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.

Novel duck reovirus exhibits pathogenicity to specific pathogen-free chickens by the subcutaneous route.

To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 µL (10-5.00 ELD50/0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 µL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

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.

The effect of Tembusu virus infection in different week-old Cherry Valley breeding ducks.

To study the effect of Tembusu virus (TMUV) infection on Cherry Valley Breeding ducks of different ages, 350 five-week-old ducks were divided into 14 groups. Ducks in seven experimental group were respectively infected with 1.265×10(5) mean embryo lethal dose (ELD50) of TMUV-AHQY strain (in 4.2mL) by intravenous route. Ducks in control groups were inoculated with Phosphate-buffered Saline (PBS) in the same way. Clinical symptoms, gross and microscopic lesions, viral loads and serum antibodies were detected and recorded for 20days after infection. Some ducks infected at 7 and 21 week s of age showed severe clinical symptoms including depression and inappetence, and no obvious clinical symptoms were seen in other week-old infected ducks. Severe gross lesions including hepatomegaly, meningeal congestion, myocardial hemorrhage, intestinal, myocardial and pulmonary edema were observed in ducks infected at 7, 18 and 21 weeks of age. No or mild gross lesions were observed in ducks infected at 14 and 16 weeks of age. The main microscopic lesions including hyperaemia, degeneration and necrosis of different cells and inflammatory cellular infiltration mainly consisting of mononuclear cells or lymphocytes were observed in ducks infected at 7 and 21 week of age. But relatively intact structures and rare lymphocytic infiltration were presented in ducks infected at 14 and 16 weeks of age. Viral antigen was more frequently observed in organ slices collected from 7 week-old infected ducks and few positive staining was found in 14 and 16 week-old infected ducks. Less viral loads in different tissues and swabs were detected by a quantitative real-time PCR assay. The level of viral loads in the tissues of ducks infected at 14 and 16 weeks of age was very lower than that of ducks infected at 7 and 21 weeks of age. Meanwhile, less viral copy numbers were detected in swab samples collected from 14 and 16 week-old infected ducks. Ducks infected at 14-week-old developed significantly higher serum neutralizing antibody titers than those infected at other week of age. These results indicated that the effect of TMUV infection on Cherry Valley ducks is partly related to weeks of age. 7-10 week-old and 18-21 week-old ducks were more susceptible to TMUV infection, but 14-16 week-old ducks were more resistant to this disease.

Development of a TaqMan-based real-time PCR assay for the detection of Novel GPV.

The newly emerged disease, duck beak atrophy and dwarfism syndrome (BADS), is caused by novel goose parovirus (N-GPV). Although N-GPV infection has severe consequences, few methods for detecting this virus have been developed. Therefore, the availability of rapid and reliable molecular diagnostic methods would aid future studies of this novel virus. Clinical specimens from 138 suspected cases of N-GPV infection and 120 cloacal swabs from breeding ducks were used in this study. The targeted sequence of N-GPV cloned into the pMD18-T vector was used to generate the N-GPV DNA standard curve. The specificity of the assay was validated using duck plague virus, GPV, duck hepatitis virus, avian influenza virus, duck reovirus, tembusu virus, and fowl adenovirus. The lowest limit of detection was 8.8×101copies/µL with a good linear standard curve (Y=-3.3682X+37.220, R2=0.9953) over a wide range of input DNA, of which the concentration was between 8.8×101 to 8.8×108copies/µL. The results show that the real-time PCR assay is a highly sensitive, specific, reproducible, and versatile method for quantitatively detecting N-GPV DNA, and thus can be used to detect this virus, thereby facilitating epidemiological investigations of animals with BADS.

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 50 µL allantoic fluid (10(4.8) ELD50/0.2 ml) respectively by the intracerebral (i.c.), subcutaneous (s.c.) and intranasal (i.n.) routes. The control group (n = 15 mice) was inoculated with 50 µL sterile phosphate-buffered saline. 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 8 dpi. 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.

Development and Characterization of Monoclonal Antibodies Against NS1 Protein of Duck Tembusu Virus.

To produce monoclonal antibodies (MAbs) against NS1 protein of duck Tembusu virus (TMUV), the NS1 gene sequence was cloned into the prokaryotic expression vector pET-28a(+). The prokaryotic protein NS1 was successfully expressed in BL21 (DE3) and used as the immunogen in mice. Six MAbs against NS1 protein were obtained by using the standard hybridoma technique. All MAbs can react with the denatured NS1 protein in the Western blot assay and the native NS1 protein from the TMUV-infected BHK-21 cells in the immunofluorescence assay. The ELISA titers of the cell supernatants and ascites of MAbs were at a high level. The subtypes of the MAbs were determined by the Rapid Mouse Isotyping Kit-Gold series. Six MAbs possessed higher specificity and sensitivity, which indicated that MAbs against NS1 protein of TMUV may be used as valuable tools for analysis of the protein functions and pathogenesis of TMUV.

Effect of age and inoculation route on the infection of duck Tembusu virus in Goslings.

Duck Tembusu virus (TMUV) is an emerging flavivirus that has caused variable levels of outbreaks in poultry in recent years. In order to study the effect of age and inoculation routes on the TMUV infection, one hundred healthy domestic 5-day-old and 20-day-old goslings were equally divided into five groups and four experimental groups of goslings were infected with the TMUV-SDSG strain by intravenous and intranasal routes, respectively. Severe clinical signs were observed in goslings infected at 5 days of age, including listlessness, growth retardation, severe neurological dysfunction and even death. However, goslings infected at 20 days of age showed mild symptoms and no mortality. The severity of gross lesions gradually reduced as goslings matured. The severe histopathological changes were observed in 5-day-old infected goslings, including cerebral edema, viral encephalitis, myocardial necrosis, hepatic steatosis, spleen lymphoid cell depletion, pancreatic epithelial cell shedding and interstitial hemorrhage. However, 20-day-old infected goslings showed mild histopathological changes. Viral loads in different tissues were detected by the SYBR Green I real-time PCR assay. The level of viral loads in most of tissues 5-day-old infected goslings was higher than that of 20-day-old infected goslings, correlating with the severity of clinical symptoms and lesions in these tissues. 20-day-old infected goslings developed significantly higher serum neutralizing antibody titers than 5-day-old infected goslings. Furthermore, goslings infected with TMUV intravenously demonstrated more severe clinical signs, lesions and higher viral loads in tissues than those of goslings infected with TMUV intranasally. Therefore, age and inoculation routes can affect the pathogenicity of TMUV in geese and younger geese are more susceptible to the virus. Age and inoculation route factors should be considered in study of the pathogenicity, pathogenesis, folumation of prevention and therapy strategies of TMUV infection in geese.

Evidence of possible vertical transmission of Tembusu virus in ducks.

In 2013, Tembusu virus (TMUV) infection was successively observed on several breeding duck farms in Shandong province, China. Affected ducks showed consistently acute anorexia, diarrhea and egg production drop. 125 hatching eggs produced by TMUV infected breeding ducks from four duck farms were collected. Among them, 35 hatching eggs were selected randomly from all before incubation for vitelline membrane samples collection. The rest of 90 hatching eggs were incubated routinely. As a result, 16 hatching eggs were found non-embryonated, 28 duck embryos died during incubation and 46 newly hatched ducklings were obtained. Vitelline membranes of non-embryonated hatching eggs, vitelline membrane, brain or liver samples of dead embryos and brain samples of newly hatched ducklings were collected for virus detection. Samples collected from one egg, embryo or duckling were treated as one. Consequently, 18 of 35 (51.43%) hatching eggs, 2 of 16 (12.50%) non-embryonated duck eggs, 17 of 28 (60.71%) dead duck embryos and 5 of 46 (10.87%) newly hatched ducklings were detected positive for TMUV using NS3-based RT-PCR. Overall, 42 of 125 (33.6%) eggs were positive for TMUV. A virus strain, designated as TMUV-SDDE, was isolated from one of these dead duck embryos which were detected TMUV positive. The results of phylogenetic analysis showed that E gene of TMUV-SDDE virus was closely related to other TMUV strains isolated in China during 2010-2013. Pathogenicity studies showed that TMUV-SDDE strain was virulent to ducklings. This is the first report that TMUV is isolated from duck embryos. The findings provide evidence of possible vertical transmission of TMUV from breeding ducks to ducklings.

Complete genome sequences of two waterfowl-origin tembusu virus strains isolated in shandong province, china.

Here, we report the complete genome sequences of two tembusu virus strains, ZC-1 and LQ-1, isolated from ducks and geese, respectively, in 2012. Phylogenetic analysis showed that the nucleotide and amino acid sequences of the two strains are closely related to those of the TMUV isolates around Shandong province. The full-length genome sequences of two waterfowl-origin TMUVs provided herein will help to understand the molecular epidemiology of tembusu virus in China, which deserves further investigation.