Literature DB >> 32426263

Genomic characteristics of classical swine fever virus strains of bovine origin according to primary and secondary sequence-structure analysis.

Massimo Giangaspero1, Shu-Qin Zhang2.   

Abstract

Background: Classical swine fever virus (CSFV), species member of the family Flaviviridae, is generally considered restricted to domestic and wild suids. The circulation of CFSV has been detected in cattle herds in China and India. Natural infection appeared associated with clinical signs in some cases. Aim: The secondary structures of the internal ribosome entry site in the 5' untranslated region (UTR) were used for the genomic characterization of bovine strains.
Methods: Sequences have been compared to the representative CSFV strains isolated from pigs, vaccines, and contaminants from porcine cell lines and an ovine strain isolated in Spain.
Results: The observed sequences from cattle showed a genetic relatedness with live-attenuated vaccine strains used in pigs. Sequence characteristics of the Chinese strain S171 are genetically distant from the previously reported CSFV genotypes, suggesting a new outgroup in the species, described for the first time, and named CSFV-d. Other Chinese strains were genetically closely related to CSFV genotype a2 (Alfort type) pig strains. Indian strains, reported from the states of Tamil Nadu and Meghalaya, were genetically closely related to CSFV genotype a1 (Brescia type) and a5 pig strains, respectively.
Conclusion: These preliminary observations are new and relevant in countries, where CSFV control and eradication strategies are applied.

Entities:  

Keywords:  Cattle; Classical swine fever virus; Pestivirus; Secondary structure

Mesh：

Year:  2020        PMID： 32426263      PMCID： PMC7193884          DOI： 10.4314/ovj.v10i1.15

Source DB:  PubMed          Journal:  Open Vet J        ISSN： 2218-6050


Introduction

The genus Pestivirus, family Flaviviridae, includes genetically related species recognized as important pathogens in veterinary medicine. The Classical swine fever virus (CSFV) has been eradicated in different countries as in Europe and North America, but still control and eradication strategies are applied against the virus in various other regions. There is a consensus concerning the capacity among pestiviruses to cross species barrier. The wide host range of these viruses may lead to the formation of virus reservoirs in other domestic or wildlife species, presenting a concern for the long-term success of eradication campaigns. Nevertheless, CSFV is generally considered to be restricted to domestic and wild suids. Current knowledge indicates that experimental infections without clinical signs have been reported in cattle, sheep, goats, and deer, but there is no evidence that these species become infected in nature (Biró ; Loan and Storm, 1968; Shimizu and Kumagai, 1989; CFSPH, 2015). In only two cases, the species has been identified in sheep (Hurtado ; Mohamed, 2004), corroborating these beliefs. One ovine isolate (strain 5440/99), which is similar to CSFV vaccine strains used in the framework of eradication campaigns in pigs, was reported from Spain (Hurtado ). A second isolate (strain 12 Ovine liver 113nt) was obtained during investigations on pestiviruses in the United Arab Emirates (Mohamed, 2004). This Pestivirus-like sequence, when aligned with corresponding sequences from a variety of pestiviruses, showed a greatest similarity to CSFV subgenotype 1.1 but raised doubts on correct classification, due to the suboptimal quality of the sequence and its short length (113 nucleotides) (Mohamed, 2004). On contrary to this apparent epidemiological static nature and apparent inability to cross species barrier in natural conditions, the recent reports indicated the isolation of CSFV strains in cattle (Bos taurus) from India and China and contaminants or associated to natural infection with reproductive disorders (abortion, stillbirth, birth defects, repeat breeding, and mastitis) (Zhang ; Giangaspero ). Twelve strains isolated from cattle aged from 6 months to 2 years have been detected in four farms, out of 19 tested, during an epidemiological survey aiming to identify bovine viral diarrhea virus (BVDV) persistently infected animals by reverse transcription-polymerase chain reaction (RT-PCR), among BVDV-seropositive enzyme-linked immunosorbent assay (ELISA) cattle and buffalo farms from four districts of the state of Tamil Nadu, India (Giangaspero ). Four strains were reported from China. Pestivirus RNA sequences have been detected from commercial fetal bovine serum batches and designated as strains S171 (KF006974), S173 (KF006975), and S112 (MK118725) (identical to S173) originated from Henan and Jiangsu provinces (Zhang ). The attempts to isolate live viruses from some of these samples have been unsuccessful. The strain HEN03 (sequence deposited by Zhang, under accession number KC176778) was detected in cattle from Henan province. In addition, during a survey for the detection and genetic characterization of 5′-untranslated region (UTR) and E2 gene of CSFV from bovine population of the northeastern region of the Indian state of Meghalaya, of 134 cattle serum samples tested, all were positive in RT-PCR for 5′-UTR region, and 10 samples were positive for CSFV antigen by a commercial antigen capture ELISA (Chakraborty et al., 2018). The full-length E2 region of CSFV was amplified from two positive samples. In this study, the 5′-UTR sequences of the reported strains have been considered for genomic characterization according to the secondary structure analysis of the internal ribosome entry site (IRES).

Material and Methods

The 5′-UTR sequences of the 16 reported strains (13 sequences detected in cattle from India and China and three sequences detected in commercial fetal bovine serum batches from China) (Zhang ; Giangaspero ; and with reference to strain HEN03 sequence deposited by Zhang) have been compared with those from other CSFV strains (n = 110) isolated from pigs, vaccines, and contaminants from porcine cell lines and an ovine strain isolated in Spain representative of identified genetic clusters in the species, with particular attention to isolates circulating in Asian countries, including India and China (Table 1), and further compared to 1,300 sequences from other Pestivirus species different from CSFV. The two Indian bovine CSFV sequences reported by Chakraborty et al. (2018) were evaluated indirectly considering the similarity of their full-length E2 region with other previously deposited pig sequences. Newly described subgenotypes reported from Brazil and Cuba (Postel ; Silva ) could not be considered, being available only E2 sequences, and apparently specific and restricted to South American and Caribbean regions.
Table 1.

List of Pestivirus strains of CSFV species (n = 126) evaluated according to palindromic secondary structure characteristics at the RNA 5′-UTR (PNS method). The nomenclature of identified genotypes is based on divergence in the genus. Clustering according to the primary sequence analysis by depositors is indicated under parenthesis. ND: not determined. Cattle (B. taurus); Pig (Sus scrofa domesticus); Sheep (Ovis aries); Wild boar (Sus scrofa scrofa).

Species/GenotypeStrainOriginYearCountryAccessionReference
CSFV-al1 IndiaCattle2016IndiaMG859286Giangaspero et al., 2017
CSFV-al10 IndiaCattle2016IndiaMK105823Giangaspero et al., 2017
CSFV-al11 IndiaCattle2016IndiaMG859287Giangaspero et al., 2017
CSFV-al13 IndiaCattle2016IndiaMK105824Giangaspero et al., 2017
CSFV-al2 IndiaCattle2016IndiaMK105825Giangaspero et al., 2017
CSFV-al39Pig2001ChinaAF407339Wu et al., unpublished
CSFV-al3 IndiaCattle2016IndiaMK105826Giangaspero et al., 2017
CSFV-al4 IndiaCattle2016IndiaMK105820Giangaspero et al., 2017
CSFV-al5 IndiaCattle2016IndiaMK105821Giangaspero et al., 2017
CSFV-al5NCR/C SF/MZ/AIZ/348Pig2011India.1X975460Rajkhowa et al., unpublished
CSFV-al5NCR/C SF/MZ/AIZ/3 52Pig2011India.1X975461Rajkhowa et al., unpublished
CSFV-al6 IndiaCattle2016IndiaMK105822Giangaspero et al., 2017
CSFV-al7 IndiaCattle2016IndiaMK109913Giangaspero et al., 2017
CSFV-al8 IndiaCattle2016IndiaMG813566Giangaspero et al., 2017
CSFV-al9 IndiaCattle2016IndiaMK105827Giangaspero et al., 2017
CSFV-al (1.1)Alfort 187PigFranceX87939Ruggli al., 1995
CSFV-alAlfort A19PigFranceU90951Smondack et al., unpublished
CSFV-alAizawl-09Pig2009IndiaHM449066Rajkhowa et al., unpublished
CSFV-alBangalore Ind-163/07Pig2007IndiaEU446419Patil et al., unpublished
CSFV-al (1.1)BresciaPigItalyM31768Moorman et al., 1990
CSFV-al (1.2)BRESCIAXPigItalyAY578687Risatti et al., 2005
CSFV-alBV-PPig2005FlungaryDQ314582Farsang et al., unpublished
CSFV-alC strainVaccine1994ChinaZ46258Moonnann et al., 1996
CSFV-al (1.1)CAPPigSwitzerlandX96550Tratschin et al., unpublished
CSFV-alcF114Pig2001ChinaAF333000Mingxiao et al., unpublished
CSFV-alCSF/MZ/KOL/73Pig2009IndiaJX094153Rajkhowa, unpublished
CSFV-alCSF/MZ/SAI/76Pig2009IndiaJX094154Rajkhowa, unpublished
CSFV-al (1.1)CSFV/1. l/dp/CSF0382/KoslovPigCzech RepublicHM237795Leifer et al., 2010
CSFV-al (2.3)CSFV/2.3/wb/0608/2005/EuskirchenWild boar2005GermanyGU233732Leifer et al., 2010
CSFV-al (2.3)C SFV/2.3/wb/C SF1046/2009/HemiefWild boar2009GermanyGU233733Leifer et al., 2010
CSFV-al (2.3)C SFV/2.3/wb/C SF 1045/2009/RoesrathPig2009GermanyLT593749Hoeper, unpublished
CSFV-al (1.1)CSFV212L-13Pig2013IndiaKY860615Tomar et al., unpublished
CSFV-alSWHPig2004ChinaDQ127910Li et al., 2006
CSFV-alVac APigUSAL42435Stadejek et al., 1996
CSFV-alYamanashi/69Pig1969JapanAB019182Harasawa and Giangaspero 1999
CSFV-a217-93PigPolandL42413Stadejek et al., 1996
CSFV-a2 (2.3)Alfort/T iibingenPigFranceJ04358Meyer et al., 1989
CSFV-a2Chiba-80PigJapanAB019659Sakoda et al., 1999
CSFV-a2 (2.3)C SFV/2.3/dp/C SF0821 /HR/No vskaPig2002CroatiaHQ 148061Leifer et al., unpublished
CSFV-a2 (2.3)CSFV/2.3/dp/CSF857/BorkenPig2006GermanyGU233731Leifer et al., 2010
CSFV-a2 (2.3)C SFV/2.3/dp/C SF864/BG/JambulPig2007BulgariaHQ 148062Leifer et al., unpublished
CSFV-a2 (2.3)C SFV/2.3/wb/XXX0609/UelzenWild boar2004GermanyGU324242Leifer et al., 2010
CSFV-a2 (2.3)CSFV/2.3/SRB/1264/2005Pig2005SerbiaKY849593Petrovic et al., unpublished
CSFV-a2 (2.3)CSFV/2.3/SRB/6168/2006Pig2006SerbiaKY849594Petrovic et al., unpublished
CSFV-a2CSF/wb/FR57/2004/0964-03Pig2004GermanyLT158404Hoeper, unpublished
CSFV-a2CSF/wb/FR67/2003/0647-l 9Pig2003GermanyLT158502Hoeper, unpublished
CSFV-a2CSF/wb/FR67/2005/0018-06Pig2005GermanyLT158405Hoeper, unpublished
CSFV-a2CSF/wb/FR67/2005/0125-05Pig2005GermanyLT158406Hoeper, unpublished
CSFV-a2C SF/wb/FR67/2005/02 3 8-02Pig2005GermanyLT158407Hoeper, unpublished
CSFV-a2CSF/wb/FR67/2006/0199-01Pig2006GermanyLT158409Hoeper, unpublished
CSFV-a2CSF/wb/FR67/2006/060003-06Pig2006GermanyLT158408Hoeper, unpublished
CSFV-a2CSF/wb/FR67/2007/0192-01Pig2007GermanyLT158410Hoeper, unpublished
CSFV-a2 (2.lg)GDI 9/2011Pig2011ChinaKU504339Gong et al., 2016
CSFV-a2HEN03Cattle2012ChinaKC176778Zhang et al., unpublished
CSFV-a2 (2.1)HNLY-2011Pig2011ChinaJX262391Jiang et al., 2013
CSFV-a2 (2.1)HNSD-2012Pig2012ChinaJX218094Jiang et al., 2013
CSFV-a2Osaka/51Pig1951JapanAB019174Harasawa and Giangaspero 1999
CSFV-a2Osaka/71Pig1971JapanAB019176Harasawa and Giangaspero 1999
CSFV-a2PaderPigGermanyAY072924Uttenthalet et al., 2001
CSFV-a2PaderbomPigGermanyGQ902941Rasmussen et al., 2010
CSFV-a2S112Contaminant2014ChinaMK118725Zhang et al., 2014
CSFV-a2S173Contaminant2014ChinaKF006975Zhang et al., 2014
CSFV-a2Shizuoka/73Pig1973JapanAB019180Harasawa and Giangaspero 1999
CSFV-a2 (2.1)SKCDKPig2009ChinaGQ923951Li et al., unpublished
CSFV-a2SpOlPig2001SpainFJ265020Mena et al., unpublished
CSFV-a2Switzerland 1/93Pig1993SwitzerlandAF045068Hofmann and Bossy, 1998
CSFV-a2Switzerland 2/93'Pig1993SwitzerlandAF045069Hofmann and Bossy, 1998
CSFV-a2Switzerland 3/93/1'Pig1993SwitzerlandAF045070Hofmann and Bossy, 1998
CSFV-a2Switzerland 3/93/2'Pig1993SwitzerlandAF045071Hofmann and Bossy, 1998
CSFV-a2Switzerland 4/93'Pig1993SwitzerlandAF045072Hofmann and Bossy, 1998
CSFV-a2VenhorstPigNetherlandsAF084049Widjojoatmodjo et al., 1999
CSFV-a2VRI4762PigMalaysiaL42437Stadejek et al., 1996
CSFV-a2 (2.3)WingenePig1994BelgiumJQ595295Haegemail et al., unpub
CSFV-a3Saitama/81Pig1981JapanAB019178Harasawa and Giangaspero 1999
CSFV-a4 (2.2)179/MIB/2014Pig2014IndiaKR350485Bhaskar et al., 2015
CSFV-a4 (2.2)181/MIB/2014Pig2014IndiaKR350486Bhaskar et al., 2015
CSFV-a4 (2.2)211/MIB/2014Wild boar2014IndiaKR350487Ravishankar et al., unpublished
CSFV-a4 (2.2)23/MIB/2014Pig2014IndiaKR149284Bhaskar et al., 2015
CSFV-a4 (2.2)322/MIB/2013Pig2013IndiaKR350488Bhaskar et al., 2015
CSFV-a4 (2.2)99/MIB/2014Pig2014IndiaKR350483Bhaskar et al., 2015
CSFV-a4 (2)C SF V-UP-BR-757-09Pig2009IndiaKC533785Tomar et al., unpublished
CSFV-a4ParambiWild boar2014IndiaKT239105Chandramohan et al., unpublished
CSFV-a5IND/AS/GHY/G4Pig2014IndiaKM362426Ahuja et al., 2015
CSFV-a6Fukuoka/72Pig1972JapanAB019150Harasawa and Giangaspero 1999
CSFV-a6FlondurasPigHondurasL42426Stadejek et al., 1996
CSFV-b5440/99Sheep1999SpainAY159514Hurtado et al., 2003
CSFV-c (3)94.4/IL/94/TWNPig1994TaiwanAY646427Lin et al., 2007
CSFV-c (3.4)Kanagawa/74Pig1974JapanAB019166Harasawa and Giangaspero 1999
CSFV-cOkinawa/86Pig1986JapanAB019172Harasawa and Giangaspero 1999
CSFV-cOkinawa-86-2Pig1986JapanABO 19663Sakoda et al., 1999
CSFV-c (3)P97PigTaiwanL49347Liu, unpublished
CSFV-dS171Contaminant2014ChinaKF006974Zhang et al., 2014
CSFVnd (2.2)Schl80PigGermanyJQ411560Postel et al., 2012
CSFV nd (1.3)VRI 4167PigMalaysiaJQ411570Postel et al., 2012
The taxonomical segregation of the genomic sequences was performed according to the genotyping based on the palindromic nucleotide substitution (PNS) method (Harasawa and Giangaspero, 1998; Giangaspero and Harasawa, 2007; Giangaspero and Apicella, 2014), through the qualitative and quantitative evaluation of relevant secondary structure regions from full-length (216–237 nt) 5′-UTR of the viral RNA, the three variable regions, and V1, V2, and V3 genomic sequences in the IRES. Genotypes were identified according to base–pair (bp) combinations at the level of low-variable positions and ranked with alphabetic nomenclature according to increasing divergence in the species. The classification among CSFV strains according to the PNS analysis based on changes in the secondary structure was compared with those based on the primary structure of the 5′-UTR performed through sequence alignment and construction of phylogenetic trees. A phylogenetic tree was constructed with Clustal X (Chenna ) by using the neighbor-joining method (Saitou and Nei, 1987) and visualized with the Newick tree format option (Mega version 7.0.26) (Kumar ). In addition, a basic local alignment search tool (BLAST; http://www.ncbi.nlm.nih.gov), which is the web-based sequence analysis tool with default values, was used to find the homologous hits for the sequence analysis of strains.

Results

The obtained secondary structures have been aligned (Table 2) for computing genetic distance among sequences, in terms of base-pairing variations at the level of the IRES in the 5′-UTR, to identify genetic groups, ordered according to the increasing values of divergence. Among Indian bovine CSFV sequences, the V2 and V3 loci are all identical. The sequence of 8 India is identical to the sequences 2, 3, 9, and 13. The sequence of 1 India was identical to 4, 5, 6, 7, 10, and 11 (11 India showed only the first nucleotide in V1 locus: adenine instead of guanine). CSFV-c strain Okinawa/86 is identical to the strains P97 and 94.4/IL/94/TWN (difference in V1/1 g-c), CSFV-a1 strain Ibaraki/66 was identical to BresciaX (difference in V2/7 g*u), and CSFV-a1 39 was identical to strains JL1(06), 5NCRCSFMZAIZ348, Aizawl-09, and RUCSFPLUM (difference in V1/1 g-c).
Table 2.

Alignment of genus Pestivirus CSFV species genotypes variable loci 5′-UTR RNA secondary structure sequences, segregated according to types of base pair combinations. Base pairing combinations were identified for genotype characterization (n = 37) and considered for identification marker definition. The different types are ordered according to increasing divergence in the species (*), and divergence in the genus (**) was expressed in a number of divergent base pairs, with reference to most common base pairs in the prevalent positions. Highly conserved base pair positions are excluded. Positions 13 in V1 locus and 2, 7 and 8 in V3 locus are species specific; positions 15 in V1 locus, 5 and 7 in V2 locus and 1 in V3 locus are genotype specific. Y: C or U; ND: not determined.

Variable locusV1
Position1235r67891213141516171819202122
Prevalent base pairsGYUAGCUGAUACGGYCGUA---
CSFVGYUAAUUGAUACGGCGCUAGCACGCCGAA--
CSFV-a.l
39.GA---
HCLV.GA---
Ibaraki/66.GA---
Ibaraki/81-115.GA---
CSFV/2.3/Hemief.CC---
CSFV2.3Roesrath.CU---
8 India.GUGA---
5NCR/C SF/MZ/AIZ/3 52.ACGA---
Alfort 187.UUGA---
Brescia.GA---
1 IndiaAUGUGA---
CSFV-a.2
Alfort.CU---
Osaka/51.CG---
Pader.GG---
Switzerland 1/93.CC---
SKCDK.GU---
Sp01.CC---
CSFV/2.3/SRB/6168/2006.CC---
CSFV/2.3/Jambul.UGCU---
17-93.GCCU---
Chiba-80.CU---
S173.GG---
HEN03.GG---
CSFwbFR672006060003-06.CU---
CSFV-a.3
Saitama/81.GA---
ND Sch 180-.-.-.-.-.-.-.GA---
CSFV-a.4
Parambi.GCGA---
211/MIB/2014.GCGA---
322/MIB/2013.GCGA---
181/MIB/2014.GCGAGA---
C SF V-UP-BR-757-09.CAGCGA---
CSFV-a.5
IND/AS/GHY/G4.AUAGGG---
CSFV-a.6
Fukuoka/72.GCGAGA---
Flonduras.UGAUGCUG---
ND VRI 4167.CAAUCU---
CSFV-b
5440/99.GCAGUA--
CSFV-c
Okinawa/86.ACAUGAGA---
Kanagawa/74.AUUGAUAUGAGA---
CSFV-d
S171.CAGG---
The sequences have been clustered into four genotypes, from CSFV-a to CSFV-d (Table 3). PNS marker characteristics to the Pestivirus CSFV species, genotype, and subgenotypes are shown in Table 4. The secondary sequence analysis of the 5’-UTR sequences of the Chinese and Indian strains suggested their appurtenance to CSFV species, genotype CSFV-a2 (Alfort type) for three of the Chinese strains and genotype CSFV-a1 (Brescia type) for all the Indian strains. The Chinese strain S171 was clustered as a new genotype in the species, named CSFV-d. Only partially related to CSFV-a2 (divergence 62.5%, mean value 9.87), with a marked difference at the level of the V3 locus nucleotide base pairings, S171 was divergent from all other CSFV genotypes (divergence values ranging from 10 to 15). Furthermore, it was significantly less related to border disease virus (BDV) (Fig. 1), a known characteristic of other CSFV strains. Base pairing comparison between strain S171 and strains of the different BDV genotypes scored the high divergence values, ranging from 17 to 25, with a mean value of 20.31. These values were very high in confront of the mean divergence value of 14.57 obtained compared to the other CSFV strains with BDV strains (Table 5). Divergence from BDV-2 tentative species is also higher with CSFV-d (divergence values ranging from 21 to 22; mean divergence value 21.33), in confront of scores obtained with other CSFV genotypes CSFV-a, CSFV-b, and CSFV-c (divergence values ranging from 15 to 20; mean divergence value 17.06) (Table 5). No relation was observed between CFSV and any other Pestivirus species (Fig. 2).
Table 3.

Clustering into genotypes of Pestivirus CSFV species strains (n = 126) evaluated according to the PNS method at the 5’ untranslated region of RNA. Nomenclature according to primary sequence analysis is indicated under parenthesis. ND: not determined.

Species genotypesStrainsDistance within species
CSFV-aCSFV-a1 (1.1; 1.2): 39; 5NCR/CSF/MZ/AIZ/348; 5NCR/CSF/MZ/AIZ/352; 1 India; 2 India; 3 India; 4 India; 5 India; 6 India; 7 India; 8 India; 9 India; 10 India; 11 India; 13 India; Alfort 187; Alfort A19; Aizawl-09; Bangalore Ind-163/07; Brescia; BRESCIAX; BV-P; C strain; CAP; cF114; CSFV212L-13; CSFV-GZ-2009; CSFV-PK15C-NG79-11; CSFV/IVRI/VB-131; CSFV/1.1/dp/CSF0382/XXXX/Koslov; CSF/MZ/KOL/73; CSF/MZ/ SAI/76; CSFV/2.3/wb/CSF1046/2009/Hennef; CSFV/2.3/wb/XXX0608/2005/Euskirchen; CSFV/2.3/Roesrath; Eystrup; GPE (-); HCLV; HCVCAD22/14; Hokkaido/66; Ibaraki/66; Ibaraki/81-115; Ibaraki/81-20; Ibaraki/81-38; Ibaraki/81-40; Ind-173/08; Ind-174/08; Ind- 175/08; Ind-176/08; Ind-239/08; Ind-243/08; Ind-272/08; JL1(06); KC; LK-VNIVViM (V1 GC); LOM; Miyazaki/81; Nakamura/66; NFP/AS-1; NFP/ML-2; NFP/ML-4; Rovac; RUCSFPLUM; Shimen; SWH; Vac A; Yamanashi/69.0.54
CSFV-a2 (2.1; 2.3): 17-93; Alfort; Chiba-80; CSFV/2.3/dp/CSF857/2006/Borken; CSFV/2.3/ dp/CSF864/2007/BG/Jambul; CSFV/2.3/dp/CSF0821/2002/HR/Novska; CSFV/2.3/wb/ XXX0609/2004/Uelzen; CSF/wb/FR5720040964-03; CSF/wb/FR672006060003-06; CSF/ wb/FR6720070192-01; CSF/wb/FR6720030647-19; CSF/wb/FR6720060199-01; CSF/wb/ FR6720050238-02; CSF/wb/FR6720050125-05; CSF/wb/FR6720050018-06; CSFV/2.3/ SRB/1264/2005; CSFV/2.3/SRB/6168/2005; CSFV/2.3/SRB/6168/2006; GD192011; HEN03; HNLY-2011; HNSD-2012; Osaka/51; Osaka/71; Pader; Pader born; S112; S173; Shizuoka/73; SKCDK; Sp01;Switzerland 1/93; Switzerland 2/93'; Switzerland 3/93/1'; Switzerland 3/93/2'; Switzerland 4/93'; Venhorst; VRI4762; Wingene.0.46
CSFV-a3 (2.2): Saitama/81.1
CSFV-a4 (2.2): Parambi; 179/MIB/2014; 181/MIB/2014; 211/MIB/2014; 23/MIB/2014; 322/ MIB/2013; 99/MIB/2014; CSFV-UP-BR-757-09.1.4
CSFV-a5 (2.1): IND/AS/GHY/G4.3
CSFV-a6 (1.3): Fukuoka/72; Honduras.3
CSFV-b5440/99.4
CSFV-c (3)94.4/IL/94/TWN; Kanagawa/74; Okinawa/86; Okinawa-86-2; P97.5.5
CSFV-dS171.7
CSFV NDSch180; VRI 4167.
Table 4.

PNS characteristics to the Pestivirus CSFV species with related genotypes and genotype variants. The position of base pairings is defined by numbering from the bottom of the variable locus. Y= C or U.

CSFV speciesLocusCharacteristic PNS markers
V1U-A in position 13 (exception U*G or C-G; C A bulge);
V3U-A in position 2; C A or U-A in position 7; U or C in position 8 (exception A).
CSFV genotypesLocusCharacteristic PNS markers
CSFV-aV1A C bulge in position 15 (exceptions G:Y and A-U);
V2U-A in position 5; G:Y in position 7;
V3A-U in position 1 (exception A G bulge).
CSFV-bV1G:Y in position 15; A G bulge in position 19; U-A in position 20;
V2A-U in position 5; A C bulge in position 7;
V3A-U in position 1; U C bulge in position 6.
CSFV-cV1A-U in position 15;
V2U-A in position 5; G:Y in position 7;
V3A G bulge in position 1.
CSFV-dV1A C bulge in position 15;
V2U-A in position 5; G:Y in position 7;
V3U U bulge in position 1.
CSFV genotype variantsLocusCharacteristic PNS markers
CSFV-a1V1AC in position 15;
V2A-U in position 1; A-U in position 6;
V3C or U in position 8.
CSFV-a2V1AC in position 15 (exception G-C);
V2A-U in position 1; G:Y in position 6;
V3A-U in position 6; U or C in position 8.
CSFV-a3V1AC in position 15;
V2AC in position 1; G:Y in position 6;
V3A in position 8.
CSFV-a4V1G:Y in position 15;
V2A-U in position 1; G:Y in position 6;
V3AC or UC in position 6; A in position 8 (exception C).
CSFV-a5V1A-U in position 15;
V2AC in position 1; A-U in position 6;
V3U in position 8.
CSFV-a6V1G:Y in position 15;
V2A-U in position 1; A-U in position 6;
V3C in position 8.
Fig. 1.

Graphical representation of PNS quantitative evaluation. CSFV shows close genetic relation to BDV and a lesser extent with BDV-2.

Table 5.

PNS quantitative evaluation. CSFV versus BDV and BDV-2 tentative species secondary structure sequence comparison divergence values (n = 2,178). Scores exceeding species determination divergence limit value (13) indicate genetic distance and different species clustering.

CSFV39Alfort 1878 IndiaBresciaHCLVIbaraki/66Ibaraki/81-11517-93AlfortChiba-80Osaka/51PaderS173HEN03Switzerland 1/93Saitama/81Fukuoka/72Honduras5440/99Okinawa/86Kanagawa/74S171
Border disease virus (BDV)
alA841/114131515141414131314121314141314151214161220Divergent
alVMM15141616151515141415131415151415161315161221Divergent
al135 66114131515141415141415131314141414151216151120Divergent
al170 33715141616151515141415131415151415161316161221Divergent
alG130516151717161616151516141516161516161417161222Divergent
alG204814131515141414131314121314141313151215151120Divergent
alD1586/214131515141414131314121314141314151215161220Divergent
alK1729/315141616151515141415141415151415161416161221Divergent
al150574416151717161616151516141416161416161517161222Divergent
al150230415141616151514131314121315151215161415161221Divergent
al106268915141616151515141415131315151315161416161221Divergent
a2X81814151515141414121314131314141314131215151120Divergent
a2Moredun ncp121317131212121011121011121211121191313918Related
a2T1789/1121317131212121011121011121211121191313918Related
a2Q1673/213141814131313111213111213131213121014141019Related
a2JH281613151814131313111213111213131213121013141019Related
a2Lyon212131713121212101112910121210121191313918Related
a2L99112131713121212101112101112121112111113141118Related
a2Moredun cp13141814131313111213111213131213121014141119Related
a2A18701213171312121210111210111212111211913131018Related
a28320-22NZ13141814131313111213111213131213121014141019Related
a2137652713141714131313111213121212121213121314151218Related
a28320-31NZ13141814131313111213111213131213121014141019Related
a2FNK2012-113141814131313111213101113131113121014141019Related
a2111821213141413131313111213101113131113121014141019Related
a2ChlEs15161616151515131415131415151415141215161221Divergent
a2Coos Bay 5 c15161615151515131414121315151315141213161221Divergent
a3BD3115151616151515151415141415151415161517171321Divergent
a4LA/5909/0915161616151515131414121315151314141314161321Divergent
bGenzkow 70113141414131313121213121213131213131215121018Related
bRentier Rudolph1415151514141413131413131414131414131613919Divergent
bBT23051415151514141413131413131414131414131613919Divergent
bST14051415151514141413131413131414131414131613919Divergent
bST15071415151514141413131413131414131414131613919Divergent
bJ10041415151514141413131413131414131414131613919Divergent
clCH-BD313141414131313131314121214141213131316131119Related
clCH-BD415161616151515141415121315151315151417151320Divergent
clItaly-10376115161616151515141413131214131315151216131319Divergent
clItaly-5898717181818171717161617151517171517171618171522Divergent
clChamois-VdA-218191919181818171718161618181618181719181623Divergent
c290-F-633516171717161616151516151516161514161517161421Divergent
c206-F-0299/35716171717161616151516151516161514161517161421Divergent
c292-F-711916171717161616151516151516161514161517161421Divergent
c210F0335616171717161616151516141416161414161517161421Divergent
c2RUPI-0517181818171717161617151517171515171618171521Divergent
dlAH12-0114151515141414121213121214141213141316141219Divergent
dlAHHX1514151515141414131314121214141213141316141219Divergent
d229716171717161616151516141416161415161516161420Divergent
elC2717181717171716131415151616161516161515181621Divergent
e2ZA1-111517181917171717151617161618181616161516181623Divergent
f10501209-052GI14151515141414131314121314141314141215141220Divergent
fl89-F-541516171716161616151516151516161515161417161421Divergent
flESP-9717181817171717161615141516151516171318151521Divergent
flAV17181817171717161615161616151616171318151521Divergent
fl93-F-728917181817171717161615161616151616171318151521Divergent
flBA/52521/0918191818181818171716151617161617181419161622Divergent
122112/9918171918181818191920181920201919181720201625Divergent
12M316151716161616171718161718181717161518181723Divergent
12050223417161817171717181819171819191818171619191624Divergent
13Rocco17181817171717161617161618171616171517171522Divergent
13C12116171716161616171718161718181717161518181723Divergent
13LE31C216171716161616171718161718181717161518181723Divergent
13Colm2417181817171717181819171819191818171619191824Divergent
f4ARAN-115161516151515161617161616161616151418171621Divergent
f4RIBAGORCA-414151515141414151516131416161415141317161521Divergent
f4Chamois 114151515141414151516151516161516141317161521Divergent
f4PALLARS-314151515141414151516131416161415141317161521Divergent
f4FRESER-215161615151515161617141517171516151418171622Divergent
f4Orlu-R4116171617161616171716171716151717161319161721Divergent
f4CADI-115161616151515161615151616151616151218151621Divergent
f4Orlu-S2415161616151515161615151616151616151218151621Divergent
f4sheep/272/FRA/200714151515141414151514141415141415141217141520Divergent
gl92019/2007/AG12131313121211910119101212912111011121018Related
gl59434/2012/EN13141414131312101112101113131013121112131119Related
gl10F0340112131312121197897911117121110911918Related
gl91-F-673212131312121110891098101091211101111917Related
gl70282/2007/EN1112121211111010111210111313101310911131119Related
gl91-F-6731131414131312119101110111212101312111212819Related
g237A13141415141415131415141314141414121114161220Divergent
g2BM01 isolate 515161616151514121314131415151315141314151121Divergent
g233S14151515141415131415141314141414131314161320Divergent
hJS12/0416171717161616151514141415141315161318141418Divergent
hJSLS12-0115161616151515141413131314131215151217131318Divergent
hJSYZ1515161616151515131314131315151315151417151319Divergent
ilTR-1318191919181818161718161618181618171618191622Divergent
i2TR-1417181818171717151617151517171517161517181621Divergent
JlInd 830-0913141414131313111213111113131013121114141218Related
J2CH-BD115161416151515141415121315151315141417151420Divergent
J206-F-008315161516151515141415131416161415151517161520Divergent
J285-F-58814151415141414141314121315151314151517171619Divergent
J290-F-622715161516151515141415131416161415151617161520Divergent
J2LA644211017181618171717161617141517171517161618171521Divergent
J206M015016171517161616151516141416161416151617161520Divergent
J289-F-537415161516151515141415141416161415151617161520Divergent
J3Gifhom14151415141414121314131315151314131415161520Divergent
J3B3000616171517161616141516141416161416141516171621Divergent
J3CH-BD217181618171717151615151516151517151417161721Divergent
kBDV/Burdur/05-TR17181618171717151617161617171617161617181523Divergent
kBDV/Aydin/04-TR17181818171717151617161617171617161616181523Divergent
Border disease virus type 2 (BDV-2)
BDV-2712/0217181718171717161617161616161616171818191821Divergent
BDV-2TO/121/0417181718171716151516151616161516171817191821Divergent
BDV-2LA/91/0518191819181817161617161717171617181918201922Divergent
Fig. 2.

Graphical representation of PNS quantitative evaluation. Genotype CSFV-d is not related to BDV or BDV-2.

By phylogenetic analysis of E2 region, the Indian bovine strains CS/ML/911/IDP/13 (KY860532) and CS/ML/AF/Umiam/14 (KY860531) (Ahuja ; Chakraborty et al., 2018) were similar to pig isolates as IND/AS/GHY/G4 (KM362426) (Ahuja ), originated from Assam, a neighboring state. By BLAST, the E2 sequence of both strains CS/ML/911/IDP/13 and CS/ML/AF/Umiam/14 showed 99% and 92% nucleotide identity with strain IND/AS/GHY/G4 (CSFV-a5) and reference strain Paderborn (GQ902941) (CSFV-a2), respectively. Taking into account the PNS evaluation of Paderborn and IND/AS/GHY/G4 5′-UTR, both bovine strains CS/ML/911/IDP/13 and CS/ML/AF/Umiam/14 were clustered into CSFV-a5. Similar to type Parambi (CSFV-a4), also this cluster was the characteristic of India. A schematic phylogenetic tree, based on secondary structure base pair variation and divergence values at the three variable loci, is shown in Figure 3. At the species level, the observed taxonomic status of the examined strains corresponded to the estimation obtained by phylogenetic tree based on the primary sequence analysis of the 5′-UTR constructed from the alignment with the representative strains from the identified genogroups (Fig. 4).
Fig. 3.

Schematic phylogenetic tree based on nucleotide changes in the secondary structure of classical swine fever virus genotypes. Mean values of base–pair divergence and variable locus location indicate the evolutionary changes.

Fig. 4.

Phylogenetic tree based on the 5′-UTR comparison, showing taxonomic relationships of the CSFV strains in the Pestivirus genus. Strains Brescia (M31768), 39 (AF407339), Eystrup (AF326963), Alfort/187 (X87939), HCLV (AF091507), Shimen (AF092448), and the bovine strain 8 India (MG813566) are references for the genotype CSFV-a variant 1. Strains Alfort/Tuebingen (J04358), Chiba-80 (AB019659), Osaka/51 (AB019174), Paderborn (AY072924), and the Chinese bovine strains S173 (KF006975), HEN03 (KC176778), and S112 (MK118725) are references for the genotype CSFV-a variant 2. Strain Saitama/81 (AB019178) is the reference for the CSFV-a variant 3, and strains Parambi (KT239105), IND/AS/GHY/G4 (KM362426), and Honduras (L42426) are the references for the CSFV-a variants 4, 5, and 6, respectively. The Spanish ovine strain 5440/99 (AY159514) is the reference for the genotype CSFV-b. The Japanese strains Kanagawa/74 (AB019166) and Okinawa/86 (AB019172) are references for the genotype CSFV-c. The Chinese bovine strain S171 (KF006974) represents the genotype CSFV-d. Distances were computed using Clustal X, version 1.8, using the neighbor-joining method. Scale bar indicates 10 nucleotide substitutions per 100 nucleotides. Nomenclature of identified genotypes is based on divergence in the genus.

Discussion

On contrary to other pestiviruses, as BVDV-1 which accounted for much more genotypic variants (Giangaspero and Apicella, 2018), the CSFV species was low heterogeneous. Only two genotypes, CSFV-c and CSFV-d, both of Asian origin, were highly divergent. Taking into account the high divergence in the species based on the primary sequence analysis, the strain Kanagawa/74 (CSFV-c) (Japan, 1974; Dreier ; Beer ) was proposed, similarly to congenital Tremor (United Kingdom, 1964), as harmonized outgroup strain for phylogenetic analyses (Paton ). Similar to the strain Kanagawa/74, the Chinese bovine strain S171 (CSFV-d) was divergent in the species (Fig. 4), suggesting its consideration as another outgroup of the species with even more enhanced divergence. The other bovine Chinese strains are less divergent, showing common genomic trait characteristic of the known reported CSFV-a2 strains, indicating the concomitant circulation of typical CSV types with others showing atypical characteristics, possibly related to the host species adaptation or a result of geographic isolation. Such occurrences have been suggested also for BVDV-1 isolates in China (Xue ; Wang ). Similarly, Indian bovine strains were genetically closely related to common CSFV genotype a1 strains and corresponded to circulate Pestivirus pig strains reported in the country. Between 2009 and 2011, Rajkhowa et al. reported three CSFV isolates from pigs (Aizawl-09, 5NCR/CSF/MZ/AIZ/348, and 5NCR/CSF/MZ/AIZ/352) (accessions numbers HM449066, JX975460, and JX975461, respectively), from the Indian state of Mizoram, located at the extreme northeastern border of Myanmar, well distant from Tamil Nadu, the area where CSFV was detected in cattle. Pig strains from Mizoram are very close to the bovine strains, belonging to the genotype CSFV-a variant 1. A distinctive guanine uracile base pairing in position 3 in the V1 locus was found only in the bovine isolates originated from Tamil Nadu. This peculiarity was different from all other CSFV strains, which present a conserved A–U pairing in this position. Interestingly, G-C or G*U pairings are conserved only among strains belonging to the BVDV species types 1, 2, and 3 (only five exceptions, out of more than 1,000 considered sequences). In the CSFV species, three genetic clusters referred specifically to Asian countries. The CSFV genotype a variant 4 (type Parambi) included only pig and wild boar strains from India [Bhaskar ; and sequences deposited by Ravishankar et al. (unpublished); Tomar et al. (unpublished); and Chandramohan et al. (unpublished)]. CSFV genotype C (type Okinawa) was reported only in pigs originating from Japan and Taiwan (Harasawa and Giangaspero, 1999; Sakoda ; Lin ; and strain P97 deposited by Liu). Furthermore, the sequence characteristics of strain S171 (Zhang ) (CSFV-d) were observed only in China. In general, apart nomenclature difference, the comparison between primary and secondary structure analyses revealed corresponding main genomic groups, defined by phylogenetic analysis, dividing CSFVs into three major lineages (Brescia, Alfort and Kanagawa/Okinawa types) and their sublineages (Lin ). Furthermore, the phylogenetic trees showed such similarity (Figs. 3 and 4). Genotype CSFV-a variant 1 (type Brescia) corresponded to 1.1 and 1.2, CSFV-a variant 2 (type Alfort/Tübingen) to 2.1 and 2.3, CSFV-a variant 4 to 2.2, and genotype CSFV-c to 3 and 3.4. The CSFV 2.2 strain Sch180 (JQ411560) from Germany and the CSFV 1.3 strain VRI 4167 (JQ411570) from Malaysia could not be determined due to incomplete V1 locus, but their other sequence fragments were compatible with PNS genotype variants CSFV-a3 (type Saitama) and CSFV-a5 (type Honduras), respectively (Table 2). However, as expected, the specific differences observable at the level of the E2 gene sequence fragment (Risatti ; Postel ) were not necessarily related to secondary structure 5′-UTR clustering. Similarly, PNS genotype CSFV-c included variants as 3.4 not discriminable from other members of genotype 3 (Lin ). Similar discrepancies may occur comparing other regions, as between 5′-UTR and Npro. With concern to the relationship between CSFV detection and clinical observations, the strain HEN03, the only among the Chinese strains for which clinical anamnestic data could be retrieved, was associated with reproductive disorders observed in many animals in the herd: abortion, stillbirth, and reduced milk yield. Furthermore, the mortality rate was high. However, concomitant infection with BVDV-1 and BVDV-2 was revealed. It is particularly interesting that also all the Indian bovine CSFV strains were isolated in herds suffering from reproductive disturbances, abortions, birth defects, repeat breeding, and mastitis. Serological positivity for bovine herpesvirus 1 (IBR) and Brucella sp. was absent or very low, manifestly not correlated to observed pathologies, and no BVDV has been isolated (Giangaspero ). Hypothetically, CSFV bovine strains detected in China and India might find their origin from vaccine prophylaxis performed in pig populations. CSFV-a1 comprises a CSFV-lapinized vaccine strain from India (EU857642–AF091507), which showed a high homology with Indian cattle strains (99% identities according to the results obtained by BLAST). Investigations on vaccination protocols applied in the research area showed that none of the cattle farms investigated in Tamil Nadu showed to have contact with pigs, and no immunization program was carried for BVDV (Giangaspero ). However, CSFV vaccine prophylaxis is carried out in the swine industry in India. At present, anti-BVDV-killed virus vaccines are available in China, but, for a long time, there were no commercial BVDV vaccines in the Chinese market. In China, and particularly in Tibet and Qinghai provinces, prophylaxis against BVDV in cattle and yaks was based on the wide use of the live-attenuated hog cholera lapinized vaccine (HCLV), administered to animals at a triple dose. This followed the experimental demonstration of efficacy and safety of the prophylactic use of HCLV for BVDV in lactating and pregnant cows, calves, and yaks undertaken by Yuan Qingzhi in 1957 (Liu ). For a long time, the HCLV was the only authorized vaccine against CSFV in China. In Tibet, the long-term use of live-attenuated HCLV not only reduced losses caused by BVDV infection but also gave the opportunity for vaccinal strain adaptation in cattle, allowing as a possible consequence of a subsequent natural diffusion. Tibet is historically a high-quality breeding region managed by a traditional seminomadic agropastoral system and appreciated by traders who seasonally purchase and move live animals and may be possibly responsible for the spread of pestiviruses, especially through immunotolerant persistently infected subjects. In Spain, the reported ovine strain 5440/99 showed the sequence similarities with CSFV vaccine strains used in pigs, suggesting spillovers from prophylactic campaigns (Hurtado ). In Belgium, some fetal calf sera positive for CSFV-like 5′-UTR sequences, experimentally not infecting or seroconverting pigs, have been detected during the 90s (Dr Koenen, Sciensano, Belgium, personal communication), suggesting circulation of unidentified virus strains with sequence similarity to CSFV within the 5′-UTR. However, in Belgium, since the last occurrence of CSFV was reported in 1997, thus, in this period, wild and vaccinal strains were present in the country, and it cannot be excluded from the occurrence of possible spillovers in cattle from CSF-infected or immunized pigs. The recent taxonomy of CSFV is based on the analysis of the E2 genomic region (Postel ). Due to the relation to envelope glycoprotein (the main immunogen of CSFV, thus essential for the development of vaccines) (Perez ) and the longer size (1,119 nucleotides), the full-length E2 encoding sequences were recommended for accurate phylogenetic analyses, assessing the viral types and subtypes. Based on the primary sequence analysis, three CSFV genotypes, each with three or six subgroups, have been identified (Paton ; Dreier ; Postel ; Beer ; Silva ). However, despite a limiting factor for the differentiation of closely related isolates (Postel ), short-length target fragments in other regions of the viral genome as 5′-UTR or NS5B have been largely applied for genotyping purposes and molecular epidemiology (Lowings ; Paton ; Beer ). Phylogenetic analysis of the sequences based on a 190-nucleotide (nt) fragment of the E2 glycoprotein proved very useful for the genetic typing of isolates in the case of new outbreaks (Dreier ). In any case, laboratory testing international standards are indicated only by the World Organization for Animal Health (Office International des Epizooties - OIE). According to the Terrestrial Manual of the World Organization for Animal Health (Office International des Epizooties—OIE) (Chapter 2.8.3 CSFV; Paragraph 1.1.5.) (OIE, 2014), as the E2 major glycoprotein gene (190 nucleotides), the fragment 5’-UTR of the CSFV genome (150 nucleotides), targeted for molecular characterization of the bovine sequences in the present study, is admitted too for genetic typing and molecular epidemiology of CSF, based on the comparison of genetic differences between virus isolates. According to the Terrestrial Code (Chapter 15.2; Article 15.2.28, Paragraph 3, Virological surveillance) (OIE, 2017) and the Terrestrial Manual (Chapter 2.8.3; Paragraph 1.1.4.) (OIE, 2014) of the World Organization for Animal Health, the RT-PCR amplification of CSFV RNA followed by nucleotide sequencing, applied for the obtention of the sequences evaluated in the present study (Zhang ; Giangaspero ), originally focused on investigations of BVDV, is admitted among recommended methods for CSFV agent detection, confirmation of clinical cases, and differentiation from other pestiviruses. It is clear that the sequence of the gene encoding the E2 glycoprotein is preferred in the recent taxonomy of the species (Postel ), but the consideration of 5′-UTR was the only possible approach to perform a comparative evaluation of CSFV with sequences of the other different species, otherwise not feasible taking into account that the majority of Pestivirus-deposited sequences are 5′-UTR (Yesilbag ; Giangaspero ). The secondary structure–sequence characterization allowed an exhaustive comparative evaluation with genomic variants in the genus, about 1,400 Pestivirus strain sequences. The particularity of the PNS method is the exclusive consideration of strategic genomic sequences corresponding to the 5’-UTR IRES, which is responsible for translational, transcriptional, and replicational events in pestiviruses. Thus, at this level, stable nucleotide variations assume a high importance in terms of virus evolutionary history. The PNS method has been successfully applied for the characterization of pestiviruses, including atypical species as Giraffe, Pronghorn, or Bungowannah (Harasawa ; Giangaspero and Harasawa, 2008; 2011), thus adequate for the scope of the present study. The presence of CSFV 5′-UTR sequences in bovines from India and China appeared atypical. However, another recent report from India (Chakraborty et al., 2018) strongly corroborated the previous observations, amplifying also full-length E2 region of CSFV in two positive samples, and suggesting a potential emerging health risk in cattle. Furthermore, the possible occurrence of similar circulation in cattle in CSFV endemic environment in Kenya is currently under evaluation (Prof VanLeeuwen, University of Prince Edward Island, Canada, personal communication).

Conclusions

These preliminary observations are new and relevant in countries, where CSFV control and eradication strategies are applied. Further investigations will be necessary to confirm adaptation and diffusion of the virus in cattle as well as provide a clear picture of genetic characteristics, in particular, to evaluate other genomic regions, E2, and eventually full-length genome, and apply other internationally recognized tests. Furthermore, it will be important to clarify the clinical suspicions and related virus differential diagnosis for correct notification obligations in compliance with the World Organization for Animal Health reference norms.
  43 in total

1.  Species characterization in the genus Pestivirus according to palindromic nucleotide substitutions in the 5'-untranslated region.

Authors:  Massimo Giangaspero; Ryô Harasawa
Journal:  J Virol Methods       Date:  2011-04-12       Impact factor: 2.014

2.  Propagation and transmission of hog cholear virus in nonporcine hosts.

Authors:  R W Loan; M M Storm
Journal:  Am J Vet Res       Date:  1968-04       Impact factor: 1.156

3.  The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

Authors:  G R Risatti; M V Borca; G F Kutish; Z Lu; L G Holinka; R A French; E R Tulman; D L Rock
Journal:  J Virol       Date:  2005-03       Impact factor: 5.103

4.  Giraffe strain of pestivirus: its taxonomic status based on the 5'-untranslated region.

Authors:  R Harasawa; M Giangaspero; G Ibata; D J Paton
Journal:  Microbiol Immunol       Date:  2000       Impact factor: 1.955

5.  Genetic heterogeneity of porcine and ruminant pestiviruses mainly isolated in Japan.

Authors:  Y Sakoda; S Ozawa; S Damrongwatanapokin; M Sato; K Ishikawa; A Fukusho
Journal:  Vet Microbiol       Date:  1999-02-23       Impact factor: 3.293

6.  Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 5' untranslated region.

Authors:  Massimo Giangaspero; Ryô Harasawa
Journal:  J Virol Methods       Date:  2007-08-23       Impact factor: 2.014

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

Authors:  Alexander Postel; Stefanie Schmeiser; Carmen Laura Perera; Lester Josué Pérez Rodríguez; Maria Teresa Frias-Lepoureau; Paul Becher
Journal:  Vet Microbiol       Date:  2012-08-03       Impact factor: 3.293

8.  Genetic typing of recent classical swine fever isolates from India.

Authors:  S S Patil; D Hemadri; B P Shankar; A G Raghavendra; H Veeresh; B Sindhoora; S Chandan; K Sreekala; M R Gajendragad; K Prabhudas
Journal:  Vet Microbiol       Date:  2009-09-26       Impact factor: 3.293

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

Authors:  Lester Josué Pérez; Heidy Díaz de Arce; Carmen Laura Perera; Rosa Rosell; Maria T Frías; Maria I Percedo; Joan Tarradas; Patricia Dominguez; Jose I Núñez; Llilianne Ganges
Journal:  Infect Genet Evol       Date:  2012-05-09       Impact factor: 3.342

10.  Genome sequence of classical Swine Fever virus genotype 1.1 with a genetic marker of attenuation detected in a continuous porcine cell line.

Authors:  N Tomar; A Gupta; R S Arya; R Somvanshi; V Sharma; G Saikumar
Journal:  Genome Announc       Date:  2015-04-30
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  1 in total

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

Authors:  Youngmin Park; Yeonsu Oh; Miaomiao Wang; Llilianne Ganges; José Alejandro Bohórquez; Soohong Park; Sungmin Gu; Jungae Park; Sangmin Lee; Jongkook Kim; Eunju Sohn
Journal:  Vaccines (Basel)       Date:  2021-04-22
  1 in total

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