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.

Implementation of point-of-care platforms for rapid detection of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. OBJECTIVES: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. METHODS: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. RESULTS: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. CONCLUSIONS: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

Antimicrobial resistance profile in Salmonella enterica serovar Choleraesuis isolates from diseased pigs in Taiwan.

This study investigated antimicrobial resistance in Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates from diseased pigs in Taiwan (2015-2020). Among 272 isolates, florfenicol (96.7%), enrofloxacin (96.3%), doxycycline (91.2%), gentamicin (84.6%), and tiamulin (80.5%) exhibited high resistance. 99.3% of the isolates were resistant to at least one antibiotic, and 97.8% of the isolates were multidrug resistant. This study illustrated that S. Choleraesuis isolates exhibited high resistance to antimicrobials currently used in the Taiwanese swine industry.

Antimicrobial susceptibility and resistome of Actinobacillus pleuropneumoniae in Taiwan: a next-generation sequencing analysis.

Actinobacillus pleuropneumoniae infection causes a high mortality rate in porcine animals. Antimicrobial resistance poses global threats to public health. The current study aimed to determine the antimicrobial susceptibilities and probe the resistome of A. pleuropneumoniae in Taiwan. Herein, 133 isolates were retrospectively collected; upon initial screening, 38 samples were subjected to next-generation sequencing (NGS). Over the period 2017-2022, the lowest frequencies of resistant isolates were found for ceftiofur, cephalexin, cephalothin, and enrofloxacin, while the highest frequencies of resistant isolates were found for oxytetracycline, streptomycin, doxycycline, ampicillin, amoxicillin, kanamycin, and florfenicol. Furthermore, most isolates (71.4%) showed multiple drug resistance. NGS-based resistome analysis revealed aminoglycoside- and tetracycline-related genes at the highest prevalence, followed by genes related to beta-lactam, sulfamethoxazole, florphenicol, and macrolide. A plasmid replicon (repUS47) and insertion sequences (IS10R and ISVAp11) were identified in resistant isolates. Notably, the multiple resistance roles of the insertion sequence IS10R were widely proposed in human medicine; however, this is the first time IS10R has been reported in veterinary medicine. Concordance analysis revealed a high consistency of phenotypic and genotypic susceptibility to florphenicol, tilmicosin, doxycycline, and oxytetracycline. The current study reports the antimicrobial characterization of A. pleuropneumoniae for the first time in Taiwan using NGS.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Background and Aim: Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Materials and Methods: Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Results: Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. Conclusion: This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Molecular Characteristics and Pathogenicity of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) 1 in Taiwan during 2019-2020.

Two variants of porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV 1 and PRRSV 2, have caused abortion in pregnant sows and respiratory distress in nursery pigs worldwide. PRRSV 2 has been thoroughly researched in Taiwan since 1993; however, the first case of PRRSV 1 was not reported until late 2018. To decipher the genetic characteristics of PRRSV 1 in Taiwan, open reading frame 5 (ORF5) genes of PRRSV 1 strains collected from 11 individual pig farms in 2019-2020 were successfully sequenced. All Taiwanese ORF5 sequences were closely related to Spanish-like PRRSV strains, which are considered to share a common evolutionary origin with the strain used for the PRRSV 1 vaccine. Analyses of amino acid (aa) and non-synonymous substitutions showed that genetic variations resulted in numerously specific codon mutations scattered across the neutralizing epitopes within the ORF5 gene. The PRRSV 1 challenge experiment disclosed the pathogenetic capability of the NPUST2789 isolate in nursery pigs. These findings provide comprehensive knowledge of the molecular diversity of the PRRSV 1 variant in local Taiwanese fields and facilitate the development of suitable immunization programs against this disease.

Phylodynamic analysis and spike protein mutations in porcine deltacoronavirus with a new variant introduction in Taiwan.

Porcine deltacoronavirus (PDCoV) is a highly transmissible intestinal pathogen that causes mild to severe clinical symptoms, such as anorexia, vomiting, and watery diarrhea in pigs. By comparing the genetic sequences of the spike glycoprotein between historical and current Taiwanese PDCoV strains, we identified a novel PDCoV variant that displaced the PDCoV responsible for the 2015 epidemic. This PDCoV variant belongs to a young population within the US lineage, and infected pigs carry high concentrations of the virus. It also has several critical point mutations and an amino acid insertion at position 52 that may enhance the affinity between the B-cell epitopes located in the N-terminal domain with its complementarity regions, consequently facilitating binding or penetration between the fusion peptide and cellular membrane. Furthermore, viral protein structure prediction demonstrated that these amino acid changes may change the ability of the virus to bind to the receptor, which may consequently alter virus infectivity. Our results hence suggest the emergence of new PDCoV strains in Taiwan with the potential for greater transmission and pathogenesis.

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.

Animal Coronavirus Diseases: Parallels with COVID-19 in Humans.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus in humans, has expanded globally over the past year. COVID-19 remains an important subject of intensive research owing to its huge impact on economic and public health globally. Based on historical archives, the first coronavirus-related disease recorded was possibly animal-related, a case of feline infectious peritonitis described as early as 1912. Despite over a century of documented coronaviruses in animals, the global animal industry still suffers from outbreaks. Knowledge and experience handling animal coronaviruses provide a valuable tool to complement our understanding of the ongoing COVID-19 pandemic. In this review, we present an overview of coronaviruses, clinical signs, COVID-19 in animals, genome organization and recombination, immunopathogenesis, transmission, viral shedding, diagnosis, treatment, and prevention. By drawing parallels between COVID-19 in animals and humans, we provide perspectives on the pathophysiological mechanisms by which coronaviruses cause diseases in both animals and humans, providing a critical basis for the development of effective vaccines and therapeutics against these deadly viruses.

Correlation of Neutralizing Antibodies (NAbs) between Sows and Piglets and Evaluation of Protectability Associated with Maternally Derived NAbs in Pigs against Circulating Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) under Field Conditions.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by a highly transmissible pathogen called porcine reproductive and respiratory syndrome virus (PRRSV), has caused severe problems, including reproductive disorders in sows and respiratory symptoms in nursery pigs worldwide, since the early 1990s. However, currently available PRRSV vaccines do not supply complete immunity to confront the viral infection. Elicitation of PRRSV-specific neutralizing antibodies (NAbs) during the preinfectious period has been deemed to be a feasible strategy to modulate this virus, especially in farms where nursery pigs are seized with PRRSVs. A total of 180 piglets in a farrow-to-finish farm that had a natural outbreak of PRRS were distributed into three groups based on the different PRRSV NAbs levels in their dams. In the present study, piglets that received superior maternal-transferred NAbs showed delayed and relatively slight viral loads in serum and, on the whole, higher survival rates against wild PRRSV infections. A positive correlation of maternal NAbs between sows and their piglets was identified; moreover, high NAbs titers in piglets can last for at least 4 weeks. These results provide updated information to develop an appropriate immune strategy for breeding and for future PRRSV control under field conditions.

Metallic glass coating for improved needle tattooing performance in reducing trauma: analysis on porcine and pig skins.

The dissemination of tattooing into mainstream culture has raised concerns pertaining to the medical implications of these practices. This paper reports on the coating of tattoo needles with metallic-glass (MG) to reduce trauma to the skin. Extensive experimentation using pork samples and live pigs demonstrated the beneficial effects of non-stick MG coatings. Following 30 insertions into pork skin, significantly less tissue adhered to the MG-coated needles than to uncoated needles. MG-coated needles were also shown to reduce the spread of pigment to the surface of surrounding skin by up to 57%. This resulted in narrower tattoo lines of higher density, indicating that MG-coated needles could be useful in high-resolution tattooing. Histopathological analysis on live pigs revealed severe trauma induced by bare needles, as indicated by the secretion of fluids immediately after tattooing. The wounds formed by coated needles closed within 2 h after tattooing; however, those formed by bare needles remained open for at least 2 h and inflammation was still observed after 3 days. At 5 days after tattooing, skin punctured by the coated needle was entirely healed, whereas skin punctured by the bare needle was still covered with scabs. In addition to the medical benefits, it appears that MG-coated needles could improve the quality of tattoos, based on the fact that the amount of pigment retained in the skin is inversely proportional to the trauma caused by needles.

Genetic characterization of feline panleukopenia virus from dogs in Vietnam reveals a unique Thr101 mutation in VP2.

BACKGROUND: Canine parvovirus type 2 (CPV-2) and feline parvovirus (FPV) are known as the main causes of several serious diseases and have a severe impact on puppies and kittens, respectively. FPV and new CPV-2 variants are all able to infect cats, causing diseases indistinguishable from feline panleukopenia. However, FPV only replicates efficiently in feline cells in vitro and replicates in dogs in the thymus and bone marrow without being shed in feces. In our previous study, the genotypes of six parvoviral isolates were unable to be identified using a SimpleProbe® real-time PCR assay. METHODS: In the present study, we characterized previously unidentified FPV-like viruses isolated from dogs in Vietnam. The six isolates were utilized to complete VP2 gene sequencing and to conduct phylogenetic analyses. RESULTS: Sequence analysis of the six parvoviral strains identified the species as being similar to FPV. Phylogenetic analysis demonstrated that the complete VP2 genes of the strains are similar to those of FPV. The FPV-like strains contain a Thr101 mutation in the VP2 protein, which is different from prototype FPV strains. DISCUSSION: Our data provide evidence for the existence of changes in the charge, protein contact potential and molecular surface of the core of the receptor-binding size with an Ile101 to Thr101 mutation. This is also the first study to provide reliable evidence that FPV may be a threat to the Vietnamese dog population.

Molecular Evidence for Intra- and Inter-Farm Spread of Porcine mcr-1-Carrying Escherichia coli in Taiwan.

From January 2013 to December 2018, 90 Escherichia coli isolates were collected from 90 sick pigs on 58 farms in seven cities in Taiwan. The minimum inhibitory concentrations (MICs) of the isolates to 14 antimicrobial agents were determined on the VITEK 2 system (bioMérieux, Marcy-l'Etoile, France), and the resistance to colistin was assessed via the reference broth microdilution (BMD) method. The mobilized colistin resistance genes (mcr) were determined for the colistin-resistant isolates, which displayed BMD MICs ≥ 4 mg/L. Genotypes of the mcr-positive E. coli isolates were determined by multilocus sequence typing, arbitrarily primed polymerase chain reaction (PCR), and pulsed-field gel electrophoresis. All of the isolates were tested for susceptibility to carbapenems. Fifty isolates (55.6%) were resistant to colistin, 39 of which (78%) were positive for the mcr-1 gene. E. coli isolates harboring mcr-1 were most frequent in 2017 (15/18, 83.3%), followed by 2018 (13/23, 56.5%), 2015 (7/21, 33.3%), and 2016 (3/24, 12.5%). A total of 18 sequence types (STs) were identified among the 39 porcine mcr-1-carrying E. coli isolates; 13 were ST2521 (33.3%) isolated in 2017 and 2018. Five genotypes (clones) were identified, and the same genotypes were in sick pigs on the same farm and different farms. This suggests intra- and inter-farm spread of porcine mcr-1-carrying E. coli. The results presented here indicate high colistin resistance and wide mcr-1 E. coli prevalence among the sick pigs sampled in 2015-2018 in different regions of Taiwan.

Cross-Sectional Study on the Sero- and Viral Dynamics of Porcine Circovirus Type 2 in the Field.

Porcine circovirus-associated diseases (PCVADs) cause considerable economic losses in industrial pork production in the field. To minimize the economic losses due to PCVAD, porcine circovirus type 2 (PCV2) vaccines have been developed, and there is widespread vaccination worldwide today. However, limited information is available concerning the current status of PCV2 infection in the field on the Asian continent. The present study aimed to assess sero- and viral dynamics of PCV2 from 12 PCV2-contaminated pig herds with vaccination against PCV2 in Southern and Central Taiwan. In particular, the level of PCV2 load during the window period for seroconversion using real-time polymerase chain reaction and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Our results revealed that pig herds showed slight or no seroconversion after three to four weeks post-PCV2 immunization. The presence of PCV2 was observed during the window period for seroconversion in all herds. In conclusion, natural exposure of PCV2 occurs in the growing to fattening period, and viremia can last until slaughter. Additionally, our findings indicate that using ELISA showed the level of antibodies and aided in the understanding and surveillance of the current PCV2 status in the field.

Serodynamic Analysis of the Piglets Born from Sows Vaccinated with Modified Live Vaccine or E2 Subunit Vaccine for Classical Swine Fever.

Classical swine fever (CSF) caused by the CSF virus (CSFV) is one of the most important swine diseases, resulting in huge economic losses to the pig industry worldwide. Systematic vaccination is one of the most effective strategies for the prevention and control of this disease. Two main CSFV vaccines, the modified live vaccine (MLV) and the subunit E2 vaccine, are recommended. In Taiwan, CSF cases have not been reported since 2006, although systemic vaccination has been practiced for 70 years. Here, we examined the sero-dynamics of the piglets born from sows that received either the CSFV MLV or the E2 vaccine and investigated in the field the correlation between the porcine reproductive and respiratory syndrome virus (PRRSV) loads and levels of CSFV antibody. A total of 1398 serum samples from 42 PRRSV-positive farms were evaluated to determine the PRRSV loads by real-time PCR and to detect CSFV antibody levels by commercial ELISA. Upon comparing the two sow vaccination protocols (CSFV MLV vaccination at 4 weeks post-farrowing versus E2 vaccination at 4-5 weeks pre-farrowing), the lowest levels of CSFV antibody were found in piglets at 5-8 and 9-12 weeks of age for the MLV and E2 groups, respectively. Meanwhile, the appropriate time window for CSFV vaccination of offspring was at 5-8 and 9-12 weeks of age in the MLV and E2 groups, respectively. There was a very highly significant negative correlation between the PRRSV load and the level of CSFV antibody in the CSFV MLV vaccination group (P < 0.0001). The PRRSV detection rate in the pigs from the MLV group (27.78%) was significantly higher than that in pigs from the E2 group (21.32%) (P = 0.011). In addition, there was a significant difference (P = 0.019) in the PRRSV detection rate at 5-8 weeks of age between the MLV (42.15%) and E2 groups (29.79%). Our findings indicate that the vaccination of CSFV MLV in piglets during the PRRSV susceptibility period at 5-8 weeks of age may be overloading the piglet's immune system and should be a critical concern for industrial pork production in the field.

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum ß-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

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.

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan.

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full-length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016-2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild-type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.

NS2B/NS3 mutations enhance the infectivity of genotype I Japanese encephalitis virus in amplifying hosts.

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle.

Phylogeographic and genetic characterization of porcine circovirus type 2 in Taiwan from 2001-2017.

Porcine circovirus type 2 (PCV2) is an important pathogen that causes significant economic losses in the swine industry worldwide. Five major PCV2 genotypes have been identified, including PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. To investigate the prevalence and phylodynamics of the different PCV2 genotypes in Taiwan, 214 PCV2 ORF2 sequences from Taiwan and other countries were analyzed. Genotypic differences were observed among PCV2a, 2b, and 2d at amino acid position 89 in ORF2, with isoleucine (I), arginine (R), and leucine (L), respectively. Similar to other countries, a genotypic shift was also observed in Taiwan, where the predominant genotype shifted from PCV2b to 2d after 2010. The estimated nucleotide substitution rate of Taiwanese strains in the ORF2 region was 8.467 × 10-4 substitutions per site per year. This rapid evolution rate of PCV2 may lead to the genotypic shift observed in Taiwan. The times to the most recent common ancestor (TMRCA) for PCV2a, -2b, and -2d-2 was dated to 1970, 1992 and 2004, respectively. Thus, the PCV2a, -2b, and -2d genotypes were already present in Taiwan before the introduction of the PCV2 vaccine.