Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

A follow-up study of 100 patients with acute brucellosis for its prognosis and prevention.

Objective: To prevent chronic brucellosis, this study analysed the changes in patient antibody titers, and the trajectories of biochemical indicators at different stages of brucellosis, identified relevant biomarkers, and explored risk factors affecting the prognosis of brucellosis patients. Methods: A prospective cohort study was conducted to follow 100 patients with acute brucellosis. Laboratory serological test results [taken with a serum (tube) agglutination test (SAT)] and biochemical parameters (liver function, renal function, and hematological system) were measured repeatedly at four-time points: 0 weeks-baseline survey, 6 weeks after the first treatment, 12 weeks after the second treatment, and 3 months after the third treatment. The changes in the antibody titres and biochemical parameters at each time point were analysed for trend changes. Results: One hundred patients with acute brucellosis were enrolled in this follow-up study, with 100% retention in follow-up. By the third follow-up, 21 patients had turned subacute and 11 had turned chronic. One-way repeated measures analysis of variance results showed statistically significant differences (p < 0.01) across the time points for the following five indicators: alanine aminotransferase, aspartate aminotransferase, total bilirubin, serum creatinine (SCr) and platelet count. The clinical symptoms of patients in the acute stage were mainly joint pain, fatigue, and fever, while those in the chronic stage complained primarily of joint pain and fatigue. The results of multivariate logistic analysis showed that joint pain [odds ratio (OR) = 3.652, 95% confidence interval (CI) =1.379-9.672], monoarticular pain (OR = 6.356, 95% CI = 4.660-8.669), elevated SCr (OR = 15.804, 95% CI = 1.644-151.966) and elevated haemoglobin (Hb) (OR = 1.219, 95% CI = 1.065-1.736) were risk factors for poor prognosis (not cured or chronic) in patients with brucellosis. Conclusion: The trajectory of changes in patient SAT posirates and antibody titers can be used to distinguish patients with chronic brucellosis. The brucellosis is preventable and treatable, and the standard treatment can be effective in reducing the clinical symptoms of affected patients. If patients are not treated in a timely manner, joint pain, monoarticular pain, and elevated SCr are risk factors for patients who are not cured. Therefore, the treatment cycle for these patients should be extended.

Evaluation of immunogenicity of gene-deleted and subunit vaccines constructed against the emerging pseudorabies virus variants.

BACKGROUND: Pseudorabies (PR) (also called Aujeszky's disease, AD) is a serious infectious disease affecting pigs and other animals worldwide. The emergence of variant strains of pseudorabies virus (PRV) since 2011 has led to PR outbreaks in China and a vaccine that antigenically more closely matches these PRV variants could represent an added value to control these infections. METHODS: The objective of this study was to develop new live attenuated and subunit vaccines against PRV variant strains. Genomic alterations of vaccine strains were based on the highly virulent SD-2017 mutant strain and gene-deleted strains SD-2017ΔgE/gI and SD-2017ΔgE/gI/TK, which constructed using homologous recombination technology. PRV gB-DCpep (Dendritic cells targeting peptide) and PorB (the outer membrane pore proteins of N. meningitidis) proteins containing gp67 protein secretion signal peptide were expressed using the baculovirus system for the preparation of subunit vaccines. We used experimental animal rabbits to test immunogenicity to evaluate the effect of the newly constructed PR vaccines. RESULTS: Compared with the PRV-gB subunit vaccine and SD-2017ΔgE/gI inactivated vaccines, rabbits (n = 10) that were intramuscularly vaccinated with SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine showed significantly higher anti-PRV-specific antibodies as well as neutralizing antibodies and IFN-γ levels in serum. In addition, the SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine protected (90-100%) rabbits against homologous infection by the PRV variant strain. No obvious pathological damage was observed in these vaccinated rabbits. CONCLUSIONS: The SD-2017ΔgE/gI/TK live attenuated vaccine provided 100% protection against PRV variant challenge. Interestingly, the subunit vaccines with gB protein linked to DCpep and PorB protein as adjuvant may also be a promising and effective PRV variant vaccine candidate.

Glycosylated protein 4-deficient PRRSV in complementing cell line shows low virus titer.

Porcine Reproductive and Respiratory Syndrome (PRRS) threats the swine industry seriously. The spread of live vaccine virus leads to the emergence of recombinant virus, which brings biosafety problems. The replication-deficient virus as a vaccine candidate would avoid this problem. In the present study, the recombinant lentiviral plasmid pLV-EF1α-EGFP-2A-ORF4 was co-transfected with lentivirus in HEK293FT cells. The transfection mixture was harvested and transduced into Marc-145 to screen a cell line stably expressing the PRRSV ORF4 with puromycin. The cell line Marc-145-GP4 was confirmed with PCR, RT-PCR, IFA, and Western blotting using a monoclonal antibody against Glycoprotein 4 (GP4) of PRRSV. To obtain a replication-deficient PRRSV, Western blotting the recombinant plasmid pNM09-ΔORF4 was constructed by Overlap PCR and DNA recombinant technology with the pNM09 as a backbone plasmid. The pNM09-ΔORF4 was transfected into Marc-145-GP4 with electroporation after transcription in vitro. The replication-deficient virus was rescued on Marc-145-GP4 cells with trans-complementation of ORF4 gene and verified by RT-PCR and IFA. The results indicated that a cell line Marc-145-GP4 stably expressed PRRSV ORF4 was obtained. The recombinant GP4 was successfully expressed and obtained a monoclonal antibody Anti-A-GP4-70, which can specifically react with the virus. Finally, the replication-deficient virus rNM09-ΔORF4 can be rescued with low titer and could only reproduce on the Marc-145-GP4 cells. Unfortunately, the rNM09-ΔORF4 showed too low virus replication titer to determine it. This study lays the foundation for the rapid detection of PRRS and the functional study of GP4 and provides experience for replication-deficient PRRSV.

Epidemiological Characteristics and Clinical Manifestations of Brucellosis and Q Fever Among Humans from Northeastern Inner Mongolia.

Objective: To investigate the distribution, epidemiology, and clinical symptoms of brucellosis and Q fever in northeastern Inner Mongolia. Methods: In this study, 64 townships of Bairin left flag and Alukerqin flag, Jarud flag and Horqin right front flag in four counties with frequent brucellosis and Q fever were selected. Epidemiological characteristics, clinical features, and exposure to risk factors were identified and descriptively analyzed in patients from these areas. Results: There were 367 brucellosis cases in the four regions and 78 positive cases of Q-fever infection. In addition, 24 cases of brucellosis and Q-fever co-infection were identified, with a co-infection rate of 1.13%. Brucellosis and Q fever were mainly concentrated in the 30-65 and 40-55 age groups. For brucellosis, the difference between age groups was statistically significant (χ2 = 29.121, P < 0.05). The sex distribution for brucellosis was 225 men (61.31%) and 142 women (38.69%), and 45 men (57.69%) and 33 women (42.31%) had Q fever. Those with brucellosis and Q fever were mainly farmers, accounting for 79.19% and 78.38% of the total number, respectively. Of the 367 cases of brucellosis infection, the main symptoms were joint pain (52.59%), fatigue (47.14%), lower back pain (38.96%), fever (33.24%), hyperhidrosis (28.88%), and muscle pain (20.44%). Of the 78 cases of Q-fever infection, the main symptoms were joint pain (35.90%), fatigue (30.77%), lower back pain (26.92%), fever (21.79%), and hyperhidrosis (17.95%). Muscle pain also accounted for 12.82%. Conclusion: Occupational distribution suggests that we should strengthen the protection measures against diseases infected through animal husbandry. Among the clinical symptoms, fever, hyperhidrosis and fatigue were associated with brucellosis, while fever, headache, and fatigue were significantly associated with Q fever.

Subunit vaccine based on glycoprotein B protects pattern animal guinea pigs from tissue damage caused by infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis (IBR) is caused by Bovine herpesvirus type 1 (BoHV-1), which seriously threatens the global cattle industry. Only vaccination to improve immunity is the most direct and effective means to prevent IBR. Attempts are being made to use subunit vaccines, deleted or recombinant viral vaccines to reduce or eradicate IBR. For investigating the immunological characteristics of glycoprotein B subunit vaccine in pattern animal guinea pigs, the partial glycoprotein B (gB) of BoHV-1 with dominant antigenic characteristic was selected. A recombinant prokaryotic expression vector pET-32a-gB with the truncated gB gene was constructed, expressed, identified and the purified proteins were used to immunize guinea pigs. The immune effect of the subunit vaccine was assessed by monitoring clinical symptoms, viral load, antibody secretion, and histopathological changes. The results indicated that guinea pigs immunized with the gB subunit vaccine produced high levels of anti-gB antibodies and virus-neutralizing antibodies. The gB subunit vaccine significantly reduced viral shedding and lung tissue damage after IBRV challenge. The animals inoculated the gB subunit vaccine also had less virus reactivation. Its protective effect on viral shedding and tissue damage was similar to that of inactivated BoHV-1 vaccine. This work is a proof-of-concept study of subunit vaccine-induced protection against BoHV-1. And it is expected to be a candidate vaccine for the prevention of IBR.

Comparative proteomic analysis of PK15 swine kidney cells infected with a pseudorabies pathogenic variant and the Bartha-K/61 vaccine strain.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease and is communicable across species. In particular, the emergence of PRV variants in 2011 have resulted in serious economic losses to the Chinese pig industry. In this study, we used tandem mass tag (TMT) quantitative protein analysis to identify differentially expressed proteins between the PRV variant strain SD-2017 and the vaccine strain Bartha-K/61 in the swine kidney cell line PK15. Overall, we identified 4690 proteins for SD-2017 infection compared with the mock-infected control cells. We found 162 differentially expressed cellular proteins including 41 up- and 121 down-regulated proteins. SD-2017-infected PK15 cells differential proteins were primarily related to gap junctions, the phagosome, antigen processing and presentation, cell adhesion molecules and peroxisome pathways. Compared to Bartha-K/61-infected PK15 cells, SD-2017-infected cells displayed differentially expressed proteins involved in tryptophan metabolism, mitophagy and Notch signaling. Western blot analysis of MARK2, TSR1 and TMED1 three representative proteins validated the reliability of the TMT data. This study is an initial at-tempt to compare the proteomes of PK15 cells infected by a PRV variant and a vaccine strain using TMT technology to provide new insights into the mechanisms of PRV pathogenesis and immune evasion.

Recent advances in the study of NADC34-like porcine reproductive and respiratory syndrome virus in China.

Since porcine reproductive and respiratory syndrome virus (PRRSV) was first described in China in 1996, several genetically distinct strains of PRRSV have emerged with varying pathogenicity and severity, thereby making the prevention and control of PRRS more difficult in China and worldwide. Between 2017 and 2021, the detection rate of NADC34-like strain in China increased. To date, NADC34-like strains have spread to 10 Chinese provinces and have thus developed different degrees of pathogenicity and mortality. In this review, we summarize the history of NADC34-like strains in China and clarify the prevalence, genomic characteristics, restriction fragment length polymorphisms, recombination, pathogenicity, and vaccine status of this strain in China. In so doing, this study aims to provide a basis for the further development of prevention and control measures targeting the NADC34-like strain.

Recombinant Bovine Herpesvirus Type I Expressing the Bovine Viral Diarrhea Virus E2 Protein Could Effectively Prevent Infection by Two Viruses.

Bovine respiratory disease complex (BRDC) is a comprehensive disease in cattle caused by various viral and bacterial infections. Among them, bovine herpesvirus type I (BoHV-1) and bovine viral diarrhea virus (BVDV) play important roles and have caused huge financial losses for the cattle industry worldwide. At present, vaccines against BRDC include trivalent attenuated BoHV-1, BVDV-1, and BVDV-2 live vaccines, BoHV-1 live attenuated vaccines, and BoHV-1/BVDV bivalent live attenuated vaccines, which have limitations in terms of their safety and efficacy. To solve these problems, we optimized the codon of the BVDV-1 E2 gene, added the signal peptide sequence of the BoHV-1 gD gene, expressed double BVDV-1 E2 glycoproteins in tandem at the BoHV-1 gE gene site, and constructed a BoHV-1 genetics-engineered vectored vaccine with gE gene deletion, named BoHV-1 gE/E2-Linker-E2+ and BoHV-1 ΔgE. This study compared the protective effects in BoHV-1, BoHV-1 ΔgE, BoHV-1 gE/E2-Linker-E2+, and BVDV-1 inactivated antigen immunized guinea pigs and calves. The results showed that BoHV-1 gE/E2-Linker-E2+ could successfully induce guinea pigs and calves to produce specific neutralizing antibodies against BVDV-1. In addition, after BoHV-1 and BVDV-1 challenges, BoHV-1 gE/E2-Linker-E2+ can produce a specific neutralizing antibody response against BoHV-1 and BVDV-1 infections. Calves immunized with this type of virus can be distinguished as either vaccinated animals (gE-) or naturally infected animals (gE+). In summary, our data suggest that BoHV-1 gE/E2-Linker-E2+ and BoHV-1 ΔgE have great potential to prevent BVDV-1 or BoHV-1 infection.

Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing.

Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5' and 3' ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3' end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP+ or EGFP- will lay the foundation for research on BoHV-1 and vaccine development in the future.

Analysis of laboratory and serological test results in patients with acute brucellosis during follow-up.

BACKGROUND: The laboratory test results and serum-specific antibodies of patients with acute brucellosis initial infection were followed up and analyzed. METHODS: 70 patients in Hohhot City, Inner Mongolia Autonomous Region, with acute brucellosis were followed up for 360 days. Serum samples were collected at 0, 15, 30, 60, 90, 180, and 360 days after diagnosis and analyzed by Rose Bengal plate test (RBPT), colloidal gold test paper (GICA), and test tube agglutination test (SAT). The serum-specific antibodies IgG and IgM were detected. RESULTS: RBPT results: False negative (-) gradually increased with the extension of the course of disease, with the largest change in 30-60 days after diagnosis, and the constituent ratio increased by 12.9%. GICA results: The false negative increased with the course of disease, and the constituent ratio of false negative was 20.0% after 180 days of diagnosis. SAT results: 1:100 positive showed a ladder like decrease with the increase in the course of disease, and the largest decrease was 90-180 days, with a decrease of 34.3% in the constituent ratio. 360 days after diagnosis, the constituent ratio of positive was only 14.3%. During the follow-up period, the IgG average value fluctuated and the average IgM value decreased. CONCLUSION: The false-negative results of RBPT, GICA, and SAT increased with the course of disease, and the false-negative rates were higher than 20% after half a year. IgM level is beneficial to the early diagnosis of brucellosis, while IgG level is helpful to the judgment of brucellosis stage.

Transcriptome sequencing analysis of porcine alveolar macrophages infected with PRRSV strains to elucidate virus pathogenicity and immune evasion strategies.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) causes a serious disease to the swine industry worldwide. To understand the mechanisms of HP-PRRSV infection, RNA-seq-based transcriptome analyses were performed on porcine alveolar macrophages (PAMs) infected with a HP-PRRSV strain (TJ), a less virulent strain of a classical lineage (CH-1a), and a vaccine strain TJM-F92. Gene ontology, Kyoto Encyclopedia of Genes and Genomes analyses indicate that TJM-F92 led to significant up-regulation of gene expression for proteins associated with membrane-bound organelles. The differentially expressed genes of HP-PRRSV TJ-infected PAM cells were up-regulated in the special G-protein coupled receptor. The six cytokines were tested by real time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The relative expression levels showed the same trend of expression difference. Significant up-regulation of TMEM173 plays an important role in the cytosolic DNA-sensing pathway and the RIG-I-like receptor signaling pathway in TJM-F92 infected PAM cells. These data provide new insight into PRRSV pathogenicity and immune evasion strategies.

Advance of African swine fever virus in recent years.

African swine fever (ASF) is one of the most devastating hemorrhagic infectious diseases that affect pigs and wild suids due to the lack of a vaccine or an effective treatment. The large dsDNA genome of African swine fever virus (ASFV) contains up to 167 ORFs that are predicted to encode proteins. Since its introduction to China in 2018, this genome has aroused the enthusiasm of researchers throughout the world. Here, we review the research progress on ASFV in recent years. Given the importance of this disease, this review will highlight recent discoveries in basic virology, focusing mainly on epidemiology, virulence, pathogenic mechanisms, diagnosis, vaccine development, and treatment; this will help in understanding virus-host interactions and disease prevention regarding ASFV.

Molecular evolutionary analysis reveals Arctic-like rabies viruses evolved and dispersed independently in North and South Asia.

BACKGROUND: Arctic-like (AL) lineages of rabies viruses (RABVs) remains endemic in some Arctic and Asia countries. However, their evolutionary dynamics are largely unappreciated. OBJECTIVES: We attempted to estimate the evolutionary history, geographic origin and spread of the Arctic-related RABVs. METHODS: Full length or partial sequences of the N and G genes were used to infer the evolutionary aspects of AL RABVs by Bayesian evolutionary analysis. RESULTS: The most recent common ancestor (tMRCA) of the current Arctic and AL RABVs emerged in the 1830s and evolved independently after diversification. Population demographic analysis indicated that the viruses experienced gradual growth followed by a sudden decrease in its population size from the mid-1980s to approximately 2000. Genetic flow patterns among the regions reveal a high geographic correlation in AL RABVs transmission. Discrete phylogeography suggests that the geographic origin of the AL RABVs was in east Russia in approximately the 1830s. The ancestral AL RABV then diversified and immigrated to the countries in Northeast Asia, while the viruses in South Asia were dispersed to the neighboring regions from India. The N and G genes of RABVs in both clades sustained high levels of purifying selection, and the positive selection sites were mainly found on the C-terminus of the G gene. CONCLUSIONS: The current AL RABVs circulating in South and North Asia evolved and dispersed independently.

SNAP25 regulates the release of the Rabies virus in nerve cells via SNARE complex-mediated membrane fusion.

Recent studies have reported that host proteins regulate Rabies virus (RABV) infection via distinct mechanisms. The abnormal neural function caused by RABV infection is related to the abnormal synaptic signal transmission in which the RABV glycoprotein (G) is involved. In the present study, two recombinant Rabies viruses (rRABVs), namely rSAD-SAD-Flag-G and rSAD-CVS-Flag-G, were established and rescued based on rSAD and verified by indirect fluorescence assay (IFA), and western blotting (WB). To investigate how the G protein interacts with synaptosomal-associated protein 25 (SNAP25), primary neuronal cells (PNC) of embryonic mice were cultured and infected with rRABVs. Immunoprecipitation (IP) and LC-MS/MS analysis of glycoprotein-binding proteins, which were flag tagged, were carried out to determine the interaction of G protein and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNARE) complex in PNC. G protein and the SNARE member SNAP25 were co-expressed in HEK293 cells or primary neuronal cells to investigate their colocalization. Knockdown of SNAP25 with small interfering RNA (siRNA) was conducted on mNA cells, and rRABV replication was observed by IFA, qRT-PCR, and virus titration. The results indicated that rRABVs were successfully rescued and grew well in PNC. Flag-tag IP and confocal microscopy demonstrated that SNAP25 works together with G protein and colocalizes with G on the cytomembrane of HEK293 cells. The downregulation of SNAP25, using RNA interference, resulted in a significant decrease in the number of viral mRNAs, viral proteins, and virus particles. Furthermore, the regression of SNAP25 did not affect the initial infection of the virus but reduced the infectivity of progeny virions.

Deficient Incorporation of Rabies Virus Glycoprotein into Virions Enhances Virus-Induced Immune Evasion and Viral Pathogenicity.

Previous studies have shown that wild-type (wt) rabies virus (RABV) evades the host immune response by restricting expression of glycoprotein (G), which blocks activation of dendritic cells (DCs) and induces production of virus-neutralizing antibodies (VNAs). In the present study, wt RABVs not only restricted G expression but also reduced incorporation of G into mature virions compared with laboratory-adapted viruses. A recombinant RABV expressing triple G was used to further determine whether G expression relates to incorporation. The recombinant virus showed higher expression and incorporation of G and activated more DCs than the virus that expressed a single copy of G. Removal of G from viruses using subtilisin or Dithiothreitol (DTT)/ Nonidet P-40 (NP40) almost completely abolishes DC activation and VNA production. Consequently, these G-depleted viruses cause lethal infection in mice. Thus, wt RABVs can subvert DC-induced antiviral immune response and maintain pathogenicity by decreasing G expression in infected cells and G incorporation into virions.

Development of an Immunochromatographic Strip for Rapid Detection of Canine Adenovirus.

Although canine adenovirus (CAdV) is highly prevalent in dogs, there is currently a lack of a quick diagnostic method. In this study, we developed a rapid immunochromatographic strip (ICS) assay using colloidal gold coupled to CAdV-2-specific monoclonal antibodies (mAbs). BALB/c mice were immunized with a purified CAdV-2 suspension, and four mAbs (belonging to two different epitopes) were generated and designated as 2C1, 7D7, 10D1, and 4G1. Western blot and protein spectral analysis indicated that the hexon protein of CAdV-2 recognized all four mAbs. The colloidal gold-coupled 7D7 and 2C1 mAbs were chosen for inclusion in the rapid ICS assay. The optimal concentrations of the coating antibody (2C1), the capture antibody (7D7), and the goat anti-mouse antibody were 1.0 mg/ml, 10 µg/ml, and 2.0 mg/ml, respectively. The limit of detection was approximately 2.0 × 102 tissue culture infective dose (TCID50)/ml. Other common canine viruses were tested to evaluate the specificity of the ICS, and positive results were observed for only CAdV-1 and CAdV-2. The ICS test was conducted on 360 samples to detect CAdV, and the results were compared with those of polymerase chain reaction (PCR) tests. The ICS test was found to be a sufficiently sensitive and specific detection method for the convenient and rapid detection of CAdV.

Development of monoclonal antibody for differentiating porcine reproductive and respiratory syndrome virus and identification of a novel non-structural protein 2 epitope peptide.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (NP) is the immunodominant region of PRRSV viral proteins. Non-structural protein 2 (Nsp2) and its hypervariable region play an essential role in the differential diagnosis of PRRSV. Western blot and immunofluorescence assay (IFA) analyses found that 2 out of 18 monoclonal antibodies (MAbs) recognized the NP and that 5 of 11 MAbs recognized Nsp2-120aa. IFA data demonstrated that 2 MAbs raised against the NP have a positive reaction to PRRSV; either HP-PRRSV, classic PRRSV or the vaccine strain at 1:100 dilution. Two MAbs raise against Nsp2-120aa also react positively with the classic PRRSV nor HP-PRRSV, but not with the PRRSV vaccine strain TJM-F92. Epitope mapping using truncated proteins identified a novel Nsp2-120aa epitope. In addition, we show that MAb BR/PNsp2-2A20 recognizes a 20 amino acid peptide (707) GRFEFLPKMILETPPPHPCG (727) of Nsp2. Based on our findings, we propose that MAb BR/PNsp2-2A20, raised against Nsp2-120aa of PRRSV, as a candidate specific diagnostic MAb for differentiation of the PRRSV virulent strains infected pig from vaccine strain TJM-F92 inoculated ones. The MAbs developed here have potential for use in diagnostic and research tools, including immunofluorescence assay, enzyme-linked immunosorbent assay and Western blotting.

Pathological and immunological characteristics of piglets infected experimentally with a HP-PRRSV TJ strain.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) remains a major threat to swine industry all over the world. The aim of this study was to investigate the mechanism of pathogenesis and immune responses caused by a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). RESULTS: All piglets experimentally infected with a HP-PRRSV TJ strain virus developed typical clinical signs of PRRS. The percentages of CD3+, CD4+, and CD8+ lymphocytes significantly decreased in the infected group as compared to the uninfected control animals (p < 0.01). Total WBC dropped in the infected animals during the experiment. The level of ELISA antibody against PRRSV increased in 7-10 days after infection and then started to decline. Pathological observations demonstrated various degree lesions, bleeding and necrosis in the lungs of the infected piglets. CONCLUSIONS: These results clearly indicated that HP-PRRSV TJ strain infection would activate host humoral immune response at the early period post infection and cause severe pathological damages on lungs and inhibit cellular immune response after infection.

Genetic analysis of porcine circovirus type 2 from dead minks.

Circovirus infection is a growing problem in the field of veterinary and public health. It is associated with enteric diseases in both mammalian and avian hosts. In this study, we detected and isolated porcine circovirus strains in the tissue samples of minks that died from diarrhoea in Shandong Province, China. We sequenced the whole genome of two porcine strains of Circovirus, designated as MiSD-1 and MiSD-2， which had a 97.34% similarity on nucleotide sequence and were closely related to porcine circovirus type 2 (PCV2), but distantly related to mink circoviral species. Phylogenetically MiSD-1 and MiSD-2 are a part of the PCV2b genotype cluster, which is a highly prevalent genotype worldwide. The closer relationship of MiSD-1 and MiSD-2 to PCV2 from pigs than to other mink circoviral species may be evidence of cross-species transmission and considerable zoonotic potential.