Molecular detection and genotyping of bovine viral diarrhea virus in Western China

Background Bovine viral diarrhea virus (BVDV) is an important global viral pathogen of cattle and other ruminants. To survey the infection rate and genetic diversity of BVDV in western China, a total of 1234 serum samples from 17 herds of dairy cattle, beef cattle and yak in 4 provinces were collected in 2019. Results All the 1234 serum samples were screened individually for BVDV by RT-PCR. Our results demonstrated that the average positive rate of BVDV was 7.2% (89/1234) in animals and 82.4% (14/17) in herds. Thirteen BVDV strains were isolated from RT-PCR positive clinical samples and they were all NCP biotype. BVDV-1a and 1c subgenotypes were identified from 22 selected virus isolates in 14 BVDV-positive herds. These results confirmed that BVDV-1a and BVDV-1c were circulating in western China, similar to the BVDV epidemics in cattle in other regions of China. Conclusions This study provides data for monitoring and vaccination strategies of BVDV in western China. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-021-02747-7.


Background
Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle worldwide and causes significant economic losses. BVDV has been detected in not only in cattle, but also in diverse domestic [1,2] and wildlife animal species [3][4][5].
Previous studies showed that a high proportion of BVDV-positive cattle came from western China [19,20], because these areas are historically the main regions with cattle production. However, studies regarding the genetic diversity of BVDV in western China remain rare [4,21,22]. Thus, this study detected and genotyped BVDV from bovines in this region. Such studies are important to understand the diversity of viral strains present in one region and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources.

Detection of BVDV in clinical serum samples
A total of 1234 serum samples were tested for BVDV by 5'UTR RT-PCR. As shown in Table 1, the average positive rate of BVDV in animals was 7.2% (89/1234). At the herd level, a herd was considered positive if one serum sample was positive in RT-PCR testing. Thus, 82.4% (14/ 17) of herds were positive and they were located in Shaanxi, Ningxia, Xinjiang, and Tibet. No BVDV infection was found in 3 herds from Shaanxi and Ningxia Provinces. The average positive rate of BVDV in Shaanxi, Ningxia, Xinjiang, and Tibet was 4.57% (44/ 963), 30% (18/60), 37.5% (6/16) and 10.77% (21/195), respectively.

Virus isolation
BVDV strains were isolated from RT-PCR positive clinical samples and they were identified by immunofluorescence, RT-PCR and sequence analysis, and transmission electron microscopy.

Sequencing and phylogenetic analysis
To investigate the extent of genetic diversity of BVDV in western China, the subgenotypes of the BVDV isolates were determined by 5'UTR sequencing and phylogenetic analysis.

Discussion
In this study, we investigated the prevalence and genetic diversity of BVDV among bovines in western China. The BVDV positive rate varies greatly among bovine herds due to the variety of detection tests, sampling methods, species, and locations. Hence, it is difficult to determine the exact extent of the BVDV prevalence in China. As previously reported, RT-PCR analysis of BVDV RNA is more sensitive than other Ag detection methods and has been widely used for BVDV detection. Primer is pivotal for the accuracy and sensitive of RT-PCR detection. In this study, the primers BP189-389 for 5'UTR region was used, which had a broad range of bovine pestivirus detection including BVDV-1, BVDV-2 and BVDV-3 [23].
Our results demonstrated that the average positive rate of BVDV in animals was 7.2% (89/1234). Previous reports showed that the RNA prevalence of BVDV was 22.64% among bovine in China [17]. A systematic review and meta-analysis showed that the RNA prevalence of BVDV was 27.1% in dairy cattle in China [19]. In our study, there were limitations in the samplings and detection methods. Most of the clinical samples were sent to our laboratory by local farmers without providing detail information on the farms such as size and position. In addition, the sampling site did not completely cover the entire regions in western China. Considering the experimental expenses, the serology test was not performed in this study. Hence, the average RT-PCR positive rate of 7.2% in animals may not reflect the accurate prevalence of BVDV among bovines in western China. Notably, however, at herd level, we found 82.4% (14/17) of herds were positive for BVDV. Recently, a high prevalence of 57.78% of herds was reported to be positive for BVDV in northwestern China [1]. Our results suggested that a high proportion of herds are at risk of BVDV infection in western China.
Virus isolation is the standard method of detection of BVDV-infected cattle. The BVDV infected animals can secrete large amounts of BVDV in their serum, especially persistent infection (PI) animals. Previous studies demonstrated that BVDV remained viable in serum under normal conditions of sample submission to a diagnostic laboratory. Hence, the serum is a valid sample for the isolation of BVDV. In this study, 13 noncytopathic (NCP) strains were successfully isolated from clinical serum samples. NCP biotype is commonly found in nature. Our results further confirmed those of other workers.
BVDV is an RNA virus with high mutation rate [24]. Study investigating the frequency and number of subgenotypes of BVDV is helpful to understand the evolution of the virus and the source of infection. This information also has important implications for the design and construction of effective vaccination strategies to control BVDV [25,26]. In this study, two subgenotypes of BVDV1a and BVDV1c were identified among 22 selected virus isolates from 14 herds. These results were in agreement with recent epidemiologic studies of BVDV in cattles in China [20,27]. BVDV2 and BVDV3 were not detected in this study.
Although the predominant subgenotype worldwide is BVDV-1b, BVDV-1a and -1c are the second and third most frequently-reported genotypes in the world, respectively [14].BVDV-1a is predominant in South Africa and is widely circulating in the United States, Korea and Japan [28], while BVDV-1c has been reported as a The sample was not successfully isolated from cell culture predominant genotype in Australia [22,28]. In China, BVDV-1a and -1c have been commonly detected in different regions of China. Recently, BVDV-1c (7/9) and BVDV-1a (1/9) strains were detected from 36 herds of dairy cattle in 5 provinces in eastern China [29]. A recent analysis of 119 BVDV sequences obtained from 92 dairy farms showed that subgenotypes of BVDV-1a (n = 37, 31.09%), BVDV-1c (n = 34, 28.57%) and BVDV-1 m (n = 25, 21.01%) were predominant in 19 provinces of China in 2017 [20]. In western China, scattered studies on the genetic diversity of BVDV among cattle and other ruminants have been reported. Subgenotypes BVDV-1b and BVDV-1c have been identified in cattle from Xinjiang Autonomous Region [22]. BVDV-1b and BVDV-1d were found predominant subgenotypes in dairy farms in Ningxia Autonomous Region [21]. Six subgenotypes of BVDV-1a, BVDV-1b, BVDV-1c, BVDV-1 m, BVDV-1o, BVDV-1p and BVDV-1q have been identified in Bactrian camels from regions of Xinjiang, Gansu and Qinghai [4]. Here, BVDV-1a was respectively detected in Shaanxi, Ningxia, Tibet and Xinjiang, and BVDV-1c was detected in Shaanxi, Ningxia, and Tibet. Taken together, our results confirmed the presence of 1a and 1c subgenotypes in western China. The genetic diversity of virus isolates hamper prevention and control of BVDV. A vaccine effective in one region may fail to protect against virus infection caused by different virus strains in another region [30]. Our findings provide important information for further characterization of the variability and geographical distribution of BVDV in China.

Conclusions
Our results demonstrated that the average positive rate of BVDV was 7.2% (89/1234) in animals and 82.4% (14/ 17) in herds. Thirteen BVDV strains were successfully isolated from RT-PCR positive clinical samples and they were all NCP biotype. BVDV-1a and 1c subgenotypes were identified from 22 selected isolates from 14 herds. These results confirmed that BVDV-1a and BVDV-1c were circulating in western China, similar to the BVDV epidemics in cattle in other regions of China. This study provides data for monitoring and vaccination strategies of BVDV in western China. Fig. 2 Phylogenetic analysis based on 5'UTR sequence. A phylogenetic tree of the 5'UTR was created using the 5'UTR sequences of 22 selected BVDV isolates and 57 reference isolates retrieved from the GenBank database. •BVDV isolates identified in provinces of Shaanxi (green dot), Ningxia (red dot), Xinjiang (yellow dot) and Tibet (blue dot) in this study.14 isolates were clustered in BVDV-1a (frame) and 8 isolates clustered in BVDV-1c (frame)

Clinical sample collection
A total of 1234 serum samples from 17 herds covering 4 provinces in western China (Shaanxi, Ningxia, Xinjiang, and Tibet) were collected in 2019. Most samples were collected from herds in which the animals showed diarrhea. Some samples were submitted from clinical healthy animals for conventional detection. The sample information is summarized in Table 1.These herds were not vaccinated against BVDV. The samples were stored at − 80°C for RT-PCR and virus isolation.

RT-PCR
The clinical serum samples or cell culture were examined for the presence of BVDV by RT-PCR. Briefly, total RNA was extracted from serum or cell culture using TRIzol Reagent (Gibco). cDNA was synthesized from 1000 ng of total RNA using RNA reverse transcription kit (invitrogen USA).

Virus isolation
To investigate the biotype of BVDV circulating in herds, BVDV strains were isolated from RT-PCR positive clinical samples using standard virus isolation techniques. Briefly, serum samples were placed on Madin-Darby bovine kidney (MDBK) cells for 1 h at 37°C in a 5% CO2 atmosphere. The cells were washed twice with PBS and then DMEM with 2% fetal bovine serum (BVDV and BVDV antibody-free) was added and incubated for 4-5 days. Then the cultures were frozen and thawed three times and the clarified supernatant was passaged five times in MDBK cells. In the absence of cytopathic effect, the cells were fixed and stained by immunofluorescence as previously described [12]. The supernatants of the infected cells were further tested by RT-PCR described as above for the presence of BVDV nucleic acid.

Transmission electron microscope
The MDBK cell culture infected by RT-PCR positive samples were examined for the presence of BVDV particles by transmission electron microscopy (TEM). Cells were fixed with 2.5% glutaraldehyde in sodium cacodylate buffer (0.2 M, pH 7.2), post-fixed with 1% buffered osmium tetroxide, dehydrated in acetone, and embedded in epoxy resin. The resin blocks were then cut into 60 nm thick ultrathin sections, stained with uranyl acetate and lead citrate, and observed in a TEM (TECNAI G2 SPIRIT BIO).

Sequencing and phylogenetic analysis
The RT-PCR amplified fragments obtained from serum samples or cell culture were directly sequenced by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Multiple sequence alignment was performed using the ClustalW program in BioEdit software. Phylogenetic analysis of the 5'UTR region was performed with the neighbor-joining method in MEGA 7.0 software. In the phylogenetic tree, the evolutionary distances were computed using the Tamura 3parameter model with 1000 bootstrap replicates.