Vet. World, 2012, Vol.5(7):437-442 REVIEW A study report on phylogenetic analysis of Classical swine fever virus isolated in different parts of the World

Classical Swine Fever Virus (CSFV) is the cause of an economically important and contagious disease in all age groups of pigs. Advances in molecular methods have facilitated genetic typing of this virus which is useful for classification, to trace patterns of virus spread and exposing the weaknesses in control strategies. Moreover, the genetic comparison of the isolates obtained from a series of outbreaks with known linkages can be used to validate and to interpret the genetic typing to determine the rate of virus mutation in the field. The CSF viruses are grouped into three groups under which there are ten subgroups. This review highlights the works on phylogenetic analysis carried out by different workers from time to time in different parts of the world including India to have better understanding of the diversified genogrouping of this virus.


Introduction
upon uncoating without the need of a cap structure.E2 is the immunogenic gene and induces high virus Classical swine fever is a contagious viral disease neutralizing titers of serum antibodies. of pigs with acute, chronic or persistent infections [1].
The possible discrimination between isolates It is caused by a member of the genus Pestivirus of the depends on the length and variability of the target family Flaviviridae.It is a globally significant disease region of the genome that is used for comparisons. of pig.The causative agent, classical swine fever virus Exchange of data from previous studies has been greatly (CSFV) is an enveloped virion with an icosahedral hampered as different genome targets for sequence nucleocapsid with glycosylated membrane proteins.determination, different phylogenetic methods for The genome of CSFV consists of a positive RNA sequence analysis and different nomenclature for molecule of about 12.3 Kb, which encodes a single demonstrated genetic clusters were used.polyprotein that contains both structural glycoproteins Global scenario as a whole and non-structural proteins [2,3].The polyprotein is processed into a mature protein by viral and host cell Three defined fragments of the genome viz., 5´ proteases [4].The envelop of pestiviruses contains NTR, E2 and NS5B were selected by Greiser -Wilke rns three glycoproteins viz., E1, E2 and E (a et al. [5] and a standardized protocol for the ribonuclease).The ORF is flanked by a 5´ and 3´ NTR.calculation of phylogenetic trees including the The large ORF encodes a single polyprotein which is nomenclature for the genetic types were established.co and post translationally cleaved into the proteins N-The isolates analyzed, showed that they could be pro Terminal protease (N ), Capsid protein C, three assigned into one of three main genetic groups (1, 2 rns envelop glycoproteins viz., E (a ribonuclease), E1, and 3).Further analysis of E2 fragment of group 1 lead E2, p7 and the non-structural proteins NS2, NS3, to three distinct subgroups.The genetic analysis of E2 NS4A, NS4B, NS5A and NS5B.The NTR at the 5´end fragment is important from the CSFV marker vaccine harbours an Internal Ribosomal Entry Site (IRES).
development point of view which induces protective Therefore, the RNA can directly be translated immunity [6,7,8,9,10,11] Most historic isolates from Europe and America and some Asian isolates were obtained from three geographically distinct regions of found in group 1 and its subgroups.Most recent the Island.Phylogenetic analysis of these viruses isolates from different regions in Europe and several indicated that the Sardinian viruses were all members from Asia were in group 2 and they were again sub of the common European sub group 2.3.Bartak and grouped into 2.1, 2.2 and 2.3 respectively.The earliest Greiser -Wilke [12] sequenced fragments of 5´ NTR appearance of Group 2 viruses was in Germany in and E2 glycoprotein gene of fourteen Czech 1982.Subsequently, these viruses have been found in Republican isolates and found that the isolates fall many different countries, up to the present time, under subgroups 2.2 and 2.3.They deduced that these including Italy, Sardinia, France, Belgium, UK, isolates were having more close relation with those of Austria, Switzerland, Hungary, The Czech Republic, Australia sequenced between the years 1992 and 1994.Poland and The Slovak Republic [12,13].Finally, The isolates in subgroup 2.3 formed a very viruses from Asia fell into one of the sub-groups in homogeneous group, although they originated from group 3.The group 3 viruses had been reported mainly different regions of the country.Epizootiological links from Korea, Japan, Taiwan, Thailand and U.K [14,15].became evident when the epidemiological data were compared.A big epidemic of classical swine fever in

Scenario in different countries
Germany in the 1990's were reported by Fritzemeier et Analysis of the nucleotide sequence data generated al. [20].They analyzed about 30 isolates from the from 5´ NTR, E2 and NS5B genes of Classical Swine outbreak by comparison of the nucleotide sequence Fever Virus (CSFV) by Lowings et al. [16] were able data generated from fragments of both the E2 to divide 115 CSFV isolates into two major groups glycoprotein gene and 5´ NTR gene.By combining viz., group 1 and 2. Most of the viruses in group 1 were epidemiological data with genetic typing, they found of US or UK origin and included the reference strains that the outbreak was related and caused by viruses Alfort/187 from France and Brescia from Italy.Group belonging to the genetic subgroup 2.1.Several 2 included the viruses isolated from the Asian outbreaks of CSF were recorded during the period of countries like Malayasia and Japan.
1997-2001 in Croatia.Jemersić et al. [21] considered The Central European isolates of CSFV were nine CSFV isolated during this period and sequenced characterized by Stadejek et al. [17] and they the viruses between 150 and 190 nucleotide fragments evaluated the applicability of molecular analysis in the of 5´ NTR and E2 glycoprotein genes respectively.epizootiology of CSFV infections.Thirty four viruses Their study revealed that these isolates fall under derived from Central European pigs or wild boars subgroups 2.1 and 2.3.Pan et al. [22] found in their were examined in their study.The discriminatory study that most of the CSFV isolated in Taiwan ability of the technique was improved by the analysis between 1989 and 2003 fall under subgroup 2.1 and of a composite data set consisting of all the sequence 2.2 and considered them as strains introduced in data from all the viruses.Using this approach they Taiwan from other places whereas a few isolates (forty were not only able to distinguish between all the three out of hundred and fifty eight) fall under viruses but also were able to group them in a manner subgroup 3.4 and were considered as endemic strains.that precisely matched their geographical origins Pereda et al. [23] performed genotyping of isolates which helped in establishing the fact that the virus is from outbreaks in domestic pigs in several countries of genetically heterogenous in Central Europe.South and Central America.Phylogenetic analysis of 15 representative CSFV For this purpose, they selected a 190 base pair isolates from Cuba targeting the E2 sequences were fragment of the E2 envelope glycoprotein gene.For conducted by De Arce et al. [18] and their finding comparison, they included European strains and showed that the viruses of Cuba clustered in subgroup isolates, and historical isolates from the United States 1.2.Moreover, it had been observed that the viruses (US) apart from their isolates.They found that all the isolated from 1996 in eastern Cuba defined a related, isolates included in their analysis belonged to group 1 but independent group.and were further resolved into three subgroups in The level of sequence variation observed in this contrast to the situation in most parts of Europe, where group didn't exclude an independent origin for the group 2 isolates predominate.The Cuban isolates and eastern isolates.Lowings et al. [19] analyzed a the isolates from Honduras and Guatemala fall under variable region of the gene encoding the major subgroup 1.2 and 1.3 respectively.The remaining glycoprotein E2 of Classical Swine Fever Virus isolates from Argentina, Brazil, Colombia and Mexico (CSFV) from 12 Sardinian isolates which had been generated four poorly resolved clusters in subgroup 1.1.This allowed in the speculation that at least in were classified into a different subgroup.Giangaspero American continent, CSFV might have appeared and Harasawa [29] analyzed 43 strains of CSFV from independently in several regions and spreading might outbreaks in pigs in Europe, Asia and America, two strains from commercial CSFV modified live vaccines have been a secondary effect.The partial E2 gene (originally isolated by Grebennikova et al., [30]) and a sequence for 10 CSF viruses isolated at different strain isolated from a diseased lamb from Spain locations of the Cuban island by Heidy et al. [24] were (originally isolated by Hurtado et al., [31]) in the 5' used for phylogenetic analyses including sequences terminal region of the genome using the Palindromic from viruses of the 1993 -1997 epizootic, previously Nucleotide Substitution (PNS) method.Nucleotide determined, as well as those from representatives of sequences were aligned using the method proposed by the different CSFV genotypes.The phylogenetic tree Higgins et al. [32].Phylogenetic trees based on the 5' obtained indicated that viruses circulating at present in NTR was constructed using the unweighed pair-group Cuba belong to the subgroup 1.2.Sabogal et al. [25] method with arithmetic averages (UPGMA) described characterized the viruses isolated from Colombia and by Sneath and Sokal [33].Nucleotide sequence found them to fall under subgroup 1.1.The results secondary structures were predicted according to the indicated that the outbreaks from the year 2002 were algorithm of Zuker and Stiegler [34].The minimum caused by a strain related to the virus CSF/Santander, free energy was calculated using the method of Freier isolated in 1980, suggesting that the current CSF et al. [35].Phylogenetic tree placed the isolates in outbreaks are the consequence of a single strain that group 1.They also found that 5´ NTR acts as an IRES continues to circulate in the field till date in Colombia.
(Internal Ribosomal Entry Site) and is responsible for Allepuz et al. [26] reported on the descriptive the genetic stability of the virion.The application of epidemiology of the outbreak of CSF in Catalonia, the PNS method was found to be an appropriate Spain.Twenty two of the infected herds were detected approach for differential diagnosis to solve crosson the basis of clinical suspicion on the part of the infections.Classical Swine fever is endemic in Peru.farmer or farm veterinarian, and the other 17 were Little was known about the genetic subgroup of CSFV detected by surveillance methods.The viruses isolated in Peru until Araínga et al. [36] phylogenetically in the two waves of the outbreak were 100 per cent characterized the viruses and found them to fall under homologous and belonged to subgroup 2.3.The origin subgroup 1.1.In Vietnam, Tung et al.
[37] conducted of the outbreak remained unknown.Cha et al. [27] study on 44 CSFV isolates from the country along with genetically characterized 24 CSFV isolates which 271 reference viruses and found that all these viruses were obtained from CSF outbreaks during 1988 and fall under subgroup 2.1.

in the Republic of Korea and compared with CSF viruses reported by other countries. Phylogenetic Indian scenario analyses classified Korean field isolates between 1988
Phylogenetic characterization of virulent (CSFV/ and 1999 into subgroups 3.2, forming an independent MP) and lapinised vaccine (CSFV/LAP) strains of clade distinct from CSF viruses identified in other CSFV at I.V.R.I, Izzatnagar placed both the strains countries.In contrast, the viruses isolated during into genogroup 1.1 [38].Especially the molecular 2002-2003 CSF epidemics were classified into characterization of CSFV/LAP strain was a landmark subgroup 2.1.The 2.1 viruses showed a close genetic as it was shown earlier that this strain of CSFV offered relationship (92.1-100 %) with CSF viruses reported a longer immunity in vaccinated animals lasting for from China and Taiwan in 1998-2001.
years upto lifelong and conferred wide range of Sang et al. [28] reported the phylogenetic immunity against different strains and genotypes characterization of CSF viruses isolated in Korea [39,40,41].between 1988 and 2003.Twenty four isolates of CSFV Barman et al. [42] found in their analysis that which were obtained from CSF outbreaks during 1988 one of the isolate from Mizoram is closely related to and 2003 in the Republic of Korea were genetically the Chinese strain Shimen-HVRI rather than other characterized for partial E2 gene (190 nucleotides) Indian isolates.They assumed that as the state of and compared with CSF viruses reported by other Mizoram is geographically nearer to China and Myanmar, countries.Phylogenetic analysis placed the Korean the virus might have spread during migration of field isolates of 1988 -1999 into subgroup 3.2, animals from these neighbouring countries.5?-NTRforming an independent clade distinct from CSF viruses identified in their countries.In contrast, the based phylogenetic analysis was conducted by Desai viruses isolated during 2002-2003 CSF epidemics et al. [43].Sequence analysis and subsequent genetic classification of nine CSFV field isolates from India References were done in their study.It was found that three 1.This study showed the prevalence of both It is important to harmonise genetic typing 11.Van Zijl M., Wensvoort G., de Kluyver E., Hulst M., methods so that the sequence data obtained in different Moennig V. (2000).Introduction to classical swine isolates belonged to genotype 2.1 and were closely fever: virus, disease and control policy.Vet.related to European CSFV strains whereas the Microbiol., 73: 93-102.remainingsix isolates belonged to genotype 1 that 2. Meyers G., Rümenapf T., Thiel H.J. (1989).contained old and new strains.Molecular cloning and nucleotide sequence of the genome of hog cholera virus.Virol., 171: 555-567.
Van der Gulden H., Gielkens A., Berns A., Moormann laboratories are comparable.Providing a large R. (1991).Live attenuated pseudorabies virus collection of sequences from one or more specific expressing envelope glycoprotein E1 of hog cholera target regions increases the likelihood of such regions virus protects swine against both pseudorabies and hog cholera.J. Virol.65(5): 2761-2765.being used in future studies also.The work of 12. Bartak P., Greiser-Wilke I. (2000).Genetic typing of Hofmann et al. [19] and Lowings et al. [26] had classical swine fever virus isolates from the territory already generated much data for the 5´ NTR, E2 and of the Czech Republic.Vet.Microbiol.,77:59-70.NS5B regions.Studies on phylogeny of current 13.Hofmann M.A., Brechtbuhl K., Stauber N. (1994).isolates of CSFV are a useful means to reveal the Rapid characterization of new pestivirus strains by origin and diversity of CSFV.More importantly, direct sequencing of PCR-amplified cDNA from the phylogenetic studies prove to be a useful approach to 5´-noncoding region.Arch.Virol., 139: 217-229.aid control and eradication programme of CSF.