Phylogenetic analysis using E2 gene of classical swine fever virus reveals a new subgenotype in China

Outbreaks of classical swine fever (CSF) have caused serious economic consequences in China. Phylogenetic analysis based on full-length E2 gene sequences showed that five classical swine fever virus (CSFV) isolates collected from Hunan province in 2011 and 2012, together with seven other isolates from neighboring provinces, Guangdong (5) and Guangxi (2), could be classified as a new subgenotype 2.1c, which may have been endemic in the south of China for at least fourteen years. Subgenotype 2.1c isolates share 90.2–94.9% and 89.9–93.8% nucleotide sequence similarity separately with those of subgenotype 2.1a and 2.1b in E2 gene, which are lower than the nucleotide identities between subgenotype 2.1a and 2.1b (91.1–95.7%). Further analysis based on a partial E2 gene sequence (216 nt) indicated that subgenotype 2.1c isolates are also circulating in Thailand. Alignment of E2 amino acid sequences showed that subgenotype 2.1c isolates exhibit a SPA → TPV substitution at positions 777 and 779 compared with subgenotypes 2.1a and 2.1b.     

Genetic diversity of subgenotype 2.1 isolates of classical swine fever virus

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.     

Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.     

Safety,efficacy, and DIVA feasibility on a novel live attenuated classical swine fever marker vaccine candidate

Classical swine fever virus (CSFV) is the etiological agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. Systemic prophylactic immunization with the live attenuated vaccine, the C-strain vaccine, is one of the effective measures for CSF control. However, one of the limitations of the C-strain vaccine is that the field strains-infected animals cannot be differentiated from the C-strain vaccinated herds by serological tests (DIVA). This constraint hampers the practical usage of the C-strain vaccine to eradicate the CSF in China. In the current study, a novel CSF modified live marker vaccine candidate was constructed based on the attenuation of the prevalent 2.1 genotype strain by the deletion of two virulence associated functional residues in the CSFV E^rns, H79, and C171. Meanwhile, four residues S14, G22, E24, and E25 were identified specifically for the 6B8 mAb binding to the CSFV E2 as the novel conformational epitope. Then four substitutions of S14K, G22A, E24R, and G25D were further incorporated in the double deletion construct as a negative serological marker. Finally, the double-deletion marker MLV candidate GD18-ddErnHC-KARD was rescued, and its safety and efficacy profiles were evaluated in piglets. The safety study results indicated that the candidate did not induce fever, clinical signs, or pathological lesions with a high dose of 10^5.0 TCID₅₀, and in addition, no virus shedding was detected until 21 days post-inoculation. Meanwhile, the efficacy study results demonstrated that at a low dose of 10^3.0 TCID₅₀, it conferred complete clinical protection and no virus shedding was detected after the challenge with a highly virulent Shimen strain. Importantly, the infected animals were differentiated using the accompanied DIVA ELISA. These results constitute a proof-of-concept for rationally designing a CSF antigenically marked modified live vaccine candidate.     

Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.     

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 E^rns 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 E^rns 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 E^rns, respectively). Likewise, amino acid-level sequence analysis showed 87.3–91.1% and 87.6–91.6% sequence identity for E2 and E^rns, 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.     

Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4(+) and cytotoxic CD8(+) T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.     

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5’- and 3’-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1^st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV E^rns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV E^rns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.     

Candidate peptide vaccine induced protection against classical swine fever virus

Former investigations demonstrated that the envelope glycoprotein E2 could protect pigs from classical swine fever virus (CSFV). Based on these findings, we prepared synthetic peptide vaccine using E2 N-terminal antigenic units B/C and hoped to induce protective activity against lethal challenge of virulent CSFV strain Shimen. Five overlapped peptides sequence-covering amino acids 693-777 on E2 of Shimen were synthesized and then conjugated with bovine serum albumin (BSA), respectively. In the vaccination course, the candidate peptide vaccines in combination (multi-peptide vaccine (MPV)) were applied for immunization of pigs (n=10) and induced strong antibody response against CSFV. It is subsequently demonstrated that this peptide vaccine could provide immunized pigs complete protection against lethal CSFV challenge as C-strain does, while all non-immunized pigs in negative control group manifested obvious typical symptoms and died during the second and third weeks after viral challenge. In order to confirm the neutralizing activity of the polyclonal antibodies induced by MPV, neutralization assay were carried out on rabbits. The live C-strain alone could ordinarily induce typical fever on rabbits. The typical fever of rabbits induced by the live C-strain could be inhibited by pre-incubation with the anti-sera (dilution 1:4 and 1:16) induced by MPV, but not inhibited by pre-incubation with the same anti-sera from which the antibodies against five peptides were removed by peptide-specific affinity chromatography, which indicates that these peptide-specific antibodies in the anti-sera induced by MPV provided protective activity against CSFV. Our finding provides a new way to develop marker vaccine against CSFV.     

Marker vaccine strategies and candidate CSFV marker vaccines

Classical swine fever (CSF) is an economically important highly contagious disease of swine worldwide. Classical swine fever virus (CSFV) is its etiological agent, and the only natural hosts are domestic pigs and wild boars. Although field CSFV strains vary in the virulence, they all result in serious losses in pig industry. Highly virulent field strains generally cause acute disease and high mortality; moderately virulent field strains raise subacute or chronic infections; postnatal infection by low virulent field strains produces subclinical infection and mortality in the new-born piglets. CSFV can cross the placental barrier, and this transplacental transmission usually results in mortality of fetuses and birth of congenitally infected pigs with a late-onset disease and death. Two main strategies to control CSF epidemic are systematic prophylactic vaccination with live attenuated vaccines (such as C-strain) and non-vaccination stamping-out policy. But neither of them is satisfying enough. Marker vaccine and companion serological diagnostic test is thought to be a promising strategy for future control and eradication of CSF. During the past 15 years, various candidate marker vaccines were constructed and evaluated in the animal experiments, including recombinant chimeric vaccines, recombinant deletion vaccines, DNA vaccines, subunit vaccines and peptide vaccines. Among them, two subunit vaccines entered the large scale marker vaccine trial of EU in 1999. Although they failed to fulfil all the demands of the Scientific Veterinary Committee, they successfully induced solid immunity against CSFV in the vaccinated pigs. It can be expected that new potent marker vaccines might be commercially available and used in systematic prophylactic vaccination campaign or emergency vaccination in the next 15 years. Here, we summarized current strategies and candidate CSFV marker vaccines. These strategies and methods are also helpful for the development of new-generation vaccines against other diseases.     

Efficacy of marker vaccine candidate CP7_E2alf in piglets with maternally derived C-strain antibodies

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain “Koslov”.     

Novel marker vaccines against classical swine fever

Classical swine fever (CSF) is one of the most devastating epizootic diseases of pigs worldwide. For eradication and control purposes, CSF vaccination is an important tool, and efficacious and safe attenuated vaccines have been available for many decades (for example, the C-strain vaccines). In addition to administering them parenterally, live attenuated vaccines are also administered orally for the control and eradication of CSF in wild boar populations. However, antibodies against live attenuated vaccines do not allow to differentiate infected from vaccinated animals (DIVA principle) and the mechanism responsible for attenuation is not known. Only a few years ago the first DIVA vaccines based on baculovirus-expressed E2 glycoprotein have been put on the market [Hulst MM, Westra DF, Wensvoort G, Moormann RJ. Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J Virol 1993;67(9):5435-42]. However, these subunit E2 marker vaccines are less efficient and more than one parenteral application is necessary. Furthermore, oral vaccination is not possible. Taking these disadvantages into account, the development of novel CSF vaccines has been focussed on five different strategies, mainly based on genetically engineered constructs: (1) immunogenic CSFV peptides, (2) DNA vaccines, (3) viral vectors expressing CSFV proteins, (4) chimeric pestiviruses, and (5) trans-complemented deleted CSFV genomes (replicons).     

山东省某地屠宰场猪肝内戊型肝炎病毒感染状况研究

目的研究商品猪肝脏内戊型肝炎（戊肝）病毒（HEV）的检出状况及其基因分型。方法采集35份山东省某个人屠宰场和某城镇大型屠宰场内的猪肝，处理后采用逆转录．巢式聚合酶链反应（nested RT·PCR）方法检测HEV RNA。并与GenBank中登录的HEV全长序列进行同源性分析。结果35份猪肝标本中共有3份检出HEVRNA，阳性率为8．57％。同源性分析显示这3株病毒均为Ⅳ型HEV。结论即将上市的商品猪肝脏内存在HEV感染，其基因型与中国大陆HEV流行株相同，均为基因Ⅳ型。     

Efficient mucosal vaccination of a novel classical swine fever virus E2-Fc fusion protein mediated by neonatal Fc receptor

Classical swine fever (CSF) remains one of the most important highly contagious and fatal viral disease of swine with high morbidity and mortality. CSF is caused by classical swine fever virus (CSFV), a small, enveloped RNA virus of the genus Pestivirus. The aim of this study was to construct the a novel CSFV Fc-fusion recombinant protein and evaluate the efficacy as a vaccine against CSFV. Here, we obtained a novel subunit vaccine expressing CSFV E2 recombinant fusion protein in CHO-S cells. Functional analysis revealed that CSFV Fc-fusion recombinant protein (CSFV-E2-Fc) could bind to FcγRI on antigen-presenting cells (APCs) and significantly increase IgA levels in serum and feces, inducing stronger mucosal immune response in swine. Additionally, CSFV-E2-Fc immunization enhanced CSFV-specific T cell immune response with a Th1-like pattern of cytokine secretion, remarkably stimulated the Th1-biased cellular immune response and humoral immune response. Further, the protective effects of CSFV-E2-Fc subunit vaccines were confirmed. The data suggest that CSFV E2-Fc recombinant fusion protein may be a promising candidate subunit vaccine to elicit immune response and protect against CSFV.     

Enhanced protective immunity to CSFV E2 subunit vaccine by using IFN-γ as immunoadjuvant in weaning piglets

The glycoprotein E2 of classical swine fever virus (CSFV) is a major immunogenic protein that induces neutralizing antibodies and protective immunity. Thus, E2 is a suitable target antigen for the development of genetically engineered CSFV vaccines. However, these vaccines cannot generate complete protective immunity in their hosts, thereby limiting the scope of applications under field conditions. IFN-γ is an immune adjuvant that has been shown to enhance antigen immune response in various experimental models. In this study, porcine IFN-γ was used to improve the immunogenicity of the CSFV E2 subunit vaccine in pigs. Pigs were immunized with E2 subunit vaccine alone or in combination with IFN-γ. Results demonstrated that porcine IFN-γ did not enhance the CSFV-specific antibody and neutralizing antibody titers compared with the E2 subunit vaccine alone. However, co-administration of the E2 and IFN-γ subunit vaccines significantly enhanced the CSFV-specific IFN-γ expression. These findings indicated that porcine IFN-γ can increase cellular immune responses to E2 protein in pigs. Furthermore, co-immunization with E2 + IFN-γ subunit vaccine and C-strain conferred complete protection against CSFV. In contrast, E2 subunit vaccines provided incomplete protection in pigs. These results indicated that using IFN-γ as an adjuvant with CSFV E2 subunit vaccines can enhance the specific protective immune response. Therefore, E2 + IFN-γ subunit vaccine is a promising marker vaccine candidate for the control and eradication of CSF.     

Positive selection pressure on the B/C domains of the E2-gene of classical swine fever virus in endemic areas under C-strain vaccination

In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each year, despite the implemented vaccination program. Interestingly, a trend towards a milder presentation of the disease has been observed among the animals during the last years. This study aimed to assess positive selection pressure acting on partial E2 gene of CSF viruses to gain insights into the mechanisms governing virulence and the driving forces of classical swine fever virus (CSFV) evolution in swine populations under regular vaccination. Selection pressure analysis were performed to detect positive selection acting on a particular lineage as well as among sites of the E2-B/C-domain of CSFV nucleotide sequences, reported in a previous study and in the present work, several models, available in the CODEML module of PAML 4.3, were used. In addition, a representative Cuban CSF isolate was assessed in an experimental infection trial for their clinical virulence in order to expand the knowledge regarding CSF viruses circulating in pig populations. The viral genomes sequenced in this study were grouped in a defined cluster within the genotype 1.2, as it has been reported previously for Cuban CSF viruses. The selection pressure analysis didn't find evidence of positive selection (dN/dS of>1) along any branch. The positive selective pressure analysis estimated six new sites under positive selection on E2 partial gene analysed. Besides, the clinical manifestations of the CSF-disease were related mainly to a mild course of the illness. The high number of positively selected sites suggests that these changes could be associated to viral evasion of the host-immune response. These observations highlight a possible association between escape viral variants and the alterations observed in the virulence and pathogenesis of the virus. Therefore, while the vaccination programs have not led to a genotype change, alterations in virulence were suggested to arise.     

Co-infection status of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018

Classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) are economically important swine viruses that cause reproductive failure and/or respiratory symptoms in pigs. However, the co-infection status of these viruses in Chinese swine herds is not well clarified. In this study, we evaluated the co-infection of these four viruses in 159 pigs collected from 63 herds in eight regions of China from 2016 to 2018. CSFV, PRRSV, PCV2 and PCV3 were detected in 14, 56, 43 and 4 of the pigs, respectively. The percentage of singular infections was 32.71%, while the percentages of dual infections and multiple infections were 15.72% and 3.15%, respectively. The E2 of CSFV, ORF5 of PRRSV, ORF2s of PCV2 and PCV3 from all positive samples were determined and used for phylogenetic analyses. E2-based phylogenetic tree showed that all 14 CSFVs identified in this study belong to 2.1b subtype. ORF5-based phylogenetic tree showed that PRRSV2 is predominant in China while PRRSV1 can also be detected. In addition, 35, 16, 4 and 1 of our PRRSVs are clustered with highly pathogenic PRRSV2, NADC30-like PRRSV2, classical PRRSV2 and PRRSV1, respectively. ORF2-based phylogenetic trees showed that our PCVs are grouped with 2 PCV2 subtypes (PCV2d and PCV2b) and 3 PCV3 subtypes (PCV3a, PCV3b and PCV3c), respectively. Our results provide the latest co-infection status and the diversity of four important swine viruses in Chinese swine herds, which is beneficial for understanding the epidemiology of these viruses.     

Analysis of the complete genome sequences of one swine and two human hepatitis E virus genotype 4 strains isolated in Beijing,China

Full-length sequences were determined and analyzed for two human (MO and W3) and one swine (W2–5) hepatitis E virus (HEV) isolates from Beijing, China. The genomes of the three strains were composed of 7242, 7239, 7239 nucleotides, respectively, excluding the poly (A) tails, and were 84% identical to each other. All were classified into genotype 4. Sequence analysis shows that the 2 human isolates have up to 91–94% nucleotide identity in full length genome with swine strains isolated in China, while the swine isolate share 92% identity with the human strain T1 from Beijing. At the amino acid level, the three strains share 94%, 97% and 89–92% identity in the ORF1, ORF2 and ORF3, proteins respectively. The human strains MO and W3 have the highest identity, 97%, 98–99% and 96–98% in ORFs 1–3, respectively, to swine strains CHN-XJ-SW13 and CHN-XJ-SW33 from Xinjiang, China, while swine strain W2–5 has highest identity with the human strain HE-JA2, 96%, 99% and 91% in ORFs 1–3, respectively. Genotype specific amino acid substitutions were found at a single site in all three ORFs by sequences alignment, and genotype specific short sequences (5–10aa in length) were found in ORF1 and the C-terminus of ORF3. However, no difference was found at any amino acid position that discriminates between human and swine HEVs within genotype 4 for any of the three ORFs. These results indicated that the genotype 4 HEV strains from humans and pigs in China may evolve from the common ancestor.     

Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus

Our previous study proved that the N-terminal (aa693-711) of glycoprotein E2 contained sequential neutralizing epitopes. In this study, four candidate epitope-vaccines (EVs) were separately prepared and evaluated. Among them, epitope-vaccine EV-BC1a (BC1a: aa693-699) induced high level of epitope-specific neutralizing antibodies and exhibited similar protective capability with that induced by Chinese vaccine strain (C-strain). These results confirmed CKEDYRY (aa693-699) as a principal sequential neutralizing epitope on E2 N-terminal. Moreover, these findings also indicate that epitope-vaccine is a potent candidate strategy for marker vaccine against classical swine fever virus (CSFV).