Genetic diversity of the envelope glycoprotein E2 of classical swine fever virus: Recent isolates branched away from historical and vaccine strains (2023)

Classical swine fever (CSF) is a highly contagious and important swine disease in China. Sporadic outbreaks with mild clinical signs are still being reported despite massive vaccination with the CSF C-strain vaccine. One possible reason for vaccine failure could be interference from maternally derived antibodies (MDAs) during vaccination in the field. The aim of this study was to evaluate the efficacy of different CSF vaccines in the presence of MDAs and to assess the different vaccination schemes in the field. The results demonstrated that vaccination with a single dose of C-strain-PK vaccine protected pigs against severe clinical signs and significantly reduced viremia. The impact of MDAs was negligible. The interference was also mild during a prime and boost vaccination scheme using the C-strain-ST vaccine. In contrast, a significant influence of MDAs on the efficacy of the subunit E2 vaccine in a one-dose vaccination scheme was observed, with pigs showing severe clinical signs, CSF-associated death, typical pathological lesions and a high level of viremia after challenge, despite robust E2 antibody induction. A field vaccination and challenge study further confirmed the superior effectiveness of a single dose of C-strain-PK vaccine in the presence of MDAs in comparison to a routine prime and boost vaccination scheme applied in the field, with pigs having fever, chronic signs, significant viremia and shedding after challenge. Delaying the vaccination time from the age of 28 days to 45 days, when MDA was low, was beneficial for improving the clinical protection and immunity induced by vaccines. Altogether, the results presented here emphasize that a high-quality vaccine and a scientific design of the vaccination scheme based on serological surveillance are essential pillars to control and eliminate CSF in China.

Classical swine fever (CSF) is a highly contagious, devastating, and transboundary viral disease that afflicts swine industries worldwide. Immunization with vaccines is one of the most effective strategies for controlling this disease. However, shifts in the antigenicity and pathogenicity of novel evolving viral strains have the potential to evade vaccination. In this study, 352 samples from swines exhibiting fever, hemorrhages, lethargy, and diarrhea in different pig farms located in 9 provinces of Vietnam were collected. CSFV was identified even within farms that had been vaccinated against CSFV. Several farms had swine which had been co-infection with CSFV and other pathogens. Copies of the E2 gene of 21 samples were isolated, cloned, sequenced, analyzed, and compared with copies of E2 in four vaccine strains. We identified a total of 42 amino acid substitutions in this glycoprotein, including 11 positions that affect the antigenic properties of E2 and 7 positions that are associated with neutralizing epitopes. The E2 glycoprotein of CSFV strains circulating in Vietnam and vaccine strains differ in their antigenicity. These findings provide deep insights into the molecular characteristics, genetic diversity, pathogenicity, antigenicity, and evolution of CSFV strains in Vietnam. Understanding the pathogenicity, antigenicity, and evolution of circulating CSFV strains will provide avenues for developing new vaccines and efficient approaches to control this disease.

Classical swine fever (CSF) remains an important pig disease in China, where it usually presents with mild or atypical clinical manifestations, with large scale outbreaks rarely seen. This has led to speculation about the possible circulation of viral strains of low virulence. To investigate this possibility, five field isolates within the predominant genotype 2 (2.1b, 2.1c, 2.1 h and 2.2) were evaluated and compared by experimental infection of naturally farrowed but colostrum-deprived piglets. All infected piglets displayed clinical signs, including persistent high fever, depression, anorexia, dyspnea, conjunctivitis, constipation, and hesitant gait. Typical pathological lesions, including pulmonary edema, hemorrhagic or cellulosic exudation, and swelling and hemorrhage of lymph nodes, were observed. Viremia and E^rns protein expression in the blood of all infected animals were detectable from 3 to 5 days post infection (DPI), their presence correlating with the onset of fever, clinical signs and leukopenia. E2 antibody did not develop in any of the field CSFV-infected piglets during the disease course, while E^rns antibody was detectable in 4–56% of infected animals at various time points. Mortalities ranged from 20 to 80% within 21 DPI, progressing to 100% by 43 DPI. Based on clinical scores and fatalities within 21 DPI, 2 of the 5 field isolates were classified as of moderate virulence and 3 of high virulence; i.e., no field isolates of low virulence were identified. The study has provided data supporting the use of these isolates as challenge viruses to evaluate the efficacy of current CSF vaccines.

Classical swine fever (CSF) continues to be a devastating infectious disease for the swine industry in China and commonly exists as wild or atypical types. From June 3rd to October 3rd, 2018, outbreaks of typical CSF cases with mortality rates of 42–86% occurred in 11 swine herds in five cities of Guangdong province, and were confirmed by RT-PCR. Phylogenetic analyses based on the nucleotide sequences of full-length E2 genes showed that the CSFV isolates collected in Guangdong, 2018 grouped into sub-subgenotype 2.1c and formed a separate clade from previously identified 2.1c isolates. Sequence comparison further confirmed the distance between the novel emergent and previously identified 2.1c isolates, with shared 94.5–98.2% and 97.8–99.7% identities at the nucleotide and amino acid levels respectively. Furthermore, 2.1c isolates collected in 2018 from Guangdong province contained a unique amino acid substitution (K174R) in the E2 protein in comparison with other 2.1c representative strains and CSFV 2.1, 2.2, 2.3 strains. Of note, the novel emergent 2.1c isolates are neutralized by sera from C-strain vaccinated sows, indicating that C-strain is still efficacious for protection against field isolates of CSFV.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV.

As the causative agent of classical swine fever, the economically devastating swine disease worldwide, classical swine fever virus (CSFV) is currently classified into the 11 subgenotypes, of which subgenotype 2.1 is distributed worldwide and showing more genetic diversity than other subgenotypes. Prior to this report, subgenotype 2.1 was divided into three sub-subgenotypes (2.1a–2.1c). To further analyze the genetic diversity of CSFV isolates in China, 39 CSFV isolates collected between 2004 and 2012 in two Chinese provinces Guangxi and Guangdong were sequenced and subjected to phylogenetic analysis together with reference sequences retrieved from GenBank. Phylogenetic analyses based on the 190-nt and/or 1119-nt full length E2 gene fragments showed that current CSFV subgenotype 2.1 virus isolates in the world could be divided into 10 sub-subgenotypes (2.1a–2.1j) and the 39 isolates collected in this study were grouped into 7 of them (2.1a–2.1c and 2.1g–2.1j). Among the 10 sub-subgenotypes, 2.1d–2.1j were newly identified. Sub-subgenotype 2.1d isolates were circulated only in India, however the rest 9 sub-subgenotypes were from China with some of them closely related to isolates from European and neighboring Asian countries. According to the temporal and spatial distribution of CSFV subgenotype 2.1 isolates, the newly classified 10 sub-subgenotypes were further categorized into three groups: dominant sub-subgenotype, minor sub-subgenotype and silent sub-subgenotype, and each sub-subgenotype can be found only in certain geographical areas. Taken together, this study reveals the complex genetic diversity of CSFV subgenotype 2.1 and improves our understanding about the epidemiological trends of CSFV subgenotype 2.1 in the world, particularly in China.

Vaccine, Volume 34, Issue 17, 2016, pp. 2021-2026

The present study aimed to evaluate the safety of the classical swine fever virus (CSFV) vaccine strain LOM in pregnant sows. Pregnant sows with free CSFV antibody were inoculated with a commercial LOM vaccine during early pregnancy (day 38; n=3) or mid-pregnancy (days 49–59; n=11). In pregnant sows vaccinated during the early stages of gestation, abortion (day 109) was observed in one case, with two stillbirths and seven mummified fetuses. The viability of live-born piglets was 34.9% in sows vaccinated during mid-pregnancy compared with 81.8% in the control group. Post-mortem examination of the organs of the sows and piglets did not reveal any pathological lesions caused by CSFV; however, CSFV RNA was detected in the organs of several vaccinated sows and their litters. The LOM strain was transmitted from sows with free CSFV antibody to their fetus, but did not appear to induce immune tolerance in the offspring from vaccinated pregnant sows. Side effects were not observed in pregnant sows with antibody to the LOM strain: transmission from sow to their litters and stillbirth or mummified fetuses. The LOM strain may induce sterile immunity and provide rapid, long-lasting, and complete protection against CSFV; however, it should be contraindicated in pregnant sows due to potential adverse effects in pregnant sows with free CSFV antibody.

Gene, Volume 531, Issue 2, 2013, pp. 199-204

Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses and, subsequently, have drawn great attention to its diagnosis and prevention. To facilitate rapid discrimination of HP-PRRSV from classical PRRSV (C-PRRSV), we developed a one-step RT-PCR assay. Primer specificities were evaluated with RNA extracted from 8 viral strains and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 25copies/μL for both HP-PRRSV and C-PRRSV. A total of 929 serum samples were identified, of which 20.45% were HP-PRRSV-positive and 1.51% were C-PRRSV-positive, which was completely consistent with that of immunochromatochemistry and sequencing method. The proposed assay can detect the virus 2days prior the onset of symptoms and it can be performed in 2h, thereby providing a rapid method to discriminate HP-PRRSV from C-PRRSV for the identification and prevention of PRRSV infections.

Journal of Virological Methods, Volume 232, 2016, pp. 16-20

Akabane disease, caused by the insect-transmitted Akabane virus (AKAV), affects livestock by causing life-threatening deformities or mortality of fetuses. Therefore, Akabane disease has led to notable economic losses in numerous countries, including Japan. In this short communication, a new T7 RNA polymerase-based AKAV reverse genetics system was developed. Using this system, in which three plasmids transcribing antigenomic RNAs were transfected into cells stably expressing T7 polymerase, we successfully reconstituted the live attenuated vaccine TS-C2 strain (named rTTT), and also generated a mutant AKAV (rTTTΔNSs) that lacked the gene encoding the nonstructural NSs protein, which is regarded as a virulence factor. Analysis of growth kinetics revealed that rTTTΔNSs grew at a much slower rate than the rTTT and TS-C2 virus. These results suggest that our established reverse genetics system is a powerful tool that can be used for AKAV vaccine studies with gene-manipulated viruses.

Virus Research, Volume 301, 2021, Article 198435

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and causes heavy economic losses to the porcine industry worldwide. In this study, PK-15 cells were infected with PCV2 for 48 h, then harvested and subjected to label-free quantitative proteomic mass spectrometry. In total, 1212 proteins were differentially expressed in PCV2-infected cells compared with mock-infected cells, including 796 upregulated and 416 downregulated proteins. Gene ontology analysis showed that these differentially expressed proteins were involved in biological processes, cellular components and molecular functions, and these categories included cellular processes, environmental information processing, genetic information processing, disease, metabolism, and body systems. Enrichment analysis of the KEGG pathway showed that innate immune responses were significantly enriched. AlphaB-crystallin (CRYAB) interacts with desmin and cytoplasmic actin to prevent protein misfolding and aggregation, helping to maintain cytoskeletal integrity and promoting cell proliferation. In this study, CRYAB was found to effect the replication of PCV2, as verified by qRT-PCR, TCID₅₀ determination and western blot analysis. Overexpression of CRYAB significantly upregulated PCV2 capsid protein and increased viral titers in both PK-15 cells and culture supernatants, whereas the opposite results were obtained in CRYAB knockdown cells. Furthermore, we revealed that the promotion of PCV2 replication by CRYAB was dependent on cell proliferation. To our knowledge, this is the first report of the effect of CRYAB on PCV2 replication and our findings contribute to a greater understanding of the mechanism of PCV2 replication and pathogenesis, as well as the host’s response to PCV2 infection.

Virus Research, Volume 283, 2020, Article 197975

Atypical porcine pestivirus (APPV) is a novel pestivirus causing congenital tremor (CT) type AII in piglets and exhibiting a broad geographical distribution. Lack of an operating system for the viral genome is one of bottlenecks which restrict further research on pathogenesis and gene functions of APPV. Reverse genetics system (RGS) is a feasible solution to this bottleneck problem, but, to-date, no RGSs have been developed for APPV. Here, for the first time, recombinant APPV CH-GD2017 were rescued using in vitro and intracellular transcription systems and the virons were observed via transmission electron microscopy. As the process of in vitro transcription is time-consuming and inefficient, a full-length cDNA clone in an intracellular transcription was further constructed using an RNA polymerase II system. Then, the rescued virus was identified via RT-PCR detection, indirect immunofluorescent assay, and transmission electron microscopy. Development of the RGS for APPV will provide an important tool for further research on this newly emerging pathogen.

Virology, Volume 533, 2019, pp. 50-58

Atypical porcine pestivirus (APPV), a newly discovered member of the genus Pestivirus, is considered to be associated with congenital tremors (CT) in piglets. From June 2016 to January 2018, 440 serum and tissue samples from CT-affected piglets in Anhui, Guangdong and Guangxi provinces were collected to detect APPV. The results showed a high level of 63.4% preference for APPV in 27 swine farms and complicated co-infection cases between APPV and other 12 swine viruses. Meanwhile, 12 novel APPV genomes were screened and identified. Results showed that complete genomes, N^pro and NS5A genes of these novel 12 APPV sequences revealed 80.5%–99.8%, 78%–100% and 76.9%–99.8% nucleotide identities, respectively. Phylogenetic analyses based on sequences of full-length genomes, N^pro and NS5A genes of APPV indicated three well-defined clades including a newly emerging branch in China. This study provides novel epidemiological information of APPV in China.