCoV), in Nelore dams and offspring. A total of 117 pregnant cows (n = 81) and heifers (n = 36) were distributed in two groups, vaccinated (VAC: cows = 40; heifers = 19) and non-vaccinated (NVAC: cows = 41; heifers = 17). Vaccination occurred between 60 to 50 days before the expected calving date with a single dose of a water-in-oil (W/O) vaccine, and NVAC group received a dose of saline solution 0.9%. Blood samples were collected before vaccination and 30 days after to evaluate the antibody (Ab) response. Specific IgG1 Abs against BoRVA and BCoV were measured by using an Enzyme Linked Immuno Sorbent Assay (ELISA). Calves' births were monitored, and the transference of passive immunity was evaluated. Diarrhea was monitored in the first 30 days of age, and fecal samples were collected for identification of the etiological agent. RESULTS: Higher titers of IgG1 Ab against BoRVA and BCoV was observed in the VAC group than NVAC group in the cow (P < 0.0001) and total dams categories (P < 0.0001). The titer of specific IgG1 Abs in the calves' serum reflected the dams response, observing higher IgG1 Ab titers for BoRVA (P < 0.0016) and BCoV (P < 0.0095) in the offspring born to VAC cows and higher IgG1 Ab titers for BoRVA(P < 0.0171) and BCoV (P < 0.0200) in the offspring born to VAC total dams. The general incidence of diarrhea observed was 18.6% (11/59) and 29.3% (17/58) in the calves born to the VAC and NVAC group, respectively. CONCLUSIONS: Prepartum vaccination with a single dose of the vaccine tested increased the titers of IgG1 Ab against BCoV and BoRVA, and it could be used as a preventive strategy to decrease the NCD occurrence in Nelore calves.

Prevalence of bovine rotavirus among cattle in mainland China: A meta-analysis.

BACKGROUND: Bovine rotavirus is the primary pathogen causing diarrhea in cattle and can be transmitted vertically through the placenta. It mainly presents with clinical signs such as depression, loss of appetite, diarrhea, vomiting, and dehydration. METHODS: A systematic review and meta-analysis were conducted to assess the prevalence of BRV infection in mainland China. We conducted a literature search on the prevalence of BRV infection in pigs between Jan 1, 1979 and Dec 31, 2021 in English and Chinese databases, including PubMed, Google Scholar, Cochrane library, Clinical Trials, VIP, CNKI, and WanFang database. Selections were made based on the title and the abstract of the paper, Search strings included if they reported the cattle samples of more than 15 cattle and provided information that allowed us to establish the prevalence of BRV. Moreover, we excluded repeated studies, reviews, other hosts. Finally, we extracted the number of cattle with BRV infection from the obtained studies and provided information that permitted us to estimate the prevalence of BRV infection in cattle in mainland China. RESULTS: The data of 29 articles (including data on 10677 cattle) are compliant with the standards. The pooled prevalence of BRV in cattle in China was 46%（6635/10677）, the pooled prevalence of BRV in cattle from Northeast China (40%) was significantly lower than those from other regions. In addition, the prevalence of BRV was associated with publication time of paper, detection methods, age of cattle, and clinical symptoms（diarrhea, etc.). CONCLUSION: Our findings suggest that BRV infection is common among cattle in China. It is, therefore, necessary to carry out further research and monitor the prevalence of BRV infection. Furthermore, powerful and effective regulatory measures should be taken to prevent the transmission and spread of BRV among cattle populations.

Prevalence of bovine rotavirus among Bovidae in China during 1984-2021: A systematic review and meta-analysis.

Bovine rotavirus (BRV) is a potential zoonotic intestinal pathogen that brings a serious threat to calf health, and has resulted in huge economic losses to China's breeding industry. Here, a systematic review and meta-analysis was conducted to estimate the prevalence of BRV among Bovidae from 1984 to 2021 in China. A total of 64 publications on BRV investigation in China were screened from the databases Chinese National Knowledge Infrastructure (CNKI), Wan Fang Database, Technology Periodical Database (VIP), PubMed, and ScienceDirect. The random-effect model was used to calculate the pooled prevalence of BRV, and the analyzed data were derived from 25 provinces in China. The estimated pooled prevalence of BRV in China was 35.7% (8176/17,292). In addition, the prevalence of BRV in Southwestern China (77.1%; 2924/3600) was significantly higher than that in other regions of China. Regarding geographic and climatic factors, the prevalence of BRV in the subgroup of latitude 30-35° (76.8%; 3303/4659) was significantly higher than that in the subgroup of latitude less than 30° (37.0%; 485/1275) or more than 35° (32.6%; 1703/5722), while the prevalence of BRV in the subgroup of longitude 100-105° (75.4%; 2513/3849) was significantly higher than that in the subgroup of longitude less than 100° (32.6%; 619/2255) or more than 105° (48.9%; 2359/5552). Rainfall was positively correlated with the prevalence of BRV, whereas temperature was negatively correlated with the positive rate of BRV (P < 0.05). Our data showed that the prevalence of BRV was strongly correlated with geographical and climatic conditions. Thus, we recommend that the corresponding prevention and control programs should be formulated according to different geographical conditions. The strengthening of BRV surveillance in areas with high altitude, low temperature, and heavy rainfall may contribute to the decrease of the incidence of BRV infection among Bovidae herds in China.

Affinity Peptide-based Electrochemical Biosensor for the Highly Sensitive Detection of Bovine Rotavirus.

Bovine diarrhea is a major concern in the global bovine industry because it can cause significant financial damage. Of the many potential infectious agents that can lead to bovine diarrhea, bovine rotavirus (BRV) is a particular problem due to its high transmissibility and infectivity. Therefore, it is important to prevent the proliferation of BRV using an early detection system. This study developed an affinity peptide-based electrochemical method for use as a rapid detection system for BRV. A BRV-specific peptide was identified via the phage display technique and chemically synthesized. The synthetic peptide was immobilized on a gold electrode through thiol-gold interactions. The performance of the BRV specific binding peptides was evaluated using square wave voltammetry. The developed detection system exhibited a low detection limit (5 copies/mL) and limit of quantitation (2.14 × 102 copies/mL), indicating that it is a promising sensor platform for the monitoring of BRV.

G and P genotype profiles of rotavirus a field strains circulating in a vaccinated bovine farm as parameters for assessing biosecurity level.

After improvement of hygiene protocols on boots in a bovine operation (farm A) in Ibaraki, Japan in September 2017, mortality of calves and the detection of 4 viral pathogen indicators, including bovine rotavirus A (RVA), became significantly low for one year. Subsequently, in the present study, these indicators and mortality were monitored and confirmed all were still low, except for the detection rate of bovine RVA in calves less than 3 weeks old. The present study aimed to investigate G and P genotypic profiles of RVAs in farm A from 2018 to 2020. Molecular analysis using semi-nested multiplex RT-PCR of positive RVAs (n=122) and sequencing of selected samples revealed the presence of G6, G8, G10, P[1], P[5] and P[11] genotypes and the prevalence of G and/or P combination and mixed infections. The most common combination of G and P types was G10P[11] (41.8%), followed by mixed infection with G6+G10P[5] (11.5%). Phylogenetic analysis of RVAs showed clustering with bovine and other animal-derived RVA strains, suggesting the possibility of multiple reassortant events with strains of bovine and others animal origins. Noteworthy as well is that vaccinated cattle might fail to provide their offspring with maternal immunity against RVA infections, due to insufficient colostrum feeding. Our findings further highlight the importance of RVA surveillance in bovine populations, which may be useful to improving effective routine vaccination and hygiene practices on bovine farms.

MicroRNA Profile of MA-104 Cell Line Associated With the Pathogenesis of Bovine Rotavirus Strain Circulated in Chinese Calves.

Bovine rotavirus (BRV) causes massive economic losses in the livestock industry worldwide. Elucidating the pathogenesis of BRV would help in the development of more effective measures to control BRV infection. The MA-104 cell line is sensitive to BRV and is thereby a convenient tool for determining BRV-host interactions. Thus far, the role of the microRNAs (miRNAs) of MA-104 cells during BRV infection is still ambiguous. We performed Illumina RNA sequencing analysis of the miRNA libraries of BRV-infected and mock-infected MA-104 cells at different time points: at 0 h post-infection (hpi) (just after 90 min of adsorption) and at 6, 12, 24, 36, and 48 hpi. The total clean reads obtained from BRV-infected and uninfected cells were 74,701,041 and 74,184,124, respectively. Based on these, 579 were categorized as known miRNAs and 144 as novel miRNAs. One hundred and sixty differentially expressed (DE) miRNAs in BRV-infected cells in comparison with uninfected MA-104 cells were successfully investigated, 95 of which were upregulated and 65 were downregulated. The target messenger RNAs (mRNAs) of the DE miRNAs were examined by bioinformatics analysis. Functional annotation of the target genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) suggested that these genes mainly contributed to biological pathways, endocytosis, apoptotic process, trans-Golgi membrane, and lysosome. Pathways such as the mammalian target of rapamycin (mTOR) (mml-miR-486-3p and mml-miR-197-3p), nuclear factor kappa B (NF-κB) (mml-miR-204-3p and novel_366), Rap1 (mml-miR-127-3p), cAMP (mml-miR-106b-3p), mitogen-activated protein kinase (MAPK) (mml-miR-342-5p), T-cell receptor signaling (mml-miR-369-5p), RIG-I-like receptor signaling (mml-miR-504-5p), AMP-activated protein kinase (AMPK) (mml-miR-365-1-5p), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling (mml-miR-299-3p) were enriched. Moreover, real-time quantitative PCR (qPCR) verified the expression profiles of 23 selected DE miRNAs, which were consistent with the results of deep sequencing, and the 28 corresponding target mRNAs were mainly of regulatory pathways of the cellular machinery and immune importance, according to the bioinformatics analysis. Our study is the first to report a novel approach that uncovers the impact of BRV infection on the miRNA expressions of MA-104 cells, and it offers clues for identifying potential candidates for antiviral or vaccine strategies.