Genetic distribution of bovine viral diarrhea virus in free-living cattle in China

doi:10.21203/rs.3.rs-3789171/v1

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Genetic distribution of bovine viral diarrhea virus in free-living cattle in China

https://doi.org/10.21203/rs.3.rs-3789171/v1

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According to sequencing of the 5’ untranslated region (5’UTR) in viral genome, five strains of bovine viral diarrhea virus (BVDV) isolated from different provinces in China were genetically typed. The phylogenetic tree for BVDV 5’UTR displays that the isolated strain 22AH-1 is clustered into the genotype 1c clade, the strain 22-Gansu-F2 is clustered into the genotype 2a clade, the strains 22Anhui-7, 22-Sichuan-B8 and 22-Gansu-F3 are clustered into the genotype 2d clade. Depending on principal component analysis for synonymous codon usage pattern, BVDV strains generally exhibit the genotype-specific model. Although most synonymous codons are selected at the highly variable levels, the strongly suppressive usages focus on synonymous codons containing CG dinucleotides in all BVDV strains. Moreover, the plot of ENC value vs. GC3 content obviously shows that apart from nucleotide usage at highly variable patterns derived from mutation pressure, strong selective forces caused by natural selection mediate synonymous codon usage pattern of BVDV. Taken together, the related results give new insight into multiple selective forces acting on synonymous codon usage beyond nucleotide usage variation during BVDV evolutionary pathway.

BVDV

phylogenetic reconstruction

genotype

synonymous codon

evolutional pathway

Since bovine viral diarrhea virus (BVDV) poses serious impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes, this virus is regarded as one of important pathogens of cattle worldwide [1]. BVDV, belonging to a member of the genus Pestivirus in the family Flaviviridae, owns a positive single-stranded RNA genome with approximately 12 kb. The viral genome encodes an open reading frame (ORF) which is translated into one single polyprotein. The polyprotein is composed of N^pro, capside, E0, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.

Currently, there have been three genotypes of BVDV identified worldwide, namely BVDV-1, BVDV-2 and BVDV-3 (also named HoBi-like pestivirus) [2]. Most of the phylogenetic analyses of BVDV strains have been performed on the 5’UTR, N^pro and E2 sequences, respectively [3–5]. In comparison of performances of genotyping classified by the three sequences mentioned above, the classification derived from 5’UTR displays the better performance than others. Genotyping methods have been applied for determining the prevalence of BVDV isolates in different geographic regions. Currently, genetic subgenotyping of BVDV-1 isolates displayed approximate 21 subgenotypes (1a-1u), while BVDV-2 ones can be divided into four subgenotypes (2a-2d) [6, 7]. Most acute infections in cattle herds with any genotype often displays mild or sub-clinical signature, and BVDV from either genotype may exhibit one of two biotypes (cytopathic (CP) or noncytopathic(NCP)) according to its ability of the production of the visible effects on cell culture. Both of BVDV strains with CP and NCP can result in acute infection; in addition, BVDV with NCP also contributes to persistence infection[8]. Of note, the BVDV-2 strains were firstly identified as viral pathogens isolated from persistently infected animals born to dams that had been vaccinated with BVDV-1 vaccines[9], suggesting that the two genotypes are antigenically distinct. The genotyping classification for BVDV isolates can be implicated in disease control as diagnostics and vaccines that work well against homologous strains. Currently, the BVDV-1b is generally predominant subgenotype in the world, followed by BVDV-1a and 1c [10]. Turning to distribution of BVDV-2 subgenotype, BVDV-2a is the most prevalent globally, while subgenotpes 2b, 2c and 2d were just prevalent in European and Asian countries[7]. In China, nine subgenotypes have been detected in cattle herds, including 1a-1d, 1m, 1o-1q and 1u [11, 12]. Moreover, Deng et al., (2020) carried out genotyping classification for BVDV strains in dairy herds in China and pointed out that BVDV-1a, 1c and 1m were the dominant strains[13]. According to the previous reports for BVDV prevalence in China, there is a higher proportion of BVDV-positive cattle with BVDV-1 than BVDV-2 in cattle herds[14–16]. With the development of cattle industry, real-time monitoring genetic diversity of BVDV strains circulating in cattle herds can benefit for clarifying the distribution of different BVDV genotypes in China. In this study, we isolated new BVDV strains from different provinces in China and analyzed genetic diversity of these strains at nucleotide and synonymous codon usages in order to clarify evolutional features of BVDV strains in China and give new insight into preventing and vaccine measures.

2.1 Clinical serum sample collection

This study was approved by the Animal Ethics Committee of College of Life Science and Engineering, Northwest Minzu University (Approval No. CLSEAEC 2020-003). A total of 300 serum samples from free-living cattle in countries from three provinces (Anhui, Gansu and Sichuan) in China were randomly collected in 2022. Some serum samples were derived from cattle which were subject from diarrhea. Most samples were collected from cattle which showed the clinical healthy status. The serum samples were stored at -20℃ for molecular detection by RT-PCR and virus isolation.

2.2 Molecular detection

Each clinical sample was detected for the BVDV presence by RT-PCR. Firstly, total RNA was extracted from serum using TIANampvirus DNA/RNA kit (TIANGEN Biotech, China). Subsequently, cDNA was synthesized from 1000ng of total RNA using the M-MLV Reverse transcriptase kit (TaKaRa, China) following the manufacturer. The synthesized cDNA were detected by PCR assay to amplify about 250bp fragment of the BVDV 5’UTR region, using the previous reported primers [17].

2.3 Virus isolation from the positive serum sample

To further estimate the biotype of BVDV circulating in free-living cattle herds, BVDV strains were isolated from the positive serum samples. Briefly, each positive serum samples was placed on Madin-Darby bovine kidney (MDBK) cells for 2 hours at 37℃ in a 5% CO₂ atmosphere. The proceed MDBK cells were washed three times with PBS and then DMEM with 1% fetal bovine serum was added and incubated for 5 days. Subsequently, the resulting cell cultures, which were detected by RT-PCR for BVDV presence, were frozen and thawed 2 ~ 3 times and the clarified supernatant was passaged 7 ~ 8 times in MDBK cells. In the absence of cytopathic effect caused by BVDV infection, the 50% tissue culture infectivity dose (TCID₅₀) method was used for the viral titer assessment [18]. Briefly, cell suspensions of 10⁵ MDBK cells per well were seeded in round bottom 96-well culture plates, and then the cells were inoculated for 2 hours with 100 µL of ten-fold dilutions of BVDV supernatant, eight replicates per dilution. Mock and medium controls were included in triplicate wells on all plates.

2.4 BVDV genome sequencing

Twelve primer sets were designed to amplify overlapping regions covering the whole BVDV genome (Table S1). The amplified nucleotide fragments needed to be harvested and purified, and then were cloned into the pMD-18T vector (TaKaRa, China). The resulting recombinant clones were sequenced by Tsingke Biotechnology Co., Ltd. The retrieved sequences were edited and trimmed with the EdiSeq program in the DNASTAR software in order to organize the whole BVDV genome.

2.5 Analyses for genetic diversity of BVDV isolates

For further genotyping the BVDV isolates, the phylogenetic tree was performed using the 5’URT region. Additional reference sequences (5’UTR) from representative isolates of previously identified subgenotypes of BVDV1 (1a-1u), BVDV2 (2a-2d) and BVDV3 were included into phylogenetic analysis. To further clarifying genetic relationship between our BVDV isolates and other reported BVDV strains emerging in China, we compared our strains with the reference BVDV ones (Table S2) on the genome level via phylogenetic tree. Nucleotide sequences from each region were aligned via the Clustal W program. The phylogenetic tree was inferred by viral genome by statistical method (Neighbour-joining tree) using MAGE 7.0 software.

2.6 Analyses for genetic features of BVDV isolates at synonymous codon usage patterns

The relative synonymous codon usage value (RSCU) for a synonymous codon is the ratio of its observed frequency to its expected frequency without biased usage (the mean frequency of all synonymous members encoding their specific amino acid) [19]. RSCU would be an objective representation of the synonymous codon usage bias without confounding the influence of amino acid composition for different BVDV strains. In general, the higher RSCU value is, the stronger bias the corresponding synonymous codon usage is [20, 21].To identify the extent of 59 synonymous codon usage bias, RSCU values of higher than 1.6 and less than 0.6 were defined as ‘overrepresented’ and ‘underrepresented’ codons, respectively [22].

To further quantify the rank of the overall codon usage bias for BVDV ORF, the effective number of codons (ENC index) was employed, which can quantify the departure of a viral ORF from the random usage of synonymous codon[23]. The ENC values, ranging from 61 (when all synonymous codon usages in random and even) to 20 (when only one synonymous codon is used per amino acid), displayed the effect of GC3 content on the overall codon usage bias. Based on ENC index estimating BVDV ORF, the plot which is composed of ENC value and GC3 content (GC content at the 3rd codon position) was used for further investigating the selective forces in synonymous codon usages during BVDV evolution. ENC value and GC3 content for BVDV ORF were calculated by CodonW software ( https://sourceforge.net/projects/codonw/ ).

2.7 Analysis for genetic diversity of BVDV circulating in China at synonymous codon usage

Principal component analysis (PCA) is a multivariate statistical method which reduces data dimensionality by performing a covariance analysis for a data matrix [24]. As for viral diversity with respect to the overall codon usage pattern of BVDV circulating in China, PCA analysis was performed, based on RSCU data of BVDV ORF. The plot which displayed genetic diversity of these strains was composed of the first variant (f₁’ data) and the second variant (f₂’ data), revealing the overall codon usage pattern derived from the 59 synonymous codon usage variants for each strain in this study.

3.1 RT-PCR for BVDV in clinical serum samples

A total of 300 serum samples from free-living cattle were detected for BVDV 5’UTR sequence via RT-PCR. The average positive rate of BVDV in cattle was about 7.7% (23/300). Moreover, the average positive rate of BVDV in Gansu, Sichuan and Anhui was 11.4% (13/114), 6.3% (6/96) and 4.4% (4/90), respectively.

3.2 Viral isolation for positive serum samples

BVDV strains were isolated from positive clinical serum samples, and these BVDV isolates needed to be checked by RT-PCR and sequence analysis. Initially the total of 23 positive serum samples can be used for viral isolation, but just only five BVDV isolates were successfully obtained and passaged in MDBK cells. The BVDV isolates were termed as 22AH-1, 22-Sichuan-B8, 22Anhui-7, 22-Gansu-F2 and 22-Gansu-F3, respectively. Among the five strains, 22AH-1, 22Anhui-7 and 22-Gansu-F3 can lead to cell lesions (identified as CP biotype), while the rest did not (NCP biotype) (Table 1). Moreover, the biotypes (CP / NCP) of these BVDV isolates did not have the correlations with cattle clinical status (health / diarrhea) (Table 1).

Table 1

The information about the eight filed BVDV isolates from serum samples
Strain	^aClinical status	Biotype	^bTCID₅₀/0.1mL
^c22-Anhui-7	clinical healthy status	CP	10^− 8.00/0.1mL
^d22-Gansu-F2	diarrhea	NCP	Not application
^d22-Gansu-F3	diarrhea	CP	10^− 6.125/0.1mL
^e22-Sichuan-B8	clinical healthy status	NCP	Not application
^c22AH-1	clinical healthy status	CP	10^− 5.25/0.1mL
^aCattle with the specific clinical status which provided the corresponding clinical serum sample.
^bthe values of TCID₅₀/mL were derived from BVDV which were processed by 8 passages in MDBK cells.
^cthe BVDV strain was collected from Anhui province
^dthe BVDV strain was collected from Gansu province
^ethe BVDV strain was collected from Sichuan province

3.3 Phylogenetic construction for the five strains

To clarify genetic diversity of the BVDV isolates in the known subgenotypes of BVDV-1, -2 and − 3, the subgenotypes of the five strains were detected by 5’UTR sequencing and phylogenetic analysis. As shown in Fig. 1, the phylogenetic tree displayed that the strain 22AH-1 was clustered into BVDV-1c subgenotype, the strain 22-Gansu-F2 belonged to BVDV-2a subgenotype and the rest strains were identified as BVDV-2d subgenotype. Furthermore, the phylogenetic construction was also performed to better give new insight into genetic profile of the five isolates among the known BVDV strains circulating in China at genome level. As shown in Fig. 2, the genetic relationship between the strain 22AH-1 and the strain GSTZ was closer than the strain GXNN1; in addition, those four isolates with BVDV-2 had a closer genetic relationship with the strain XJ-04 than the strains JZ05-1 and HLJ-10.

3.4 The genotype-specific pattern of synonymous codon usage in the BVDV isolates

For further analyzing the pattern of synonymous codon usage in the five isolates, RSCU values for 59 synonymous codons were calculated for each viral ORF (Table 2). Generally, the comparisons of synonymous codon usage patterns among the five BVDV isolates exhibited the effect of the genotype-specific usage pattern on synonymous codon usages in the BVDV ORF. As for the synonymous codon usage variants of the five isolates, the underrepresentive synonymous codons (CUU & CUC for Leu, UGG for Ser, CCG for Pro, ACG for Thr and GCG for Ala) and the overrepresentative ones (CCA for Pro) shared with the five BVDV isolates (Table 2). Interestingly, there were two extreme usage patterns (overrepresentation and underrepresentation) in the synonymous codon family of amino acid Arg, namely CGC, CGU, CGA and CGG being the underrepresentive codons and AGA and AGG being the overrepresentative ones (Table 2). Of note, all synonymous codons with CG dinucleotides were also subject to strongly suppressive selection in the five strains.

Table 2

Synonymous codon usage patterns for the five BVDV isolates
	22AH-1	22Anhui-7	22-Gansu-F2	22-Gansu-F3	22-sichuan-B8
UUU(F)	0.97	1.10	1.10	1.10	1.10
UUC(F)	1.03	0.90	0.90	0.90	0.90
UUA(L)	0.88	1.06	1.05	1.06	1.06
UUG(L)	1.36	1.23	1.24	1.23	1.23
CUU(L)	0.48	0.53	0.52	0.53	0.53
CUC(L)	0.50	0.58	0.58	0.58	0.56
CUA(L)	1.36	1.39	1.41	1.39	1.40
CUG(L)	1.41	1.22	1.21	1.20	1.22
AUU(I)	0.63	0.46	0.46	0.45	0.46
AUC(I)	0.71	1.04	1.04	1.04	1.05
AUA(I)	1.67	1.49	1.50	1.51	1.49
GUU(V)	0.56	0.75	0.75	0.75	0.74
GUC(V)	1.01	0.66	0.66	0.66	0.68
GUA(V)	0.96	1.30	1.30	1.30	1.30
GUG(V)	1.47	1.29	1.29	1.29	1.28
UCU(S)	0.77	0.60	0.63	0.60	0.60
UCC(S)	0.71	0.79	0.78	0.79	0.79
UCA(S)	1.75	1.45	1.44	1.45	1.45
UCG(S)	0.09	0.09	0.09	0.09	0.09
AGU(S)	1.28	1.67	1.69	1.73	1.70
AGC(S)	1.40	1.39	1.38	1.35	1.38
CCU(P)	1.03	0.99	0.99	0.99	0.98
CCC(P)	0.91	0.81	0.81	0.84	0.81
CCA(P)	1.65	1.85	1.85	1.82	1.84
CCG(P)	0.41	0.35	0.35	0.35	0.37
ACU(T)	0.91	0.91	0.93	0.91	0.91
ACC(T)	1.03	0.87	0.85	0.87	0.87
ACA(T)	1.60	1.67	1.67	1.67	1.67
ACG(T)	0.46	0.55	0.55	0.55	0.55
GCU(A)	0.89	1.28	1.27	1.28	1.27
GCC(A)	1.27	0.98	0.99	0.97	0.99
GCA(A)	1.53	1.46	1.47	1.45	1.47
GCG(A)	0.31	0.28	0.28	0.30	0.28
UAU(Y)	0.94	0.98	0.98	0.98	0.99
UAC(Y)	1.06	1.02	1.02	1.02	1.01
CAU(H)	0.68	0.84	0.82	0.84	0.84
CAC(H)	1.32	1.16	1.18	1.16	1.16
CAA(Q)	0.97	1.02	1.02	1.00	1.03
CAG(Q)	1.03	0.98	0.98	1.00	0.97
AAU(N)	0.87	0.88	0.88	0.86	0.88
AAC(N)	1.13	1.12	1.12	1.14	1.12
AAA(K)	0.99	1.04	1.04	1.04	1.03
AAG(K)	1.01	0.96	0.96	0.96	0.97
GAU(D)	1.15	0.91	0.91	0.91	0.93
GAC(D)	0.85	1.09	1.09	1.09	1.07
GAA(E)	1.05	1.06	1.06	1.06	1.06
GAG(E)	0.95	0.94	0.94	0.94	0.94
UGU(C)	1.11	0.94	0.94	0.94	0.94
UGC(C)	0.89	1.06	1.06	1.06	1.06
CGU(R)	0.08	0.06	0.06	0.06	0.06
CGC(R)	0.19	0.09	0.09	0.09	0.09
CGA(R)	0.19	0.16	0.16	0.19	0.16
CGG(R)	0.30	0.19	0.19	0.19	0.19
AGA(R)	2.73	2.94	2.94	2.92	2.92
AGG(R)	2.51	2.56	2.56	2.54	2.58
GGU(G)	0.78	1.05	1.04	1.02	1.06
GGC(G)	0.73	0.70	0.70	0.70	0.70
GGA(G)	1.03	0.92	0.93	0.94	0.93
GGG(G)	1.46	1.33	1.33	1.33	1.31
The RSCU value with more than 1.6 or less than 0.6 needs to be marked by bold text.

Based on synonymous codon usage variants of the five isolates and of BVDV strains circulating in China (Table S2), PCA was carried out for revealing evolutionary distance between each BVDV isolate and the reference strains at codon usage level. Generally, BVDV-1 strains displayed the stronger dispersion than those of BVDV-2 and − 3 with respect to the overall codon usage pattern (Fig. 3). As shown in Fig. 3, the evolutionary distance between the strain 22AH-1 (f’₁=0.977, f’₂=-0.762) and GSTZ (f’₁=0.886, f’₂=-0.620) was closer than that between the strain 22AH-1 and GXNN1 (f’₁=0.647, f’₂=-0.765); in addition, other four isolates, 22-Anhui-7 (f’₁=-1.845, f’₂=-0.325), 22-Sichuan-B8 (f’₁=-1.806, f’₂=-0.345), 22-Gansu-F2 (f’₁=-1.839, f’₂=-0.346) and 22-Gansu-F3 (f’₁=-1.803, f’₂=-0.451) shared the closer genetic relationship than other BVDV-2 strains (JZ05-1, HLJ-10 and XJ-04). The genetic feature of codon usage pattern for the five BVDV isolates was consistent with alignment of viral genome.

3.5 Strong selective forces suppressing the overall codon usage for the BVDV isolates

According to the genetic features displayed in Figs. 2 and 3, it was necessary to further estimate whether nucleotide composition variations of viral ORF served as an evolutionary dynamic for the overall codon usage patterns of BVDV strains circulating in China or not. In Fig. 4, the plots composed of ENC vs. GC3 content per BVDV strains were obviously located below the expected curve line, suggesting that the overall codon usage of BVDV strain was strongly suppressed by natural selection. Moreover, all BVDV strains isolated from China did not show the strong overall codon usage bias, due to the ENC values ranging from 50 to 55 (Fig. 4). Thus, the below-curve scattering dots implied that natural selection dominated the mutation pressure derived from nucleotide composition constraint. However, the influence of mutation pressure is not completely absent and the interplay between mutation pressure and natural selection shapes the evolutionary trend of BVDV in China.

With the development of cattle industry, BVDV, serving as RNA virus with a high mutation rate, has became more and more prevalence in China. In our study, the prevalence and genetic diversity of BVDV were investigated in three different geographical regions (Sichuan, Gansu and Anhui province) in China. The BVDV positive rate varies largely in different regions, due to sampling measure, bovine species or clinical status. Compared with ELISA assay for viral antigen, RT-PCR assay for viral sequence is more sensitive and has been broadly applied for viral detection.

According to the previous studies, the prevalence of BVDV RNA was 27.1% in dairy cattle in China [25]; in addition, that was about 8.14% in newborn calves in northern China [17]. However, we revealed that the average positive rate of BVDV in free-living cattle was 7.7% (23/300) and the prevalence of BVDV is different in the three different geographic regions (Gansu, Sichuan and Anhui provinces) in China. Although limitations in the sampling and detection methods influence the positive rate of BVDV RNA to some degree, geographic factor and local cattle industry play important roles in BVDV prevalence in the particular region in China. Based on the positive rate of BVDV RNA in the target regions, the effective viral isolation plays an important role in further investigating the genetic and antigenic characterizations in BVDV-infected cattle. Cattle with BVDV-infection tends to secrete a huge number of virions in serum, especially persistent infection (PI) cattle [26, 27]. Thus, the clinical serum sample of cattle has been regarded as a valid source for isolation of BVDV. Here, the five BVDV strains were successfully obtained from the serum samples. Moreover, the isolates with CP or NCP biotype not only existed in cattle with diarrhea but also in cattle with clinical healthy status (Table 1), strongly suggesting that BVDV-infected cattle with clinical healthy status pose threat to the healthy cattle herds. RT-PCR detection for cattle herds with clinical healthy sign can effectively monitor BVDV prevalence in cattle herds and give new insight into designing preventive measures.

Serving as an RNA virus, BVDV owns high mutation rates and has evolved into multiple subgenotypes. Real-time investigation of the frequency and number of BVDV subgenotypes benefits for clarify genetic diversity of viral isolates and the infection source. In this study, BVDV strain 22AH-1 has been identified as subgenotype 1c and displays a closer genetic relationship with strain GSTZ than GXNN1 (Fig. 2), despite the strains GSTZ and GXNN1 belonging to BVDV-1c. In agreement with the previous reports [16, 17, 28, 29], the result further demonstrates that BVDV-1c is commonly found in China. In addition, one BVDV-2a strain (22-Gansu-F2) and three BVDV-2d strains (22-Anhui-7, 22-Sichuan-B8 and 22-Gansu-F3) have the close genetic relationships (Fig. 2). These results also give new insight into epidemiologic trends of BVDV in China. The related information has valuable implications for the design and construction of effective vaccination strategies to control BVDV spread.

With the development of sequencing techniques for viral genome, more and more observations are able to indicate the nucleotide composition constraint caused by mutation pressure and natural selection derived from the various outside environments, serving as the important evolutionary dynamics in shaping synonymous codon usage patterns for viral genome [30–33]. Although the strain 22AH-1 has the divergent patterns from the strains (22-Anhui-7, 22-Sichuan-B8, 22-Gansu-F2 and 22-Gansu-F3) with respect of synonymous codon usages, all synonymous codons including CG dinucleotides are strongly suppressed at usage and synonymous codon family for amino acid Arg display the conserved usage pattern to a high degree (Table 2). These results strongly reflect that selective forces derived from natural selection participate in evolutionary pathway of BVDV, apart from mutation pressure. This result is consistent with the observations that natural selection derived from host-dependent selection plays an important role in eliminating CG dinucleotides in the viral genome [34–36]. According to the overall codon usage patterns for BVDV strains in China (Fig. 3), these BVDV strains also highlighted the genotypic-specific characterization. Moreover, the overall codon usages of BVDV-1 strains also displayed more various patterns than BVDV-2 and − 3. This result is consistent with the previous reports for BVDV-1 strains with high mutation variations [13, 37, 38]. The most obvious evolutionary dynamic which determines synonymous codon usage is nucleotide composition constraint caused by mutational pressure. This evolutionary dynamic is most significant in viral genomes with extreme nucleotide composition: most RNA and DNA viruses with high GC or AT content have strong trends for selecting the nucleotide with richer concentration in genome as the ending base in the code[22, 39–43].

Translation selection, as one of the natural selections, is responsible for the biased usage of synonymous codons in coding sequences in a wide variety of organisms [21, 44, 45]. Based on the overall codon usage pattern represented by ENC of BVDV strains in China (Fig. 4), those codon usage outliers that obviously deviate from the expected curve line strongly reflect that evolutionary dynamics related to natural selection have impact on usage trend of synonymous codons. This genetic phenomenon also accounts for a few overrepresented synonymous codon in despite of low GC3 content in BVDV ORF. These interesting genetic characteristics could reflect the selection pressure on the overall nucleotide usage pattern of BVDV and nucleotide usage at the third codon position could modulate viral codon usage with the specific biological functions (e.g. fine-tuning of translation kinetics). Additional evidences have verified that usage of synonymous codons in protein coding sequences is necessarily biased and the overall codon usage pattern could match the tRNA pool of the host organism[46–53]. Generally, higher species (i.e. human beings) appear to shape their the overall codon usage patterns impacted solely by isochore composition or genomic GC organization, whereas lower species such as the flies or worms, appear to present an intermediate magnitude of selection partly determining their synonymous codon usage pattern [54–56]. These observations can be plausibly explained by the interplay between mutation pressure and natural selection.[57–59]

This study revealed that the average positive rate of BVDV was 7.7% (23/300) in free-living cattle. One BVDV-1c strain, one BVDV-2a strain and three BVDV-2d strains were successfully isolated from RT-PCR positive serum samples. Apart from confirming BVDV-1c circulating in China, these BVDV-2 strains also highlight that BVDV-2 strains have circulated in China. Moreover, the evolutionary dynamics derived from mutation pressure and natural selection drive BVDV evolution and sustain the genotype-specific trail at synonymous codon usages. Our results give new insight into monitoring genetic diversity and vaccination strategies of BVDV in China.

BVDV

bovine viral diarrhea virus

5’UTR

5’ untranslated region

ORF

open reading frame

cytopathic

NCP

noncytopathic

MDBK

Madin-Darby bovine kidney

TCID50

50% tissue culture infectivity dose

RSCU

relative synonymous codon usage value

ENC

effective number of codons

PCA

principal component analysis

Conflict of interest:

All authors have no conflict of interest.

Author Contribution

HHW: Methodology, Data curation, Writing- Original draft preparationXYW: Investigation, Writing- Original draft preparationXYH: Software, ValidationJQW: Software, ValidationXAC: Conceptualization, Methodology, Writing- Reviewing and Editing.JHZ: Conceptualization, Methodology, Writing- Reviewing and Editing.

Acknowledgement:

The work was supported by the Natural Science Foundation of Gansu province, China (No. 23JRRA715), the Fundamental Research Funds for the Central Universities (No. 31920220069) and the Fundamental Research Funds for Introduction of Talent Research Fund of Northwest Minzu University (No. xbmuyjrc202225).

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Genetic distribution of bovine viral diarrhea virus in free-living cattle in China

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Clinical serum sample collection

2.2 Molecular detection

2.3 Virus isolation from the positive serum sample

2.4 BVDV genome sequencing

2.5 Analyses for genetic diversity of BVDV isolates

2.6 Analyses for genetic features of BVDV isolates at synonymous codon usage patterns

2.7 Analysis for genetic diversity of BVDV circulating in China at synonymous codon usage

3. Results

3.1 RT-PCR for BVDV in clinical serum samples

3.2 Viral isolation for positive serum samples

3.3 Phylogenetic construction for the five strains

3.4 The genotype-specific pattern of synonymous codon usage in the BVDV isolates

3.5 Strong selective forces suppressing the overall codon usage for the BVDV isolates

4. Discussion

5. Conclusion

Abbreviations

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