Duck Tembusu Virus Exhibits Pathogenicity to Kunming Mice by Intracerebral Inoculation

In this study, Kunming mice were used as the animal models to study the pathogenicity of TMUV. Three groups of 3-week-old female Kunming mice (n=15 mice per group) were infected with the SDSG strain of TMUV in 50μL allantoic fluid (104.8 ELD50/ 0.2ml) respectively by the intracerebral (i.c.), subcutaneous (s.c.) and intranasal (i.n.) routes. The control group (n=15 mice) was inoculated with 50μL sterile phosphate-buffered saline (PBS). Clinical signs, gross and microscopic lesions, viral loads in different tissues, and serum antibody titers were examined and recorded. Kunming mice infected intracerebrally showed typical clinical symptoms, including severe hindlimb paralysis, weight loss and death. Only dead mice presented severe intestinal mucosal edema. No gross lesions were observed in mice sequentially euthanized. However, microscopic lesions in the brain, spleen, liver, kidney and lung were very typical including varying degrees of viral encephalitis, lymphocytes depletion, liver cell necrosis and nephritis, etc. Viral loads in different tissues were detected by the SYBR Green I real-time PCR assay. Viral loads in the brain, liver and spleen were first detected and maintained a longer time, which indicated that these organs may be the target organs of TMUV. The level of viral loads was consistent with the severity of clinical signs and microscopic lesions in different tissues. The neutralizing antibody began to seroconvert at 8dpi. Clinical signs, microscopic lesions, viral loads and serum neutralizing antibodies weren’t observed in other groups. In summary, TMUV can cause systemic infections and death in Kunming mice by i.c., which provides some experimental basis for further study of the significance of TMUV in public health

Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb) 3G2 by indirect enzyme-linked immunosorbent assay (ELISA). In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays

Western-blot identification of epitope.

<p>The 16-AA polypeptide of NS1-27 reacted with mAb 3G2 in Western-blot assay. Lane M, PageRuler Prestained Protein Ladder (Fermentas, Canada); Lane 1, GST-tag didn’t react with mAb 3G2; Lane 2, The band of NS1-27-GST fusion protein was visualized with mAb 3G2.</p

Sequences of the overlapping polypeptides from TMUV NS1 (SDSG strain, Accession number: KJ740747.1).

<p>Sequences of the overlapping polypeptides from TMUV NS1 (SDSG strain, Accession number: KJ740747.1).</p

Sequences of the truncated NS1-27 polypepide.

<p>Sequences of the truncated NS1-27 polypepide.</p

IFA and western-blot identification of mAb 3G2.

<p>A: Western-blot identification Lane 1, Control, GST-tag didn’t react with mAb 3G2; Lane 2, The band of NS1-GST fusion protein was reacted with mAb 3G2; Lane M, Blue plusIIprotein Marker (14-120kda, Transgen Biotech). B: IFA identification Monoclonal antibody against TMUV NS1 protein was used to perform IFA on TMUV-infected BHK-21 cells. BHK-21 cells infected with TMUV yielded significant fluorescence with six MAbs in the cytoplasm; Control BHK-21 cells didn’t yield any fluorescence.</p

The accurate mapping of one B cell epitope with mAb.

<p>NS1-27 polypeptide was truncated from the carboxy and amino terminals. After the truncated peptides were expressed as a GST fusion protein, they were probed with mAb 3G2 by indirect ELISA respectively. The minimal unit of the peptide was the sequence of 8 AA and 3 AA truncated from the carboxy and amino terminals of NS1-27.</p

Primary screening of epitope with mAb 3G2.

<p>One 16-AA polypeptide of TMUV NS1 protein (NS1-27) was screened with mAb 3G2 by indirect ELISA. Mouse serum against TMUV NS1 protein and normal mouse serum were used as positive and negative controls, respectively. Each sample was detected in triplicate. Error bars were expressed as standard deviation of the means (n = 3). The mean value was statistically significant, calculated by the two-tailed Student’s unpaired t-test (*P < 0.05).</p

BST2 negatively regulates porcine reproductive and respiratory syndrome virus replication by restricting the expression of viral proteins

Porcine reproductive and respiratory syndrome virus (PRRSV) has seriously affected the viability of swine industries worldwide, and effective measures to control PRRSV are urgently required. Understanding the mechanisms of action of antiviral proteins is crucial for developing antiviral strategies. Interferon-induced bone marrow stromal cell antigen 2 (BST2) can inhibit the replication of various viruses via different pathways. However, little is known about the effects of BST2 on PRRSV. Therefore, this study aimed to evaluate whether the interferon-induced BST2 can inhibit PRRSV replication. We used western blotting and RT-qPCR techniques to analyze the effect of BST2 overexpression and knockdown on PRRSV replication. Overexpression of BST2 inhibited the replication of PRRSV, whereas knockdown of BST2 by small interfering RNA promoted PRRSV replication. Additionally, the expression of BST2 was upregulated during the early phase of PRRSV infection in porcine alveolar macrophages. Analysis of PRRSV proteins showed that BST2 restricted the expression of several non-structural viral proteins. BST2 downregulated the expression of Nsp12 through a proteasome-dependent pathway and downregulated the expression and transcription of E protein. These findings demonstrate the potential of BST2 as a critical regulator of PRRSV replication