Classical swine fever virus isolates from Cuba form a new subgenotype 1.4

Identification and classification of classical swine fever virus (CSFV) on the basis of nucleotide sequencing and phylogenetic analysis have become an important tool to study the epidemiology and to control CSF disease. According to phylogenetic analyses of short sequences from the 5′nontranslated region (150 nt) and the E2 (190 nt), most CSFV isolates from South and Central America have been assorted to the subgenotypes 1.1 and 1.3, while CSFV isolates from Cuba have been allocated to subgenotype 1.2. Here we demonstrate that determination and comparison of full-length E2 sequences as well as of the sequences encoding for N^pro, C, E^rns, E1 and E2 (3361 nt) do not support segregation of Cuban CSFV isolates to subgenotype 1.2. In fact, our analysis revealed that the Cuban isolates are more divergent from other so far known CSFV subgenotype 1 isolates and form a novel separate subgenotype that is proposed to be designated subgenotype 1.4.     

Molecular epidemiology of classical swine fever in the Russian Federation

The ability to discriminate between various classical swine fever virus (CSFV) strains and isolates is a prerequisite for following the spread of the virus after an outbreak. To determine the relatedness between Russian CSFV isolates from different geographical regions, three fragments of the viral genome (5' NTR, the variable region of the E2 gene and a fragment of the NS5B gene) were sequenced and used for genetic typing. Thirty-one field isolates were obtained from CSF outbreaks which occurred between 1994 and 1999. In addition, three attenuated strains were included in the study, namely the LK and CS vaccine strains, and the moderately virulent 238H isolate. The vaccine strains have been used in Russia for more than 30 years. Our results showed that all field isolates are in subgroup 1.1 together with Alfort 187 and with the highly virulent strain Shimen. In contrast, the CS and LK vaccine strains belong to subgroup 1.2. While there is no evidence for the reversion of the two vaccine strains to wild type, it is feasible that the highly virulent Shimen strain, which has been used as a challenge strain for many years, contributed to field strain generation. The Russian field isolates from the 1990s can be distinguished from the CSF virus isolates which occurred in the EU Member States in the same decade, as here all outbreaks were caused by CSF viruses belonging to subgroup 2.     

Phylogenetic analysis of recent classical swine fever virus (CSFV) isolates from Assam, India

Classical swine fever (CSF), a highly contagious viral disease of pigs, is endemic in India. As there is no information concerning the accurate genetic typing of classical swine fever virus (CSFV) isolates in India, 16 CSF viruses isolated during 2005-2007 from domestic pigs in different districts of Assam were typed in 5′ UTR (150 nucleotides). To confirm the genetic typing results and to study the genetic variability, selected viruses were also analyzed in E2 (190 nt) and NS5B gene (409 nt) regions. Phylogenetic analysis revealed that all the 16 CSFV isolates analyzed belonged to group 1 and subgroup 1.1 in contrast to the situation in other Asian countries. Additionally, analysis in E2 and NS5B region placed the Indian isolates in a clearly separated clade within subgroup 1.1. The results suggest that subgroup 1.1 CSF viruses are currently circulating in India, which is important for epidemiology and control of CSF.     

Comparative sequence analysis of classical swine fever virus isolates from the epizootic in The Netherlands in 1997-1998.

Sixteen classical swine fever virus (CSFV) field isolates from outbreaks of classical swine fever from the period between February 1997 and March 1998 in the Netherlands were sequence analysed. Parts of the 5' noncoding region (5'NCR) and the E1/E2 gene were sequenced after RT-PCR. The obtained sequences were compared with isolates of recent outbreaks in Europe and those of former outbreaks in the Netherlands. Sequence alignment of the 5'NCR region (321 bp) revealed that the isolates of the Dutch outbreak of 1997-1998 were closely linked to an isolate of the CSF outbreak that started in Paderborn, Germany in 1996. A relatively large fragment of the E1/E2 gene of 850 bp, including the antigenic region of E2, which is one of the most variable regions of the CSFV genome, was sequenced to determine whether this region can be used for epidemiology within an epizootic. Epidemiological tracing of transmission of virus was followed, starting from the first isolate and a line of five generations of viruses was analysed. Besides this, new isolates which could not be epidemiologically linked to preceding ones were also characterised. Differences between the isolates of the Dutch outbreak were minor both for the linked as well as for the non-linked isolates, indicating that all isolates have a common origin. Furthermore, our data show for the first time the genetic stability of CSFV even in the highly variable antigenic region of the E2 gene during a major epidemic lasting more than 1 year.     

Determination of the sequence of the complete open reading frame and the 5'NTR of the Paderborn isolate of classical swine fever virus

The classical swine fever (CSF) epidemic in the Netherlands in 1997-1998 lasted 14 months, during which 429 infected and 1300 at risk herds were culled, at an estimated economical cost of 2 billion US dollars. Despite the overwhelming scale of the epizootic, the CSF virus (CSFV) strain causing the outbreak has remained largely uncharacterized. The Dutch epizootic is epidemiologically linked to a small CSF outbreak in 1997, in Paderborn in Germany. E2 and partial 5' NTR sequencing has shown that the index Paderborn isolate, and several Dutch isolates taken during the 1997-1998 epizootic, are virtually identical, confirming that the Paderborn isolate triggered the Dutch outbreak, and furthermore showing that this single isolate was stable throughout the whole Dutch outbreak (the above reviewed in [C. Terpstra, A. J. de Smit, Veterinary Microbiol. 77 (2000) 3-15]). We determined the nucleotide sequence of the 5' NTR (by 5' RACE) and the complete open reading frame of the Paderborn isolate (GenBank AY072924). Our sequence was identical to previously published partial 5'NTR and E2 sequences for the index Paderborn 1997 and Dutch 1997 (Venhorst) isolates, confirming the identity of the virus we sequenced. Phylogenetic analysis based on the complete open reading frame showed that Paderborn is genetically very different from common European laboratory reference strains. Neutralization studies showed that Paderborn is also antigenically very different from common laboratory strains such as Alfort 187. Paderborn is the only recent European CSFV field isolate for which a complete sequence is available, and given Paderborns genetic and antigenic uniqueness, the Paderborn sequence may have practical use for diagnostic and vaccine antigen development.     

Emergence of 2.1. subgenotype of classical swine fever virus in pig population of India in 2011

Background: Limited studies are available on molecular epidemiology of classical swine fever virus (CSFV) in India and are restricted to domestic pigs. These studies show the presence of 1.1. genotype.

Hypothesis/objectives: The aim of the present study was to subgenotype four CSFV isolates, two each from the outbreaks of CSF in wild (Sus scrofa) and domestic pigs of Mizoram state, India, in 2011.

Animals and methods: CSFV isolates were subjected to nucleotide sequencing in E2 and NS5B genomic regions. Phylogenetic analysis of the isolates in both genomic regions was carried out with 39 Indian isolates (4 isolates from the present study of Mizoram state and 35 isolates from the other states of India) and 57 reference sequences retrieved from the GenBank database. Two of the 39 isolates from India were collected from wild boar and were subgenotyped as 2.1. Out of 37 isolates from domestic pigs, only two were subgenotyped as 2.1.

Results: The analysis revealed the emergence of 2.1. subgenotype of CSFV in both wild and domestic pigs in India.

Conclusions and clinical importance: The isolates from domestic pigs of Mizoram state (CSF/MZ/KOL/73 and CSF/MZ/AIZ/115) were grouped in genotype 1 and subgenotype 1.1., thus confirming that the source of CSF outbreaks in domesticated pigs in Mizoram was not from wild pigs. The current study forms an essential step for better understanding of the epidemiology of 2.1 subgroup as well as the movement and spread of the disease in India.     

2017-2019年山东省部分地区猪瘟流行病学调查

为掌握近年来山东地区猪瘟病毒（CSFV）的流行及其遗传变异情况，本研究对2017年1月-2019年12月从山东省济南市、泰安市、聊城市、青岛市等10个地区采集的1 687份疑似猪瘟感染猪病料运用反转录PCR方法进行了病毒核酸检测，将CSFV检测为阳性的样品进行E2基因和CSFV全基因扩增和测序，利用生物信息学软件DNAStar和Mega 7.0对CSFV的E2蛋白以及全基因序列进行遗传进化分析。另外，将这些地区采集的4 866份血清样本进行了抗体ELISA检测。结果显示，1 687份病料中病原检测有188份阳性，总阳性率为11.1%，其中2017-2019年病原检测阳性率分别为8.5%、13.1%、15.1%，抗原阳性率逐年上升且呈不稳定性，提示规模化猪场猪瘟兔化弱毒疫苗的免疫存在一定的风险和压力；采集的4 866份血清样本中4 146份呈现抗体阳性（85.2%），统计得到2017-2019年抗体阳性率分别为84.3%、83.0%、92.3%，抗体阳性率呈现上升趋势，说明山东省规模化养殖场猪群猪瘟疫苗免疫保护的整体水平逐年提高；从阳性病料中扩增获得了5株CSFV全基因组序列和5株病毒E2基因序列，经过序列比对发现，5株临床分离毒株与2.1d亚型的CSFV全基因组序列同源性在97.5%~99.0%之间，表明5株分离毒株与2.1d亚型的CSFV亲缘性较近，分离毒株氨基酸位点的突变集中在102位（L→M）以及159位（K→R）氨基酸处。本研究分析了2017年1月-2019年12月山东部分地区猪群中CSFV毒株的流行与变异情况，为指导山东省CSFV预防与控制提供了有力的参考。     

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

The envelope glycoprotein E2 of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle, interaction with host and pathogenesis. We sequenced the E2 gene of 34 CSFV isolates from southeastern China for analysis of genetic diversity in this particular region. Phylogenetic analysis revealed that genotype 2.1b viruses became predominant in southeastern China with 33 isolates clustered in 2.1b and only 1 isolate belonged to 2.2. Pairwise comparisons demonstrated isolates in this study showed a homology of 78.4-82.6% to Chinese C-strain in the 190nt of the E2 fragment. The minimum similarity within the 2.1b isolates was 91.1%. Variability of the full length E2 amino acid sequences of 45 CSFV isolates representative of three main groups of CSFV including 19 from southeastern China during 2004--2007 and 26 from other parts of China and other countries was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. Two variable and three conserved regions as well as some specific positions under positive selection were defined. Our results suggest that recent isolates from southeastern China have shifted away from historical CSFV isolates including the vaccine strains probably as a result of their adaptive abilities to the selection forces within the host.     

鸡传染性支气管炎病毒SD060311株和GD080122(肾型)株的全基因组序列分析

通过对鸡传染性支气管炎病毒(IBV)分离株SD060311和GD080122(肾型)的基因组进行RT-PCR扩增和基因序列测定,结果表明其基因组大小分别为27 788 nt和27 407 nt,与已报道的IBV全基因组序列大小不完全一致,但基因组编码基因的顺序与已报道的IBV一致,均为5′cap-Replicase-S-3a-3b-3c-M-5a-5b-N-poly (A)3 ′.与已报道的IBV毒株和火鸡冠状病毒进行比较,绘制系统进化树,结果显示,SD060311和GD080122(肾型)分离株自成一支,与中国分离株BJ株和A2株的亲缘关系最近.本研究不仅丰富了冠状病毒的生物信息数据库,且为进一步研究IBV致病机理和变异机制等奠定了基础.     

非典型性型猪瘟病毒云南株的分离鉴定及其E0／E2基因序列分析

从云南10个地州13个大型猪场采集到的17份样品中分离得到5株猪瘟病毒。经测序鉴定昆明、玉溪、曲靖地区分离株的核苷酸序列99．99％同源,大理和宝山地区的核苷酸序列99．99％同源,5株分离毒均属于基因二群。5株分离毒在PK-15细胞上的平均毒价约为2×10^6TCID50／mL,应用荧光抗体染色可以检测到CSFV。通过RT-PCR扩增猪瘟病毒约1200bp和700bp的E2和E0蛋白全部抗原编码区序列,并将其分别克隆到pMD18-T载体中。序列分析结果表明,5株分离株的E0和E2基因片段为697bp和1173bp,与猪瘟病毒Shimen株的核苷酸同源性仅为95．4％和96．6％、82．5g和84．3％,与C-株的核苷酸同源性分别为94．1％和95．2％、83．3％和85．4％。动物试验结果表明,分离的2株猪瘟野毒不能引起典型的猪瘟症状,但是能持续带毒。     

Genotypic characterization of Chilean llama (Lama glama) and alpaca (Vicugna pacos) pestivirus isolates

Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities worldwide where they have been introduced. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus, in particular Bovine Viral Diarrhea (BVDV), but there is little data available on Pestivirus infections in SACs. In this study we aimed to detect and identify Pestivirus genotypes and subgroups infecting SACs in both wild and confined environments. Samples were collected from 136 llamas and 30 alpacas from different areas in the Chilean Altiplano (wild animals), and from 22 llamas and 26 alpacas diagnosed as Pestivirus positive from the Metropolitana region in Chile (confined animals). Seroneutralization tests showed titers lower than 2 in all 166 samples from Chilean Altiplano. These samples were also negative to BVDV isolation, indicating that these animals have not been exposed to Pestivirus. After reactivation of positive samples from the Metropolitana region, the 5′ non-codifying region (5′NCR) and E2 glycoprotein were amplified by RT-PCR from the Pestivirus genome. Viral sequences were pairwise compared and phylogenetic trees were constructed. The 5′NCR analysis showed that all 12 sequenced isolates belonged to BVDV-1. Of particular interest, isolates from eight llama and two alpaca were BVDV-1j and two alpacas were BVDV-1b. In agreement with these results, E2 phylogenetic analysis rendered a similar grouping indicating that all 16 isolates belong to BVDV-1. However, the lower availability of E2 sequences determines the creation of a smaller number of sub-groups than the 5′NCR sequences. Based on the E2 sequences, the 5′NCR BVDV 1j group consisting of all the llamas and 3 alpacas are completely included in the E2 BVDV 1e group. Due to the universal availability of the 5′NCR segment, we propose the classification of these Chilean llamas and alpacas Pestivirus isolates as BVDV 1j and BVDV 1b respectively. Thus, this is the first time BVDV-1j is obtained in SACs. In addition, these results indicate Pestivirus infection in llamas and alpacas is associated with bovine population as genotypes and sub-groups are the same as those affecting Chilean livestock.     

猪瘟病毒一步法RT PCR检测方法的建立

本研究根据NCBI公布的猪瘟病毒石门毒株的E2基因序列，设计、合成了1对检测猪瘟病毒E2基因的RT-PCR引物。自疑似猪瘟病料及接猪瘟F114毒的PK-15细胞中提取总RNA，经一步法RT-PCR扩增E2基因。建立了猪瘟病毒E2基因RT-PCR检测方法，RT-PCR从26份疑似猪瘟病料中检出阳性病料24份，阳性检出率为92.3%；结果表明，建立的 RT-PCR方法具有良好的特异性和敏感性，可用于CSFV的分离鉴定、临床病料的检测和分子流行病学调查。     

Molecular epidemiology of classical swine fever in Italy

To gain an insight into the molecular epidemiology of classical swine fever (CSF) in Italy, virus isolates originating from outbreaks that occurred between 1985 and 2000 in wild boar or in domestic pigs in mainland Italy and in Sardinia were analysed by genetic typing. For this, a fragment (190 nucleotides) of the E2 glycoprotein gene was sequenced and phylogenetic analyses were performed, including older Italian isolates and isolates from recent outbreaks in Europe for comparison. The results show that in mainland Italy, several independent epidemiological events occurred in the last decade. In the north of the country, viruses of genotype 2.2 have persisted in wild boar, causing sporadic outbreaks in domestic pigs. In contrast, viruses of subgroups 2.1 and 2.3 appeared only intermittently in different regions of the mainland. In 1997, classical swine fever virus (CSFV) isolates belonging to the subgroup 2.1 and genetically and epidemiologically related to the Dutch isolate in Venhorst, affected domestic pigs exclusively. The isolates of subgroup 2.3, derived from wild boar as well as from domestic pigs were closely related to isolates from Germany and Poland. In Sardinia, CSF is an endemic in wild boar and affects domestic pigs also. Genetic typing showed that viruses of subgroups 1.1 and 2.3 have been present, the last ones being unrelated to the mainland viruses of the same subgroup. Due to the large quantities of pig and wild boar meat imported in some parts of Italy, it cannot be established if these viruses were always present in either the mainland or Sardinia, or if they represent recent introductions.     

猪瘟病毒基因分型寡核苷酸芯片方法的建立

根据猪瘟病毒（CSFV）E2基因序列,设计41条针对CSFV 3个基因群共10个亚群各亚群寡核苷酸探针。利用欧盟猪瘟诊断手册推荐的CSFV E2基因套式RT-PCR方法,在内套PCR过程中进行Cy3-dCTP掺入荧光标记,制备芯片杂交样品。用标记的PCR产物与寡核苷酸探针阵列杂交,置于GenePix 4100A扫描仪中扫描,利用Ge-nePix Pro 6.0软件分析杂交图像。特异性和灵敏度试验显示,芯片方法与本室发表的CSFV real-time RT-PCR方法的灵敏度相近,芯片探针与猪繁殖与呼吸综合征病毒（PRRSV）、猪2型圆环病毒（PCV2）、猪伪狂犬病毒（PRV）样品无非特异性杂交。以包括1.1、2.1、2.2、2.3亚群的8份CSF阳性样品进行芯片的检测验证,结果表明,通过特异性的杂交图谱或杂交信号分析可准确判定样品所属的基因亚群,寡核苷酸芯片的检测结果与测序的分型结果全部符合。本研究为将寡核苷酸芯片技术用于猪瘟病毒的基因分型和分子流行病学研究奠定了基础。     

A common neutralizing epitope on envelope glycoprotein E2 of different pestiviruses: implications for improvement of vaccines and diagnostics for classical swine fever (CSF)?

The Pestivirus genus within the family of Flaviviridae consists of at least three species; classical swine fever virus (CSFV) found in swine and wild boar, bovine viral diarrhoea virus type 1 and type 2 (BVDV-I and BVDV-II) mainly isolated from cattle and border disease virus (BDV) preferably replicating in ovine species. Many features demonstrate differences between CSFV and other pestiviruses, BVDV-I, BVDV-II and BDV, here defined as nonCSFV, whereas other features show similarities between all different species of pestiviruses. Focussing on the major envelope glycoprotein E2, the immunodominant protein of pestiviruses, CSFV seems to be a more distinct species within the Pestivirus genus. Here we confirm on one hand the more separated grouping of CSFV by isolation of monoclonal antibodies (MAbs) raised against E2 of BVDV-I and BVDV-II. None of these MAbs recognize E2 of CSFV strains. On the other hand, only one MAb, MAb 912, was isolated against E2 of BDV. MAb 912 binds to E2 of CSFV strains and partly neutralizes CSFV. The epitope of MAb 912 is mapped in antigenic domain B of CSFV-E2. This common epitope of CSFV strains and nonCSFV strains could have implications for development of DIVA vaccines and serological diagnostics for CSF.     

Genetic characterization of Indian peste des petits ruminants virus (PPRV) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments

Peste des petits ruminants (PPR) is an important viral disease of sheep and goats, endemic in India. The study was undertaken to characterize the local PPRV by sequencing fusion (F) protein and nucleoprotein (N) gene segments and phylogenetic analysis, so as to focus on genetic variation in the field viruses. Selected regions of PPRV genome were amplified from clinical samples collected from 32 sheep and goats by RT-PCR and the resulting amplicons were sequenced for phylogenetic analysis. The phylogenetic tree based on the 322bp F gene sequences of PPRV from five different locations clustered them into lineage 4 along with other Asian isolates. While the 425bp N gene sequences revealed a different pattern of branching, yielding three distinct clusters for Nigerian, Turkey and Indian isolates. Thus, classification of PPRV into lineages based on the N gene sequences appeared to yield better picture of molecular epidemiology for PPRV.     

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.     

2018年中国部分地区猪瘟病毒2.1d亚型新流行株的基因组特征分析

为了解中国猪瘟病毒（classical swine fever virus，CSFV）的分子流行病学及遗传变异情况，本研究应用RT-PCR方法对2018年采集自河南、河北、山东、黑龙江和辽宁5个省份的350份疑似CSFV感染的病料进行E2和NS5B基因扩增，并对PCR产物进行测序和序列分析。结果显示，350份样品中21份为CSFV阳性，共获得14株CSFV的E2基因序列和7株CSFV的部分NS5B基因序列。E2全基因、NS5B部分基因序列分析表明，21份阳性样品均属于近年来在中国流行的CSFV 2.1d亚型，且新发现的2.1d亚型CSFV与中国较早2.1d亚型CSFV毒株间同源性差异不大，新发现的2.1d亚型CSFV分离株在E2基因的6个氨基酸（R³¹、S³⁴、W¹⁸²、K²⁰⁵、K³⁰³、A³³¹）上具有相同的分子特征，E2蛋白中15个位点上的半胱氨酸均未发生变异。韩国2.1d亚型CSFV在E2蛋白上具有3个独特的氨基酸（N⁹⁷、K¹⁵⁹、R²⁰⁵）特征，并且发现了韩国毒株YC11WB可能作为2.1b和2.1d亚型CSFV过渡毒株的证据，流行于中国和韩国的2.1d亚型CSFV可能分别来自于本国早期2.1b亚型CSFV的衍化。本研究证实，2018年中国及周边国家CSFV较为活跃，且流行毒株依然以2.1d亚型为主，为中国科学防控CSFV提供了依据。