In vivo characterization of the superior fitness of classical swine fever virus genotype 2.1 to genotype 3.4.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease in pigs. In Taiwan, the emerging genotype 2.1 (G2.1) CSFV caused sporadic outbreaks in 1994 and replaced the previous G3.4 CSFV in the field. The shift of CSFV genotypes to G2 CSFV was also observed in several CSFV-affected countries. The present study aimed to explore the mechanism of the genotype shift of CSFV. Two groups of specific pathogen-free (SPF) pigs were first inoculated with either G2.1 or G3.4 CSFV (single-inoculated group) and housed together with naïve SPF pigs (cohabitating group). The results showed that peak viremia, viral loads in blood and tissues, and viral shedding of G2.1 were consistently higher than those of G3.4 CSFV in single-inoculated and cohabitating pigs. The phenomenon of superinfection exclusion (SIE), characterized by the prevention of secondary infection by a primary infection, was readily observed in CSFV single-inoculated pigs. Interestingly, coinfection of both genotypes of CSFV was observed in 3 out of 4 cohabitating pigs, while only one pig was infected with G2.1 CSFV alone. These findings suggest that the genetic shift in CSFV in the field may be in part the consequence of SIE.

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

Transmission of Classical Swine Fever Virus in Cohabitating Piglets with Various Immune Statuses Following Attenuated Live Vaccine.

Classical swine fever (CSF) is a systemic hemorrhagic disease affecting domestic pigs and wild boars. The modified live vaccine (MLV) induces quick and solid protection against CSF virus (CSFV) infection. Maternally derived antibodies (MDAs) via colostrum could interfere with the MLV's efficacy, leading to incomplete protection against CSFV infection for pigs. This study investigated CSFV transmission among experimental piglets with various post-MLV immune statuses. Nineteen piglets, 18 with MDAs and 1 specific-pathogen-free piglet infected with CSFV that served as the CSFV donor, were cohabited with piglets that had or had not been administered the MLV. Five-sixths of the piglets with MDAs that had been administered one dose of MLV were fully protected from contact transmission from the CSFV donor and did not transmit CSFV to the piglets secondarily exposed through cohabitation. Cell-mediated immunity, represented by the anti-CSFV-specific interferon-γ-secreting cells, was key to viral clearance and recovery. After cohabitation with a CSFV donor, the unvaccinated piglets with low MDA levels exhibited CSFV infection and spread CSFV to other piglets through contact; those with high MDA levels recovered but acted as asymptomatic carriers. In conclusion, MLV still induces solid immunity in commercial herds under MDA interference and blocks CSFV transmission within these herds.

First detection and phylogenetic analysis of lumpy skin disease virus from Kinmen Island, Taiwan in 2020.

Lumpy skin disease is an arthropod-borne bovine disease caused by lumpy skin disease virus. A suspect lumpy skin disease case in a breeding cattle farm on Kinmen Island, Taiwan was reported on July 8, 2020 and later confirmed the first occurrence of lumpy skin disease in the country by molecular biological detections, electron microscopy, and sequence comparison. Implementation of control measures including blanket vaccination on the island effectively ceased the outbreaks. Phylogenetic analyses revealed that the virus discovered in the outbreaks was most similar to those identified in China in 2019. Identifying this virus in the coastal areas in East Asia indicated the rapid eastward spread of lumpy skin disease in Asia.

Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

Deletion in the S1 Region of Porcine Epidemic Diarrhea Virus Reduces the Virulence and Influences the Virus-Neutralizing Activity of the Antibody Induced.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and a high rate of mortality in suckling pigs. The epidemic of PEDV that occurred after 2013 was caused by non-insertion and deletion of S gene (S-INDEL) PEDV strains. During this epidemic, a variant of the non-S-INDEL PEDV strain with a large deletion of 205 amino acids on the spike gene (5-17-V) was also found to co-exist with a non-S-INDEL PEDV without deletion (5-17-O). Herein, we describe the differences in the complete genome, distribution, virulence, and antigenicity between strain 5-17-O and variant strain 5-17-V. The deletion of 205 amino acids was primarily located in the S1O domain and was associated with milder clinical signs and lower mortality in suckling pigs than those of the 5-17-O strain. The 5-17-V strain-induced antibody did not completely cross-neutralize the 5-17-O strain. In conclusion, the deletion in the S1 region reduces the virulence of PEDV and influences the virus-neutralizing activities of the antibody it induces.

In Vivo Demonstration of the Superior Replication and Infectivity of Genotype 2.1 with Respect to Genotype 3.4 of Classical Swine Fever Virus by Dual Infections.

In Taiwan, the prevalent CSFV population has shifted from the historical genotype 3.4 (94.4 strain) to the newly invading genotype 2.1 (TD/96 strain) since 1996. This study analyzed the competition between these two virus genotypes in dual infection pigs with equal and different virus populations and with maternally derived neutralizing antibodies induced by a third genotype of modified live vaccine (MLV), to simulate that occurring in natural situations in the field. Experimentally, under various dual infection conditions, with or without the presence of maternal antibodies, with various specimens from blood, oral and fecal swabs, and internal organs at various time points, the TD/96 had consistently 1.51-3.08 log higher loads than those of 94.4. A second passage of competition in the same animals further widened the lead of TD/96 as indicated by viral loads. The maternally derived antibodies provided partial protection to both wild type CSFVs and was correlated with lower clinical scores, febrile reaction, and animal mortality. In the presence of maternal antibodies, pigs could be infected by both wild type CSFVs, with TD/96 dominating. These findings partially explain the CSFV shift observed, furthering our understanding of CSFV pathogenesis in the field, and are helpful for the control of CSF.

Development of a quantitative real-time RT-PCR assay for detecting Taiwan ferret badger rabies virus in ear tissue of ferret badgers and mice.

In 2013, the first case of Taiwan ferret badger rabies virus (RABV-TWFB) infection was reported in Formosan ferret badgers, and two genetic groups of the virus were distinguished through phylogenetic analysis. To detect RABV-TWFB using a sensitive nucleic acid-based method, a quantitative real-time reverse transcription polymerase chain reaction targeting the conserved region of both genetic groups of RABV-TWFB was developed. This method had a limit of detection (LOD) of 40 RNA copies/reaction and detected viral RNA in brain and ear tissue specimens of infected and dead Formosan ferret badgers and mice with 100% sensitivity and specificity. The mean viral RNA load detected in the ear tissue specimens of ferret badgers ranged from 3.89 × 108 to 9.73 × 108 RNA copies/g-organ, which was 111-fold to 2,220-fold lower than the concentration detected in the brain specimens, but 2,000-fold to 5,000-fold higher than the LOD of the assay. This highly sensitive technique does not require facilities or instruments complying with strict biosafety criteria. Furthermore, it is efficient, safe, and labor-saving as only ear specimens need be sampled. Therefore, it is a promising technique for epidemiological screening of Taiwan ferret badger rabies.

Competitive replication kinetics and pathogenicity in pigs co-infected with historical and newly invading classical swine fever viruses.

Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field.

Rabies Virus Infection in Ferret Badgers (Melogale moschata subaurantiaca) in Taiwan: A Retrospective Study.

Fifteen ferret badgers (Melogale moschata subaurantiaca), collected 2010-13 and stored frozen, were submitted for rabies diagnosis by direct fluorescent antibody test and reverse transcription PCR. We detected seven positive animal samples, including some from 2010, which indicated that the ferret badger population in Taiwan had been affected by rabies prior to 2010.

The identification of frequent variations in the fusion protein of canine distemper virus.

Canine distemper (CD) is a highly contagious disease with a worldwide distribution. Genetic diversity in genes encoding the haemagglutinin (H) and fusion (F) virus envelope proteins have been implicated in the increasing incidence of CD. Unlike the H gene, little is known about the genetic variability of the F gene in this virus. In the present study sequence analysis of the complete coding region of the F protein from CD virus isolates from Taiwan were carried out. Phylogenetic analysis demonstrated that the majority of isolates were similar to those found in neighbouring China and Japan, but were genetically distinct from vaccine strains. Remarkable variations were found scattered throughout the pre-peptide region (residues 1-135). The sequence identity of this region between locally sourced strains and between these strains and vaccine strains was 89% and 64 to 67%, respectively. Analysis suggested a novel strain of distant genetic lineage was present in dogs in the geographically isolated city of Hualien.