Combination of an enzyme-linked immunosorbent assay and an indirect fluorescence assay for a nationwide sero-survey of Toxoplasma gondii infection in pig herds in Taiwan.

Toxoplasma gondii is a zoonotic pathogen that can infect farm animals, companion animals, and humans, sometimes causing public health issues. In Taiwan, the pig industry is a vital agricultural industry, with a self-sufficiency rate of 91 %, and pigs are also food-producing animal reservoirs of Toxoplasma gondii. Infected pigs are usually asymptomatic, and abortions and death may occur in severe cases. We combined an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescence assay (IFA) to investigate the seroprevalence of Toxoplasma gondii among pig populations in Taiwan. A stratified sampling approach to determine the number of sample farms proportional to the number of pig farms in each county was employed, with 15 blood samples collected at each farm between July and September 2017. With the tested results, empirical Bayesian smoothing was utilized to assess the proportion of Toxoplasma-positive farms at the county level. Bayesian mixed-effects logistic regression models, incorporating farm and county as random effects, were employed to investigate associations between Toxoplasma test results and potential risk factors. A total of 930 serum samples from 62 pig farms were collected and tested. An overall herd prevalence of 27.4 % was shown with the seroprevalence in northern Taiwan being greater than that in southern Taiwan. The sampling month and companion dog density in 2017 were significantly associated with Toxoplasma infections in pigs. With every increase in the number of companion dogs per km² at the county level, the odds of Toxoplasma infection in pigs increased by 4.7 % (95 % CI: 1.7-8.9 %). This study demonstrated that combining ELISA for screening with IFA for confirmation is a cost-effective and time-saving method for conducting a large-scale sample investigation. This was also the first nationwide, cross-sectional study in Taiwanese pig herds to investigate Toxoplasma gondii infection.

[Influencing factors of traumatic arthritis after ankle fracture surgery and construction of risk prediction model].

OBJECTIVE: To explore risk factors of post-operative traumatic arthritis in patients with ankle fracture,and to establish risk prediction model. METHODS: Totally 550 patients with ankle fracture treated from May 2020 to May 2022 were selected as research objects and divided into modeling group (385 patients) and verification group (165 patients) according to 7:3. In modeling group,patients were classified as occurrence group (112 patients) and non-occurrence group (273 patients) according to whether traumatic arthritis occurred after opertaion. Age,body mass index(BMI),gender,smoking history,diabetes history,injury type,fracture type,operation time,manual labor,open injury,osteoporosis,poor reduction,postoperative weight-bearing time,vascular injury,and surgical method were recorded; risk factors of traumatic arthritis in ankle fracture patients were analyzed by single factor and multi factor logistic regression analyses; R software was used to build the prediction model of line graph;receiver operating characteristic (ROC) curve and calibration graph were applied to verify the discrimination and consistency of the model. RESULTS: One hundred and twelve of 385 patients with ankle fracture were developed to post-operative traumatic arthritis,and 275 did not. Univariate analysis showed that there were significant differences in age,BMI,fracture type,operation time,physical labor aboveⅡ,open injury,osteoporosis and poor reduction between two groups (P<0.05). Multivariate Logistic regression analysis showed that age (OR=2.887),BMI (OR=4.042),fracture type (OR=4.244),operation time (OR=2.665),physical labor above gradeⅡ(OR=5.099),osteoporosis (OR=10.219),and poor reduction (OR=3.112) were independent risk factors for traumatic arthritis after ankle fracture (P<0.05). Based on the above risk factors,an nomogram model was established to predict the risk of postoperative traumatic arthritis in ankle fracture patients,and internal and external verification was conducted. The results showed calibration curve of modeling group and verification group showed a good fit between correction curve and ideal curve,indicating that the predicted risk of postoperative traumatic arthritis by the model was basically consistent with actual risk. Area runder ROC curve analysis results showed 0.867[(95%CI(0.826,0.908)] and 0.882 [95%CI(0.827,0.938)],respectively,indicating that the prediction model had good prediction ability. CONCLUSION: Age,BMI,fracture type,operation time,physical labor above gradeⅡ,osteoporosis and poor reduction are all risk factors for post-operative traumatic arthritis in patients with ankle fracture. The prediction model based on the above risk factors could effectively evaluate risk of post-operative traumatic arthritis in patients with ankle fracture.

Identification of subgenomic mRNAs derived from the coronavirus 1a/1b protein gene: Implications for coronavirus transcription.

Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.

Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry.

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA-protein pull-down assay, six DVG-encoded proteins were identified by LC-MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.

Targeting the conserved coronavirus octamer motif GGAAGAGC is a strategy for the development of coronavirus vaccine.

BACKGROUND: Coronaviruses are pathogens of humans and animals that cause widespread and costly diseases. The development of effective strategies to combat the threat of coronaviruses is therefore a top priority. The conserved coronavirus octamer motif 5'GGAAGAGC3' exists in the 3' untranslated region of all identified coronaviruses. In the current study, we aimed to examine whether targeting the coronavirus octamer motif GGAAGAGC is a promising approach to develop coronavirus vaccine. METHODS: Plaque assays were used to determine the titers of mouse hepatitis virus (MHV)-A59 octamer mutant (MHVoctm) and wild-type (wt) MHV-A59 (MHVwt). Western blotting was used for the determination of translation efficiency of MHVoctm and MHVwt. Plaque assays and RT-qPCR were employed to examine whether MHVoctm was more sensitive to interferon treatment than MHVwt. Weight loss, clinical signs, survival rate, viral RNA detection and histopathological examination were used to evaluate whether MHVoctm was a vaccine candidate against MHVwt infection in BALB/c mice. RESULTS: In this study, we showed that (i) the MHVoctm with mutation of coronavirus octamer was able to grow to high titers but attenuated in mice, (ii) with the reduced multiplicity of infection (MOI), the difference in gene expression between MHVoctm and MHVwt became more evident in cultured cells, (iii) MHVoctm was more sensitive to interferon treatment than MHVwt and (iv) mice inoculated with MHVoctm were protected from MHVwt infection. CONCLUSIONS: Based on the results obtained from cultured cells, it was suggested that the synergistic effects of octamer mutation, multiplicity of infection and immune response may be a mechanism explaining the distinct phenotypes of octamer-mutated coronavirus in cell culture and mice. In addition, targeting the conserved coronavirus octamer motif is a strategy for development of coronavirus vaccine. Since the conserved octamer exists in all coronaviruses, this strategy of targeting the conserved octamer motif can also be applied to other human and animal coronaviruses for the development of coronavirus vaccines, especially the emergence of novel coronaviruses such as SARS-CoV-2, saving time and cost for vaccine development and disease control.

Development of Polycistronic Baculovirus Surface Display Vectors to Simultaneously Express Viral Proteins of Porcine Reproductive and Respiratory Syndrome and Analysis of Their Immunogenicity in Swine.

To simultaneously express and improve expression levels of multiple viral proteins of a porcine reproductive and respiratory syndrome virus (PRRSV), polycistronic baculovirus surface display vectors were constructed and characterized. We engineered polycistronic baculovirus surface display vectors, namely, pBacDual Display EGFP(BacDD)-2GP2-2GP4 and pBacDD-4GP5N34A/N51A (mtGP5), which simultaneously express and display the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP4-gp64TM-CTD, and His-tagged mtGP5-gp64TM-CTD fusion proteins of PRRSV on cell membrane of Sf-9 cells. Specific pathogen-free (SPF) pigs were administered intramuscularly in 2 doses at 21 and 35 days of age with genetic recombinant baculoviruses-infected cells. Our results revealed a high level of ELISA-specific antibodies, neutralizing antibodies, IL-4, and IFN-γ in SPF pigs immunized with the developed PRRSV subunit vaccine. To further assess the co-expression efficiency of different gene combinations, pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs were designed for the co-expression of the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP3-gp64TM-CTD, and His-tagged GP4-gp64TM-CTD proteins as well as the ectodomain of His-tagged mtGP5-gp64TM-CTD and His-tagged M-gp64TM-CTD fusion proteins of PRRSV. To develop an ELISA assay for detecting antibodies against PRRSV proteins, the sequences encoding the ectodomain of the GP2, GP3, GP4, mtGP5, and M of PRRSV were amplified and subcloned into the pET32a vector and expressed in E. coli. In this work, the optimum conditions for expressing PRRSV proteins were evaluated, and the results suggested that 4 × 105 of Sf-9 cells supplemented with 7% fetal bovine serum and infected with the recombinant baculoviruses at an MOI of 20 for three days showed a higher expression levels of the protein. Taken together, the polycistronic baculovirus surface display system is a useful tool to increase expression levels of viral proteins and to simultaneously express multiple viral proteins of PRRSV for the preparation of subunit vaccines.

Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

Synthesis and Anticancer Evaluation of 4-Anilinoquinolinylchalcone Derivatives.

A series of 4-anilinoquinolinylchalcone derivatives were synthesized and evaluated for antiproliferative activities against the growth of human cancer cell lines (Huh-7 and MDA-MB-231) and normal lung cells (MRC-5). The results exhibited low cytotoxicity against human lung cells (MRC-5). Among them, (E)-3-{4-{[4-(benzyloxy)phenyl]amino}quinolin-2-yl}-1-(4-methoxyphenyl) prop-2-en-1-one (4a) was found to have the highest cytotoxicity in breast cancer cells and low cytotoxicity in normal cells. Compound 4a causes ATP depletion and apoptosis of breast cancer MDA-MB-231 cells and triggers reactive oxygen species (ROS)-dependent caspase 3/7 activation. In conclusion, it is worth studying 4-anilinoquinolinylchalcone derivatives further as new potential anticancer agents for the treatment of human cancers.

The Use of Distinctive Monoclonal Antibodies in FMD VLP- and P1-Based Blocking ELISA for the Seromonitoring of Vaccinated Swine.

The serum neutralization (SN) test has been regarded as the "gold standard" for seroconversion following foot-and-mouth disease virus (FMDV) vaccination, although a high-level biosafety laboratory is necessary. ELISA is one alternative, and its format is constantly being improved. For instance, standard polyclonal antisera have been replaced by monoclonal antibodies (MAbs) for catching and detecting antibodies, and inactive viruses have been replaced by virus-like particles (VLPs). To the best of current knowledge, however, no researchers have evaluated the performances of different MAbs as tracers. In previous studies, we successfully identified site 1 and site 2 MAbs Q10E and P11A. In this study, following the established screening platform, the VLPs of putative escape mutants from sites 1 to 5 were expressed and used to demonstrate that S11B is a site 3 MAb. Additionally, the vulnerability of VLPs prompted us to assess another diagnostic antigen: unprocessed polyprotein P1. Therefore, we established and evaluated the performance of blocking ELISA (bELISA) systems based on VLPs and P1, pairing them with Q10E, P11A, S11B, and the non-neutralizing TSG MAb as tracers. The results indicated that the VLP paired with S11B demonstrated the highest correlation with the SN titers (R2 = 0.8071, n = 63). Excluding weakly positive serum samples (SN = 16-32, n = 14), the sensitivity and specificity were 95.65% and 96.15% (kappa = 0.92), respectively. Additionally, the P1 pairing with Q10E also demonstrated a high correlation (R2 = 0.768). We also discovered that these four antibodies had steric effects on one another to varying degrees, despite recognizing distinct antigenic sites. This finding indicated that MAbs as tracers could not accurately detect specific antibodies, possibly because MAbs are bulky compared to a protomeric unit. However, our results still provide convincing support for the application of two pairs of bELISA systems: VLP:S11B-HRP and P1:Q10E-HRP.

A stochastic assessment to quantify the risk of introduction of African swine fever virus to Taiwan via illegal pork products carried by international travellers.

The current study quantified the risk of releasing African swine fever virus (ASFV) into Taiwan from pork products illegally carried by international travellers from 157 countries or territories through six international airports and three international seaports. The association between various factors and the number of pork products detected by the border control authorities was also examined. The risk was estimated with a stochastic process after modelling the number of undetected illegal pork products, probability of pork product detection at international airports and seaports and probability of ASFV contamination of pork products from various countries. The overall annual probability of ASFV release to Taiwan was estimated to be 1 [95% confidence interval (CI): 1-1] under no enhanced mitigation measures. All the median airport-level risks were higher than .921, and four of them reached 1. The total annual risk was .570 (95% CI: .109-.937) for international seaports. The country or territory level risk was estimated to be 1 for Vietnam, China, Hong Kong, the Philippines and South Korea, .999 (95% CI: .628-1) for Macao and .967 (95% CI: .359-1) for Indonesia. After the total number of travellers was factored in, the number of detected illegal pork products was the highest in January and February, and travellers from Vietnam [risk ratio to Japan (RR): 80.45; 95% CI: 58.68-110.3], the Philippines (RR: 37.67; 95% CI: 26.9-52.74) and Cambodia (RR: 28.39; 95% CI: 12.69-63.51) were most likely to bring pork products to Taiwan. Our study indicated a high risk of ASFV introduction through international travellers and also identified the factors associated with the risk. This information can be used as empirical evidence for cost-effective risk mitigation practices.

The Effects of Freshwater Clam (Corbicula fluminea) Extract on Activated Hepatic Stellate Cells.

BACKGROUND: The extract of freshwater clams has been used to protect the body against liver diseases in traditional folk medicine. This study aims at investigating the effects of freshwater clam extract on activated hepatic stellate cells (aHSCs), which are critical contributors to liver fibrosis. METHODS: The aHSCs used in this study were derived from hepatic stellate cells that were isolated and purified from the livers of male Wistar rats and then transformed into the activated phenotype by culturing on uncoated plastic dishes. Freshwater clam extract (CE) was collected after the outflow from the live freshwater clams in a water bath at 100°C for 60 min. The effects of CE on aHSCs were analyzed by MTT assay, flow cytometry, Oil Red O (ORO) staining, western blot, and real-time RT-PCR. RESULTS: The results indicated that CE suppressed the proliferation of aHSCs through G0/G1 cell cycle arrest by downregulating cyclin D1 and upregulating p27. The expression levels of a-SMA, collagen I, TGF-ß, and TNF-α were inhibited in the CE-treated aHSCs. In addition, the CE treatment increased the lipid contents in aHSCs by promoting PPARγ expression. Furthermore, CE modulated the expression of ECM-related genes, i.e., by upregulating MMP-9 and downregulating TIMP-II. CONCLUSIONS: These data revealed that CE could induce the deactivation of aHSCs. We therefore suggest that CE has potential as an adjuvant therapeutic agent against hepatic fibrosis.

Phylogenetic Classification of Global Porcine Deltacoronavirus (PDCoV) Reference Strains and Molecular Characterization of PDCoV in Taiwan.

Porcine deltacoronavirus (PDCoV), a highly transmissible intestinal pathogen, causes mild to severe clinical symptoms, such as anorexia, vomiting and watery diarrhea, in piglets and/or sows. Since the first report of PDCoV infection in Hong Kong in 2012, the virus has readily disseminated to North America and several countries in Asia. However, to date, no unified phylogenetic classification system has been developed. To fill this gap, we classified historical PDCoV reference strains into two major genogroups (G-I and G-II) and three subgroups (G-II-a, G-II-b and G-II-c). In addition, no genetic research on the whole PDCoV genome or spike gene has been conducted on isolates from Taiwan so far. To delineate the genetic characteristics of Taiwanese PDCoV, we performed whole-genome sequencing to decode the viral sequence. The PDCoV/104-553/TW-2015 strain is closely related to the G-II-b group, which is mainly composed of PDCoV variants from China. Additionally, various mutations in the Taiwanese PDCoV (104-553/TW-2015) strain might be linked to the probability of recombination with other genogroups of PDCoVs or other porcine coronaviruses. These results represent a pioneering phylogenetic characterization of the whole genome of a PDCoV strain isolated in Taiwan in 2015 and will potentially facilitate the development of applicable preventive strategies against this problematic virus.

Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability.

Coronaviruses are able to establish persistence. However, how coronaviruses react to persistence and whether the selected viruses have altered their characteristics remain unclear. In this study, we found that the persistent infection of bovine coronavirus (BCoV), which is in the same genus as SARS-COV-2, led to alterations of genome structure, attenuation of gene expression, and the synthesis of subgenomic mRNA (sgmRNA) with a previously unidentified pattern. Subsequent analyses revealed that the altered genome structures were associated with the attenuation of gene expression. In addition, the genome structure at the 5' terminus and the cellular environment during the persistence were responsible for the sgmRNA synthesis, solving the previously unanswered question regarding the selection of transcription regulatory sequence for synthesis of BCoV sgmRNA 12.7. Although the BCoV variants (BCoV-p95) selected under the persistence replicated efficiently in cells without persistent infection, its pathogenicity was still lower than that of wild-type (wt) BCoV. Furthermore, in comparison with wt BCoV, the variant BCoV-p95 was not able to efficiently adapt to the challenges of alternative environments, suggesting wt BCoV is genetically robust. We anticipate that the findings derived from this fundamental research can contribute to the disease control and treatments against coronavirus infection including SARS-CoV-2.

The Impacts of Antivirals on the Coronavirus Genome Structure and Subsequent Pathogenicity, Virus Fitness and Antiviral Design.

With the global threat of SARS-CoV-2, much effort has been focused on treatment and disease control. However, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics are also unanswered questions with medical importance. To this end, bovine coronavirus (BCoV), which is in the same genus as SARS-CoV-2, was used as a test model and the findings were as follows. With the treatment of antiviral remdesivir, the selected BCoV variant with an altered genome structure developed resistance, but its pathogenicity was not increased in comparison to that of wild type (wt) BCoV. Under the selection pressure of innate immunity, the genome structure was also altered; however, neither resistance developed nor pathogenicity increased for the selected BCoV variant. Furthermore, both selected BCoV variants showed a better efficiency in adapting to alternative host cells than wt BCoV. In addition, the previously unidentified feature that the spike protein was a common target for mutations under different antiviral treatments might pose a problem for vaccine development because spike protein is a common target for antibody and vaccine designs. The findings derived from this fundamental research may contribute to the disease control and treatments against coronaviruses, including SARS-CoV-2.

Outbreak of Porcine Reproductive and Respiratory Syndrome Virus 1 in Taiwan.

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic lossesin the swine industry worldwide. The PRRS virus (PRRSV) can be divided into two species, PRRSV1 (European) and PRRSV 2 (North American). In Taiwan, PRRSV 2 isolates are dominant and causerespiratory symptoms in nursing pigs. From October to November 2018, in a pig herd in centralTaiwan, pregnant sows had abortions and stillbirths, and piglets suffered from respiratorydisorders. Laboratory tests identified the presence of PRRSV 1 in serum from sows and sucklingpiglets in this scenario. The complete genome of the identified PRRSV 1 strain was geneticallyclosely related to that of a European PRRSV vaccine strain (98.2%). This local European isolate isdesignated as PRRSV/NPUST-2789-3W-2/TW/2018 (NPUST2789). This report is the first to indicatean outbreak in Taiwan of a PRRSV 1 strain that shares a common evolutionary ancestor with theEuropean PRRSV vaccine strain.

Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan.

BACKGROUND: Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis strains can exist in single animals. Typing techniques are required for identifying G. parasuis isolates. Different strains within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence-associated trimeric autotransporters (vtaA) gene is an indicator of virulence. The aim of this study was to characterize Taiwanese G. parasuis isolates via molecular serotyping, vtaA PCR and ERIC-PCR. METHODS: One hundred and forty-five strains were collected between November 2013 and March 2017 in Taiwan and further examined by molecular serotyping, vtaA PCR and ERIC-PCR. RESULTS: The dendrogram revealed heterogeneous genetic diversity within many clusters. Partial correlation between the ERIC-PCR clusters of different strains, serovars and lesion patterns was observed. Twelve herds (8.3%) infected with more than one strain. Group 1 vtaA positive rate reached 98.6%. DISCUSSION: This study showed the high genetic diversity of G. parasuis in Taiwan by a high discriminatory capability of ERIC-PCR. Group 1 vtaA commonly exists in G. parasuis isolates and may play important roles in the pathogenesis of Taiwanese G. parasuis isolates.

Emergence of a virulent porcine reproductive and respiratory syndrome virus in Taiwan in 2018.

In March 2018, an abortion storm caused by porcine reproductive and respiratory syndrome virus was confirmed in a farrow-to-finish pig herd in Taiwan. Open reading frame 5 and non-structural protein 2 of the virus confirmed that the virus is closely related to the virulent strains circulating in the United States.

Discovery of 2-Substituted 3-Arylquinoline Derivatives as Potential Anti-Inflammatory Agents Through Inhibition of LPS-Induced Inflammatory Responses in Macrophages.

We describe herein the preparation of certain 2-substituted 3-arylquinoline derivatives and the evaluation of their anti-inflammatory effects in LPS-activated murine J774A.1 macrophage cells. Among these newly synthesized 2-substituted 3-arylquinoline derivatives, 2-(4-methoxy- benzoyl)-3-(3,4,5-trimethoxyphenyl)quinoline (18a) and 2-(4-fluorobenzoyl)-3-(3,4,5-trimethoxy- phenyl)quinoline (18b) are two of the most active compounds which can inhibit the production of NO at non-cytotoxic concentrations. Our results have also indicated that compounds 18a and 18b significantly decrease the secretion of pro-inflammatory cytokines (TNF-á and IL-6), inhibit the expression of iNOS, suppress the phosphorylation of MAPKs, and attenuate the activity of NF-êB by LPS-activated macrophages. Through molecular docking analysis, we found that 18b could fit into the middle of the TNF-á dimer and form hydrophobic interactions with Leu55, Leu57 chain A and B, Tyr59, Val123 chain B and D, Ile 155. These results suggest that both 18a and 18b are potential lead compounds in inhibiting LPS-induced inflammatory responses. Further structural optimization to discover novel anti-inflammatory agents is ongoing.

Molecular serotyping of Haemophilus parasuis isolated from diseased pigs and the relationship between serovars and pathological patterns in Taiwan.

BACKGROUND: Haemophilus parasuis is the etiological agent of Glässer's disease, and causes severe economic losses in the swine industry. Serovar classification is intended as an indicator of virulence and pathotype and is also crucial for vaccination programs and vaccine development. According to a polysaccharide biosynthesis locus analysis, H. parasuis isolates could be classified by a molecular serotyping assay except serovars 5 and 12 detected by the same primer pair. The aim of this study was to identify H. parasuis isolates from diseased pigs in Taiwan by using a molecular serotyping assay and to analyze the relationship between serovars and pathological patterns. METHODS: From August 2013 to February 2017, a total of 133 isolates from 277 lesions on 155 diseased animals from 124 infected herds serotyped by multiplex PCR and analyzed with pathological data. RESULTS: The dominant serovars of H. parasuis in Taiwan were serovars 5/12 (37.6%), 4 (27.8%) and 13 (15%) followed by molecular serotyping non-typable (MSNT) isolates (13.5%). Nevertheless, the serovar-specific amplicons were not precisely the same sizes as previously indicated in the original publication, and MSNT isolates appeared with unexpected amplicons or lacked serovar-specific amplicons. Most H. parasuis isolates were isolated from nursery pigs infected with porcine reproductive and respiratory syndrome virus. The percentage of lung lesions (30.4%) showing H. parasuis infection was significantly higher than that of serosal lesions. DISCUSSION: Collectively, the distribution of serovars in Taiwan is similar to that found in other countries, but MSNT isolates remain due to genetic variations. Furthermore, pulmonary lesions may be optimum sites for H. parasuis isolation, the diagnosis of Glässer's disease, and may also serve as points of origin for systemic H. parasuis infections in hosts.

Human Exposure to Ferret Badger Rabies in Taiwan.

On 17 July 2013, Taiwan confirmed multiple cases of the rabies virus (RABV) in the wild Taiwan Ferret badger (TFB) (Melogale moschata) member of the family Mustelidae. This study aims at investigating the risk factors for human exposure to rabid TFBs. Statistical inference based on Pearson correlation showed that there was a strong positive correlation between the total number of positive TFB rabies cases and the number of rabid TFBs involved with human activities in 81 enzootic townships (r = 0.91; p < 0.001). A logistic regression analysis indicated that the risk probability of a human being bitten by rabid TFBs was significantly higher when there were no dogs around (35.55% versus 6.17% (indoors, n = 171, p = 0.0001), and 52.00% versus 5.26% (outdoors, n = 44, p = 0.021)), and whether or not there was a dog around was the only crucial covariate that was statistically significantly related to the risk of a human being bitten. In conclusion, this study showed the value of having vaccinated pets as a deterrent to TFB encounters and as a buffer to prevent human exposure to rabid TFBs. The presence of unvaccinated pets could become a significant risk factor in the longer term if rabies isn’t controlled in TFBs because of the spillover between the sylvatic and urban cycles of rabies. Consequently, raising dogs, as well as keeping rabies vaccinations up-to-date for them, can be considered an effective preventive strategy to reduce the risk for human exposure to rabid TFBs.