Isolation of BVDV-1a, 1m, and 1v strains from diarrheal calf in china and identification of its genome sequence and cattle virulence

Abstract

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5'-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

Keywords: BVDV-1v; bovine viral virus diarrhea (BVDV); calf diarrhea; genetic variation; novel genotype; pathogenicity.

Figure 1

The animal experiment protocol. Calves were intranasally infected with bovine viral diarrhea virus (BVDV). All animals were euthanized and necropsied at 28 dpi, except for one animal (BVDV-1a HN-03 group) that died at 21 dpi. The markers below indicate the measures and sample collection on the day.

Figure 2

Viral isolation and characterization. The specific immunofluorescent signals were detected in the cytoplasm of cells inoculated with BVDV-1a HN-03 (A), BVDV-1m HB-01 (B), and BVDV-1v HB-03 (C). Madin-Darby bovine kidney (MDBK) cells inoculated with BVDV-1b AV-69 (as a positive control) cells showed specific fluorescence (D), and mock-infected cells showed no bright fluorescence (E). Median tissue culture infectious dose (TCID₅₀) of the three bovine viral diarrhea virus (BVDV) isolates inoculated with MDBK in passages (F). One-step growth curves of BVDV-1a HN-03, BVDV-1m HB-01, and BVDV-1v HB-03 isolates (G).

Figure 3

Schematic diagram of the genomic structure and phylogenetic analysis. Schematic diagram of the annotated bovine viral diarrhea virus (BVDV) genome (A). Phylogenetic tree of the nucleotide sequences of the 5-untranslated region (5′-UTR) (B) and N^pro (C). Molecular evolutionary genetics analysis was performed with MEGA 7 (Molecular Evolutionary Genetics Analysis) using the neighbor-joining (NJ) and bootstrap analysis (n = 1,000) method. Unrooted trees described the relationship between sequences retrieved pestivirus [BVDV-1, BVDV-2, classical swine fever virus (CSFV), and border disease virus (BDV)] from the GenBank database and the three BVDV isolates analyzed in this work (red circle). Information on the reference strains is shown in Supplementary Table 3.

Figure 4

Phylogenetic tree of the nucleotide sequences of the complete gene and six segments (Erns, E1, E2, NS2-3, NS4, and NS5). Molecular evolutionary genetics analysis was performed with MEGA 7 (Molecular Evolutionary Genetics Analysis) using the neighbor-joining (NJ) and bootstrap analysis (n = 1,000) method. Unrooted trees described the relationship between sequences retrieved from the GenBank database and the three bovine viral diarrhea virus (BVDV) isolates analyzed in this work (red circle). Information on the reference strains is shown in Supplementary Table 3.

Figure 5

Nucleotide sequence identity (%) comparison of each segment of the three bovine viral diarrhea virus (BVDV) isolates to other strains of BVDV-1 and 2. Information on the reference strains is shown in Supplementary Table 3.

Figure 6

Recombination analysis of the BVDV-1v HB-03 strain genome. Similarity plots and boot scanning analyses were performed using the SimPlot (Similarity Plotting) software package (version 3.5.1). Recombination breakpoints are shown with red lines, and the locations are shown at the side. (A,C) Show that there were two potential recombination events in BVDV-1v HB-03, respectively. Phylogenies of the major and minor parent regions are shown below the similarity plot (B,D).

Figure 7

Unique mutation analyses in the E2. (A) Sequence alignment of BVDV E2 sequences. Two highly conserved regions within the E2 sequence were revealed to locate at the amino acid positions of 116–136 and 286–364, respectively (black rectangle). (B) The cartoon schemes of the BVDV-1v HB-03 strain E2 protein structure. The domains of BVDV E2 modified from El Omari et al. (35). Domain DA, comprising residues 4–87, is in orange, and domain DB, comprising residues 88–164, is in blue. Domain DC, comprising residues 165–271, is in light purple, and domain DD, comprising 272–333, is in dark fluorescent green.

Figure 8

Evaluation of the three bovine viral diarrhea virus (BVDV) isolates' pathogenic and serological characters. (A) Fever (B), clinical signs (C), average daily weight gain (D), and survival rate (E) of all calves were monitored daily. Hematology of calves infected with BVDV isolates was measured at 0–14 dpi. Mean number expressed as mean ± standard error for white blood cell (WBC) (E) and lymphocytes (LYM) (F). Relative quantification of virus load in blood (G), nasal swabs (NSs) (H), and rectal swabs (RSs) (I) provided insights into the viral load of the virus following intranasal infection. Serum neutralizing antibody titers were tested at 0, 7, 14, and 28 dpi (J). Significant differences are indicated with asterisks (*p < 0.05, **p < 0.01).

Figure 9

Histopathological changes of lung, duodenum, and spleen. (A–L) Represent groups of the negative control, BVDV-1a HN-03, BVDV-1m HB-01, and BVDV-1v HB-03, respectively. Compared with the normal tissue structure (A), the alveolar structure of calves infected with bovine viral diarrhea virus (BVDV) was destroyed (B–D). Compared with the normal duodenal tissues (E), the duodenum of the calves infected with bovine viral diarrhea virus (BVDV) (F–H) had a large amount of small intestinal chorion shed in the intestine. Compared with the normal spleen tissues (I), the spleen of the calves infected with BVDV (J,K) showed a significantly wide periarterial lymphatic sheath, with increased bleeding points and lymphocytes depletion in red pulp. The horizontal line on the bottom right of each figure is the scale bar (100 μm scale).