J Vet Sci.  2015 Dec;16(4):491-500. 10.4142/jvs.2015.16.4.491.

Genetic characterization of bovine viral diarrhea virus strains in Beijing, China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

• Weng XG
• Song QJ
• Wu Q
• Liu MC
• Wang ML
• Wang JF

• KMID: 2133632
• DOI: http://doi.org/10.4142/jvs.2015.16.4.491

Abstract

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5'-untranslated region (5'-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-alpha (IFN-alpha), IFN-beta, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-alpha, and IFN-beta mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.

Keyword

bovine viral diarrhea virus; interferon regulatory factor; persistent infection; phylogenetic analysis; Toll-like receptor

MeSH Terms

Animals
Cattle*
China*
Diarrhea*
Immunity, Innate*
Immunoblotting
Interferon Regulatory Factor-3
Interferon-alpha
Interferons
Orthomyxoviridae
RNA, Messenger
Toll-Like Receptors
Interferon Regulatory Factor-3
Interferon-alpha
Interferons
RNA, Messenger
Toll-Like Receptors

Figure

• 

  Fig. 1 Phylogenetic analysis of 245 nucleotides of the 5'-UTR or 385 nucleotides from the Npro genomic region in diverse strains of bovine viral diarrhea virus (BVDV). The 5'-UTR fragment (A) was determined in 18 of the analyzed strains, and a fragment of the Npro region (B) was determined in another five strains. Numbers over branches indicate the percentage of one thousand bootstrap replicates. The strains sequenced in this study are labeled with black triangles. GenBank accession nos. of reference strains in the 5'-UTR region were as follows: ZM-95 (AF526381), Shihezi148 (EU159700), Manasi (EU159702), SH1051 (JN248740), NX0803 (GU120254), DG07 (GU120250), TJ06 (GU120246), BJ0701 (GU120247), JS10116 (JN248734), camel-6 (KC695810), SD0803 (JN400273), FJ1003 (JN248728), ZJ1003 (JN248744), SH0710 (JN248738), SH1060 (JN248741), NADL (AJ133738), CP7 (U63479), KA07 (GQ495691), TJ0802 (GU120256), TrangieY546 (AF049222), KA01 (GQ495677), KB01 (GQ495676), 10-84 (AF298054), IT99-7101 (AJ318618), cell-con-1 (KC695816), F-Au (AF298065), J-Au (AF298067), W-Au (AF298073), A-Au (AF298064), L-Au (AF298069), G-Au (AF298066), KS86-1ncp (AB078950), Rebe (AF299317), SuwaCp (AF117699), 06z127 (DQ973181), XJ-04 (FJ527854), and 890 (U18059). Accession nos. of the BVDV reference strains in the Npro region are as follows: NADL (AJ133738), SD-1 (M96751), Deer-NZ1 (U80903), Shitara/01/05 (AB359926), F-Au (AF287284), 3186V6 (AF287282), J-Au (AF287286), W-Au (AF287290), A-Au (AF287283), L-Au (AF287287), G-Au (AF287285), 24/15 (AF287280), DeerGB1 (U80902), TR27 (EU163975), ZM-95 (AF526381), TJ0801 (GU120262), IS25CP/01 (AB359931), Shitara/02/06 (AB359930), SoCP/75 (AB359929), 871 (AF144462), Osloss (M96687), and BVDV-2 strain 890 (U18059).

• 

  Fig. 2 Expression of mRNA encoding (A) TLR-2, (B) TLR-3, (C) TLR-4, (D) TLR-7, (E) TLR-8, (F) RIG-I, and (G) MDA5 in peripheral blood mononuclear cells (PBMCs) collected from healthy cattle and PI cattle based on quantitative real-time PCR. Data presented are the means ± SEM (n = 6 per group). P < 0.05 is considered statistically significant.

• 

  Fig. 3 Expression of mRNA encoding (A) IFN-α, (B) IFN-β, (C) MX1, and (D) ISG-15 in PBMCs collected from healthy and PI cattle based on quantitative real-time PCR. Data presented are the means ± SEM (n = 6 per group). P < 0.05 is considered statistically significant.

• 

  Fig. 4 Immunoblotting detection of IRF-3 and IRF-7 in PBMCs from healthy and PI cattle. (A) Representative panels of IRF-3 and IRF-7 are shown. The expression of (B) IRF-3 and (C) IRF-7. Protein lysates were prepared with protease inhibitors and the expression of IRF-3 and IRF-7 was analyzed by immunoblotting, detected with enhanced chemiluminescence, and then analyzed with the Quantity One densitometric software. Simultaneous staining of β-actin on the same membrane was used as a control for protein loading in the individual lanes. Results are presented as the ratio of the intensity of the IRF-3 or IRF-7 band to the intensity of the β-actin band. Data presented are the means ± SEM (n = 6 per group). P < 0.05 is considered statistically significant.

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