Classical Swine Fever Virus Strain Research Articles

Circulation of classical swine fever virus (CSFV) strains of bovine origin in China and India.

The classical swine fever virus (CSFV) is a species member of the family Flaviviridae. CSFV is widely distributed in the world causing a severe impact on pig industry. This pathogen is considered restricted to domestic and wild suids. However, some reports from 2014 to 2018 showed the presence of the CFSV antigen in the bovine species. The virus was found in commercialized batches of fetal bovine serum (FBS) of Chinese origin and in bovine herds in in the provinces of Henan and Jiangsu, China, and in Tamil Nadu and Meghalaya, southern and north‑eastern states of India, respectively. Detection was done using antigen capture ELISA and RT‑PCR tests. In certain cases, animals with natural infection showed clinical signs and reproduction was also affected. Genetic characterization was performed considering the 5'‑UTR sequences of the bovine strains. In addition, the entire CSFV E2 genomic region could be amplified from two positive animals. The bovine strains were genetically related to the Chinese CSFV live attenuated hog cholera lapinized vaccine (HCLV) strain used in pigs, sharing sequence characteristics. The vaccine strain HCLV was widely used in China to protect bovines and yaks from bovine viral diarrhea, and, as a possible consequence, inducing an adaptation in cattle and a further natural diffusion. Furthermore, a contaminant strain from China was genetically distant from all other previously described genotypes of the CSFV. This suggests also the occurrence of micro evolutive step in the species related to geographical segregation. These observations deserve attention and further investigations, especially relevant in countries where CSFV control and eradication strategies are applied.

Neutralizing antibodies as a correlate of protection against classical swine fever in Porvac® vaccinated pigs

Porvac is a classical swine fever (CSF) subunit vaccine. It is safe and induces a robust neutralizing antibody response, sterilizing immunity, and early protection, and it prevents vertical transmission in pregnant sows. The methodology to approve Porvac batches is a challenging experiment in pigs with a virulent CSF virus strain. However, there is an ethical reason to reduce, at minimum, the use of animals in these lethal experiments. The knowledge indicates that neutralizing antibody titers in the blood could be a good correlate of protection. The results of 22 challenge experiments involving 116 Porvac vaccinated and 38 unvaccinated animals were analyzed. All vaccinated animals remained free from CSF clinical signs and pathological lesions and were negative for viral isolation after the challenge. In contrast, all unvaccinated pigs developed clinical and pathological signs of the disease and had to be euthanized eight days post-challenge. All vaccinated pigs exhibited high neutralizing antibody titers, with a geometric mean value of 1: 5153. The lower titer registered was 1: 800. A complete correspondence between neutralizing antibody titers and protection was demonstrated. These results support substituting the viral challenge test for the neutralizing peroxidase-linked assay in the release of Porvac® batches. Keywords. Classical swine fever; virus; subunit vaccine; viral challenge; neutralizing antibodies

The FlagT4G Vaccine Confers a Strong and Regulated Immunity and Early Virological Protection against Classical Swine Fever.

Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination, mostly using vaccines that do not allow for differentiation of vaccinated from infected animals (DIVA). FlagT4G vaccine is a novel candidate that confers robust immunity and shows DIVA capabilities. The present study assessed the immune response elicited by FlagT4G and its capacity to protect pigs for a short time after vaccination. Five days after a single dose of FlagT4G vaccine, animals were challenged with a highly virulent CSFV strain. A strong, but regulated, interferon-α response was found after vaccination. Vaccinated animals showed clinical and virological protection against the challenge, in the absence of antibody response at 5 days post-vaccination. Upon challenge, a rapid rise in the titers of CSFV neutralizing antibodies and an increase in the IFN-γ producing cells were noticed in all vaccinated-challenged pigs. Meanwhile, unvaccinated pigs showed severe clinical signs and high viral replication, being euthanized before the end of the trial. These animals were unable to generate neutralizing antibodies and IFN-γ responses after the CSFV challenge. The results from the present study assert the fast and efficient protection by FlagT4G, a highly promising tool for CSFV control worldwide.

Classical swine fever (CSF): towards new challenges

Classical swine fever (CSF) is a highly contagious swine-specific disease which may have a huge economic impact for porcine production. CSF is caused by a virus belonging to the Pestivirus genus, which has expanded for the past 5 years with the discovery of new species whose genetic proximity to the CSF virus could further complicate laboratory diagnosis. The various forms of the disease, and in particular the increased frequency of attenuated forms, linked to an evolution of CSF virus strains towards a reduction in their virulence, delay clinical diagnosis. Thus, a long period may elapse before an outbreak is detected, allowing the virus to circulate longer, with the risk of spreading to distant geographical areas. Efforts must be maintained in terms of surveillance and diagnostic tools development in order to detect CSF virus infection early and thus limit the spread of the disease and facilitate control measures.

Epidemiological Survey of Four Reproductive Disorder Associated Viruses of Sows in Hunan Province during 2019-2021.

Simple SummaryPorcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV) are major causative agents contributing to the reproductive disorders in sows in China. This is the first research investigating the epidemiology and molecular characteristics of these four viruses in sows with reproductive disorders in China. In this study, 407 aborted-fetus samples were obtained from 89 pig farms in Hunan province from 2019 to 2021 for molecular detection and phylogenetic analysis of PRRSV, PRV, PCV2, and CSFV. Collectively, various patterns of singular infections or co-infections were identified in pig herds; PRRSV and PCV2, especially, were frequently detected in the collected specimens. Further phylogenetic analysis revealed the complex genotypes of each virus prevalent in pig populations in Hunan province. In conclusion, this study provides the latest information on the epidemiology and genotype diversity of four viruses in sows with reproductive diseases in Hunan province of China, which would contribute to developing effective strategies for disease control.Porcine reproductive disorders have been considered as the major factors that threaten pig industries worldwide. In this study, 407 aborted-fetus samples were obtained from 89 pig farms in Hunan province, to investigate the prevalence of four viruses associated with porcine reproductive disease, including porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV). Meanwhile, the target gene sequences of representative PRRSV (ORF5), PCV2 (ORF2), CSFV (E2), and PRV (gE) strains were amplified, sequenced, and analyzed. The results showed that the positive rates of PRRSV, PCV2, PRV, and CSFV among the collected samples were 26.29% (107/407), 52.83% (215/407), 6.39% (26/407), and 12.29% (50/407), respectively. Moreover, co-infection with two and three pathogens were frequently identified, with PCV2/PRRSV, PRRSV/CSFV, PRRSV/PRV, PCV2/CSFV, PCV2/PRV, and PRRSV/PCV2/CSFV mix infection rates of 9.09%, 3.19%, 2.95%, 3.69%, 2.21%, and 0.49%, respectively. Moreover, ORF5-based phylogenetic analysis showed that 9, 4, and 24 of 37 PRRSV strains belonged to the PRRSV2 lineages 1, 5, and 8, respectively. ORF2-based phylogenetic analysis revealed that PCV2d and PCV2b were prevalent in Hunan province, with the proportions of 87.5% (21/24) and 12.5% (3/24), respectively. An E2-based phylogenetic tree showed that all 13 CSFV strains were clustered with 2.1 subgenotypes, these isolates were composed of 2.1b (10/13) and 2.1c (3/13) sub-subgenotypes. A gE-based phylogenetic tree showed that all six PRV strains belonged to the genotype II, which were genetically closer to variant PRV strains. Collectively, the present study provides the latest information on the epidemiology and genotype diversity of four viruses in sows with reproductive diseases in Hunan province, China, which would contribute to developing effective strategies for disease control.

Search for additional tests for immunobiological evaluation of the candidate vaccines against African swine fever.

The spread of African swine fever (ASF) in Eurasia has forced a return to the development of live vaccines based on naturally or experimentally attenuated strains of the virus including those resulting from genetic manipulations. This process includes evaluation of the immunomodulating properties of the vaccines. In this report we provide our assessment of two tests for immunobiological evaluation of a candidate live vaccine against ASF from the attenuated ASF virus (ASFV) strain KK-202: (i) investigation of the effect of the attenuated ASFV strain KK-202 on the protectiveness of the vaccine ASFV strain FK-32/135 and a vaccine against classical swine fever (CSF) from the strain LK-VNIIVViM; (ii) determination of the phagocytic activity of blood neutrophils in pigs inoculated with ASFV strains differing in virulence. A simultaneous or sequential inoculation of attenuated strain KK-202 (seroimmunotype II) and vaccine strain FK-32/135 (seroimmunotype IV) into pigs resulted in the loss of protection against the virulent strain France-32 (seroimmunotype IV). Following the simultaneous or sequential inoculations of the ASFV strain KK-202 and the CSF virus (CSFV) vaccine produced from the strain LK-VNIIVViM, the neutralizing antibody titers against the CSFV observed in the experimental groups (after vaccination and after the challenge infection with the virulent CSFV strain Shimen) were not different from those found in animals of the control group. The phagocytic activity of blood neutrophils was shown to increase from 30% in the norm to 50%–94% depending on the virulence of the ASFV strains inoculated into pigs. The results of this work demonstrate the ability of the attenuated ASFV strains to modulate the development of the cellular link of protective immunity without negative impact on the humoral immune response. The informative value of the described immunobiological tests in vivo and in vitro seems to be a more preferable alternative in comparison to the commonly used in vitro tests, which do not always correlate with the development of protection against ASF.

Antiviral Effects of 5-Aminolevulinic Acid Phosphate against Classical Swine Fever Virus: In Vitro and In Vivo Evaluation.

The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 107.0 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.

Determination of reproductive properties of virulent and vaccine classical swine fever virus strains in primary and continuous cell cultures

Determination of reproductive properties of virulent and vaccine classical swine fever virus strains in primary and continuous cell cultures

Epidemiological investigation and phylogenetic analysis of Classical Swine Fever virus in Yunnan province from 2015 to 2021.

BackgroundClassical swine fever virus (CSFV), the causative agent of classical swine fever (CFS), is a highly contagious disease that poses a serious threat to Chinese pig populations.ObjectivesMany provinces of China, such as Shandong, Henan, Hebei, Heilongjiang, and Liaoning provinces, have reported epidemics of CSFV, while the references to the epidemic of CSFV in Yunnan province are rare. This study examined the epidemic characteristics of the CSFV in Yunnan province.MethodsIn this study, 326 tissue samples were collected from different regions in Yunnan province from 2015 to 2021. A reverse transcription-polymerase chain reaction (RT-PCR), sequences analysis, and phylogenetic analysis were performed for the pathogenic detection and analysis of these 326 clinical specimens.ResultsApproximately 3.37% (11/326) of specimens tested positive for the CSFV by RT-PCR, which is lower than that of other regions of China. Sequence analysis of the partial E2 sequences of eleven CSFV strains showed that they shared 89.0–100.0% nucleotide (nt) and 95.0–100.0% amino acid (aa) homology, respectively. Phylogenetic analysis showed that these novel isolates belonged to the subgenotypes 2.1c and 2.1d, with subgenotype 2.1c being predominant.ConclusionsThe CSFV was sporadic in China’s Yunnan province from 2015 to 2021. Both 2.1c and 2.1d subgenotypes were found in this region, but 2.1c was dominant.

Genetic diversity and molecular characterization of classical swine fever virus envelope protein genes E2 and Erns circulating in Vietnam from 2017 to 2019.

Classical swine fever virus (CSFV) is an RNA virus that incurs severe economic costs to swine industries worldwide. This study was conducted to investigate the genetic diversity among CSFV strains circulating in Vietnam, with a focus on their genetic variants relative to four vaccine strains. Samples from clinical cases were collected from different provinces of Central and Southern Vietnam from 2017 to 2019. 21 CSFV-positive samples were selected for amplification and sequencing of the full-length Erns and E2 genes. Phylogenetic analyses of these two genes showed that most CSFV strains circulating in Central and Southern Vietnam from 2017 to 2019 belong to subgroup 2.1c, whereas the remaining strains cluster into subgroup 2.2. All CSFV field strains in this study were genetically distant from group 1 strains. Analysis of the E2 and Erns genes indicated that all CSFV field strains have low sequence identity with the vaccine strains (80-83.5% and 82.3-86% sequence identity for E2 and Erns, respectively). Likewise, amino acid-level sequence analysis showed 87.3-91.1% and 87.6-91.6% sequence identity for E2 and Erns, respectively. Together, our findings indicate that CSFV strains circulating in Vietnam belong to subtypes 2.2 and 2.1c, and we also provide novel insights into the epidemiology, molecular characteristics, genetic diversity, and evolution of these circulating CSFV strains.

Immunohistochemical analysis of the distribution of classical swine fever (CSF) viral antigen in boar-pig hybrids and pigs four weeks after infection

We detected the classical swine fever virus (CSFV) antigen in three boar-pig hybrids (hybrids) and three pigs. All animals were experimentally infected with CSFV strain JPN/27/2019 to optimize diagnostic sampling and risk assessment of virus dissemination. Two hybrids died 17- and 19-days post-inoculation (dpi). The other animals were euthanized at 28 dpi. The detection of CSFV antigen at 28 dpi in epithelial cells of the apocrine sweat and sebaceous glands in the skin, salivary glands, mucosal epithelial cells in the rectum, and epithelial cells in the kidney and urinary bladder, suggests that CSFV persists in these tissues and spreads via sweat, saliva, feces, and urine for at least 4 weeks. These findings reveal that hybrids and pigs represent a high risk of virus dissemination four weeks after infection with CSFV strain JPN/27/2019. Prominent CSFV antigens were also detected in hair follicles of the skin. These results suggest that postmortem sampling of animal skin may be effective for CSF diagnosis and can be used to develop a rapid and easy diagnostic method using hair follicles.

Development of a Dendrimeric Peptide-Based Approach for the Differentiation of Animals Vaccinated with FlagT4G against Classical Swine Fever from Infected Pigs.

Classical swine fever virus (CSFV) causes a viral disease of high epidemiological and economical significance that affects domestic and wild swine. Control of the disease in endemic countries is based on live-attenuated vaccines (LAVs) that induce an early protective immune response against highly virulent CSFV strains. The main disadvantage of these currently available LAVs is the lack of serological techniques to differentiate between vaccinated and infected animals (DIVA concept). Here, we describe the development of the FlagDIVA test, a serological diagnostic tool allowing for the differentiation between animals vaccinated with the FlagT4G candidate and those infected with CSFV field strains. The FlagDIVA test is a direct ELISA based on a dendrimeric peptide construct displaying a conserved epitope of CSFV structural protein E2. Although FlagDIVA detected anti-CSFV anti-bodies in infected animals, it did not recognize the antibody response of FlagT4G-vaccinated animals. Therefore, the FlagDIVA test constitutes a valuable accessory DIVA tool in implementing vaccination with the FlagT4G candidate.

Abrogation of the RNase activity of Erns in a low virulence classical swine fever virus enhances the humoral immune response and reduces virulence, transmissibility, and persistence in pigs

ABSTRACT The prevalence of low virulence classical swine fever virus (CSFV) strains makes viral eradication difficult in endemic countries. However, the determinants for natural CSFV attenuation and persistence in the field remain unidentified. The aim of the present study was to assess the role of the RNase activity of CSFV Erns in pathogenesis, immune response, persistent infection, and viral transmission in pigs. To this end, a functional cDNA clone pPdR-H30K-36U with an Erns lacking RNase activity was constructed based on the low virulence CSFV field isolate Pinar de Rio (PdR). Eighteen 5-day-old piglets were infected with vPdR-H30K-36U. Nine piglets were introduced as contacts. The vPdR-H30K-36U virus was attenuated in piglets compared to the parental vPdR-36U. Only RNA traces were detected in sera and body secretions and no virus was isolated from tonsils, showing that RNase inactivation may reduce CSFV persistence and transmissibility. The vPdR-H30K-36U mutant strongly activated the interferon-α (IFN-α) production in plasmacytoid dendritic cells, while in vivo, the IFN-α response was variable, from moderate to undetectable depending on the animal. This suggests a role of the CSFV Erns RNase activity in the regulation of innate immune responses. Infection with vPdR-H30K-36U resulted in higher antibody levels against the E2 and Erns glycoproteins and in enhanced neutralizing antibody responses when compared with vPdR-36U. These results pave the way toward a better understanding of viral attenuation mechanisms of CSFV in pigs. In addition, they provide novel insights relevant for the development of DIVA vaccines in combination with diagnostic assays for efficient CSF control.

Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Phylogenetic analysis of classical swine fever virus isolates from China.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a severe disease that causes huge economic losses in the swine industry worldwide. In China, CSF has been under control due to extensive vaccination since 1954. However, there are still sporadic CSF outbreaks in China. Here, we isolated 27 CSFV strains from three Chinese provinces (Shaanxi, Gansu, and Ningxia) from 2011 to 2018. Phylogenetic analysis based on the full-length envelope glycoprotein E2 coding region revealed that 25 out of 27 CSFV isolates clustered within subgroups 2.1 and 2.2, while two strains from Gansu belonged to subgroup 1.1. The sequence identity among these 27 isolates varied from 79.3% to 99.8% (nucleotides) and from 83.1% to 99.7% (amino acids). Further analysis based on the E2 amino acid sequences showed that these new isolates have consistent amino acid substitutions, including R31K and N34S.

Early and Solid Protection Afforded by the Thiverval Vaccine Provides Novel Vaccination Alternatives Against Classical Swine Fever Virus.

Classical swine fever virus (CSFV) remains a challenge for the porcine industry. Inefficient vaccination programs in some endemic areas may have contributed to the emergence of low and moderate virulence CSFV variants. This work aimed to expand and update the information about the safety and efficacy of the CSFV Thiverval-strain vaccine. Two groups of pigs were vaccinated, and a contact and control groups were also included. Animals were challenged with a highly virulent CSFV strain at 21- or 5-days post vaccination (dpv). The vaccine induced rapid and strong IFN-α response, mainly in the 5-day immunized group, and no vaccine virus transmission was detected. Vaccinated pigs showed humoral response against CSFV E2 and Erns glycoproteins, with neutralising activity, starting at 14 days post vaccination (dpv). Strong clinical protection was afforded in all the vaccinated pigs as early as 5 dpv. The vaccine controlled viral replication after challenge, showing efficient virological protection in the 21-day immunized pigs despite being housed with animals excreting high CSFV titres. These results demonstrate the high efficacy of the Thiverval strain against CSFV replication. Its early protection capacity makes it a useful alternative for emergency vaccination and a consistent tool for CSFV control worldwide.

A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination.

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

Subgrouping and analysis of relationships between classical swine fever virus identified during the 2018-2020 epidemic in Japan by a novel approach using shared genomic variants.

Classical swine fever (CSF) is a worldwide devastating disease of the pig industry caused by classical swine fever virus (CSFV). In September 2018, an outbreak of CSF occurred in Japan where the disease had been eradicated and was officially designated a CSF-free country since 2015. Following the detection of the first 2018 case on a farm in Gifu Prefecture, the disease spread among both farm pigs and wild boars and still continues. Epigenome analysis using whole-genome information is helpful in identifying the infection route, but the current approaches provide an insufficient resolution. In this study, a novel method of using single-nucleotide variants (SNVs) was employed to identify the associations among 158 isolates (65 from farms and 93 from wild boars). The identified groups of CSFV strains were plotted in different colours on a map, identifying the location where each strain was collected. The lack of an SNV set shared between the index case and the other strains suggested the first infection in Japan during the outbreak occurred in wild boars, not at the index farm. For the Atsumi Peninsula outbreaks, where nine farms were found infected within a 10-km radius area, the farm strains were assembled into three groups, suggesting these outbreaks resulted from at least three different infection events in this area. For the infections in the area around Saitama Prefecture, an area remote from the epicentre, strains from both the farms and wild boars were identified as being in the same group, suggesting they resulted from one viral introduction. Likewise, seven infected farms in Okinawa Prefecture, almost 1,500km from Gifu Prefecture, were identified as being in a common, but separate group. By demonstrating the variety of transmission routes and possibility of long-distance infection, these results will help improve disease control measures.

Investigation of congenital tremor associated with Classical swine fever virus genotype 2.2 in an organized pig farm in north-eastern India.

The present study describes an outbreak of Classical swine fever (CSF) in an organized pig farm followed by an episode of CSF virus (CSFV) associated congenital tremors in piglets. The outbreak was recorded in a newly procured herd of Hampshire pigs housed adjacent to the existing pigs of the farm. The recorded CSF outbreak caused a mortality of 100% in the newly procured and 54.28% in the existing herd. As the disease subsides, the clinically recovered boars were served naturally with Tamworth gilts. Though, the sows farrowed on usual gestation period, litters born to each sow showed congenital tremors and eventually died within 24 h of birth. Necropsy analysis of affected piglets was indicative of CSFV infection and was further confirmed using RT-PCR signifying a transplacental infection. The CSFV strains from the initial outbreak and post outbreak episode of congenital tremors were successfully isolated in PK-15 cells and detected in indirect FAT and RT-PCR. Phylogenetic analysis based on E2 gene and 5′NTR of CSFV grouped the isolates within the genotype 2.2 and revealed close resemblance with previously reported Indian isolates of CSFV genotype 2.2 origin. To the best of our knowledge, this is the first report of CSFV induced congenital form reported from India under natural conditions.

Broad neutralization of CSFV with novel monoclonal antibodies in vivo

Classical swine fever is a highly contagious disease in China. Although vaccination against Classical swine fever virus (CSFV) has been widely carried out in China, CSFV cases still emerge in an endless stream. Therefore, it is necessary to take new antiviral measures to eliminate CSFV. Glycoprotein E2 of CSFV is the major vaccine candidate that confers protective immunity. Thus, in this study, a batch of neutralizing monoclonal antibodies (mAbs) against E2, as alternative antiviral strategies, were produced. Among them, mAbs 6D10, 8D8 and 3C12 presented neutralizing reactivity against CSFV in a dose-dependent manner. Based on truncated overlapping fragments of E2 and mutants, three linear neutralizing epitopes were identified highly conserved in various CSFV strains. Epitopes 8YRYAIS13 and 254HECLIG259 were reported for the first time. All the three epitopes are involved in virus internalization and attachment as shown in pre- or post-attachment neutralization. Recombinant polypeptides carrying epitopes successfully inhibit virus infection in PK-15 cells, indicating epitopes were located in receptor-binding domain (RBD). Further, both prophylactic and therapeutic functions of neutralizing antibody were evaluated in rabbits upon CSFV challenge, confirming the efficacy in vivo. These findings provide alternative antiviral strategies against CSFV and deepen the understanding in E2 function during virus entry.