Phylogenetic analysis of classical swine fever virus in Taiwan

Summary. Two envelope glycoprotein (E^rns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the E^rns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (E^rns–E2) displayed better resolution than that based on the complete open reading frame.     

Phylogenetic analysis of classical swine fever virus (CSFV) field isolates from outbreaks in South and Central America

To date, there is little information concerning the epidemiological situation of classical swine fever (CSF) in the Americas. Besides summarizing the available data, genotyping of isolates from outbreaks in domestic pigs in several countries of South and Central America was performed. For this, a 190 base fragment of the E2 envelope glycoprotein gene was used. European strains and isolates, and historical isolates from the United States (US) were included for comparison. In contrast to the situation in most parts of Europe, where group 2 isolates predominate, it was found that all the isolates from the American continent analyzed belonged to group 1 and were further resolved into three subgroups. The Cuban isolates clustered in subgroup 1.2, whereas the isolates from Honduras and Guatemala clustered in subgroup 1.3. The remaining isolates from Argentina, Brazil, Colombia and Mexico generated four poorly resolved clusters in subgroup 1.1, together with the vaccine strains, with historical European and US isolates, and with a recent Russian isolate. While the vaccine strains and the historical European isolates formed a relatively distinct cluster, one of the US isolates clustered together with the Mexican, and another one with Colombian isolates. Historically, CSF (hog cholera) was observed almost simultaneously in the US and in Europe in the first half of the 19th century, and its origin remains a matter of discussion. Our results showed that the US isolates are closely related to isolates from South America, while appearance of isolates in Cuba on one hand and in Honduras and Guatemala on the other hand, seems to have been due to unrelated events. This allows to speculate that at least in the American continent, CSF virus may have appeared independently in several regions, and spreading may have been a secondary effect.     

Phylogenetic comparison of classical swine fever virus in China.

An N-terminal fragment of the E2 gene of classical swine fever (CSF) virus encoding major immunogenic sites was amplified by RT-PCR directly from 110 clinical specimens representing 109 epizootic sites during the last decade in China. Phylogenetic relationships between these viruses as well as 20 reference strains were determined by comparison of their nucleotide sequences. A phylogenetic tree showed that 103 of the 110 field viruses (93.6%) were clustered within group 2 and subdivided into three subgroups, while the remaining seven viruses (6.4%), along with two Chinese reference strains, Shimen and HCLV (attenuated vaccine strain), were clustered into subgroup 1.1 within group 1. However, none of the Chinese CSF viruses were members of subgroup 1.2 (represented by reference strain Brescia). This is the first report on the distribution of CSF virus genotypes in China. Results indicated that CSF viruses predominating in recent epizootics within China are genetically divergent from the reference strain Shimen and the vaccine strain HCLV.     

Phylogenetic analysis of swine fever virus strains

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.     

First molecular identification and characterization of classical swine fever virus isolates from Nepal

Classical swine fever (CSF) is a major constraint to pig production worldwide, and in many developing countries, the epidemiological status is unknown. Here, for the first time, molecular identification and characterization of CSFV isolates from two recent outbreaks in Nepal are presented. Analysis of full-length E2-encoding sequences revealed that these isolates belonged to CSFV subgenotype 2.2 and had highest genetic similarity to isolates from India. Hence, for CSFV, Nepal and India should be regarded as one epidemiological unit. Both Nepalese isolates exhibited significant sequence differences, excluding a direct epidemiological connection and suggesting that CSFV is endemic in that country.     

Phylogenetic analysis of NS5B gene of classical swine fever virus isolates indicates plausible Chinese origin of Indian subgroup 2.2 viruses

Twenty-three CSFV isolates recovered from field outbreaks in various parts of India during 2006–2009 were used for genetic analysis in the NS5B region (409 nts). Seventeen of these were studied earlier [16] in the 5′UTR region. Phylogenetic analysis indicated the continued dominance of subgroup 1.1 strains in the country. Detailed analysis of a subgroup 2.2 virus indicated the plausible Chinese origin of this subgroup in India and provided indirect evidence of routes of CSFV movement within South East Asia region.     

Genetic diversity and positive selection analysis of classical swine fever virus isolates in south China

Classical swine fever virus (CSFV) causes a highly contagious disease that leads to significant economic losses in the pig industry worldwide. However, there is a paucity of knowledge on the accurate genotyping of CSFV isolates in south China. This study genotyped the E2 gene of 14 CSFV strains isolated during 2008-2010 from domestic pigs in different districts of south China. Phylogenetic analyses revealed that all of the 14 CSFV isolates were clustered into genetic subgroup 1.1. This contrasts with most parts of China, where group 2 isolates are predominant. Furthermore, the positive selection pressures acting on the E(rns) and E2 envelope protein genes of CSFV were assessed and a site-by-site analysis of the dN/dS ratio was performed to identify specific codons that undergo diversification under positive selection. While no significant evidence for positive selection was observed in E(rns), two positively selected sites at amino acid residues 49 and 72 in the E2 encoding region were identified. Our results revealed that a predominance of subgroup 1.1 CSFV isolates is currently circulating in some districts of south China, which appear to be unrelated to the Chinese C-strain vaccine. Moreover, the envelope protein gene, E2, has undergone positive selection in 14 CSFV strains and two positively selected sites have been identified in this study. Understanding the molecular epidemiology and functional importance of these positively selected amino acid positions could help to predict possible changes in virulence, the development of vaccines and disease control.     

Phylogenetic analysis of the E2 gene of classical swine fever viruses from Lao PDR

The E2 genes of 21 classical swine fever viruses (CSFV) were genetically characterized and compared with reference CSF viruses. The viruses originated from CSF outbreaks that occurred in the Lao People's Democratic Republic (Lao PDR) during 1997 though to 1999. All viruses characterized belonged to genogroup 2 and were members of subgroups 2.1 and 2.2. Results demonstrated a geographic delineation between subgroups 2.1 that was only found in the North-Central region, and subgroup 2.2 that was mostly found in the South-Central regions of Lao PDR. Although it was not possible to determine the origin of these viruses, it is probable that they may have been introduced to Lao PDR following cross-border trade. Alternatively, they have evolved independently of other viruses in the region.     

Evidence of natural recombination in classical swine fever virus

Classical swine fever (CSF) virus, one member of the family Flaviviridae is the pathogen of CSF, an economically important and highly contagious disease of pigs. Although homologous recombination has been demonstrated in many other members of the family, it is unknown whether there is recombination in natural populations of CSFV. To detect possible recombination events, we performed a phylogenetic analysis of 25 full-length CSFV strains isolated all over the world. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning. A mosaic virus, CSFV 39 (AF407339) isolated in China was found. And its two putative parental-like strains CSFV Shimen (AF333000) and GXWZ02 (AY367767) were identified. Our work revealed that homologous recombination occurred in natural CSFV populations, generating genetic diversity. This would provide some insights for the role homologous recombinant plays in CSFV evolution.     

Analysis of synonymous codon usage in classical swine fever virus

Using the complete genome sequences of 35 classical swine fever viruses (CSFV) representing all three genotypes and all three kinds of virulence, we analyzed synonymous codon usage and the relative dinucleotide abundance in CSFV. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in CSFV. Furthermore, we observed that the relative abundance of dinucleotides in CSFV is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as the subgenotypes and aromaticity, also influence the codon usage variation among the genomes of CSFV. This study represents the most comprehensive analysis to date of CSFV codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete sequence of a subgroup 3.4 strain of classical swine fever virus from Taiwan

Classical swine fever viruses from Taiwan have been classified into two subgroups (3.4 and 2.1). Outbreaks caused by 3.4 viruses were reported in Taiwan prior to 1996 and which mainly distributed in the geographic range from southern Japan to Taiwan. We have determined the complete sequence of a reference strain, 94.4/IL/94/TWN. The genome contains 12,296 nucleotides, encoding 3,898 amino acids flanked by a 372-nt region at the 5' untranslated region (UTR) and a 227-nt region at the 3'-UTR. Similarities of nucleotides among 3.4 viruses isolated from Taiwan and Japan (Kanagawa/74; Okinawa/86) maintained in 94.2-97.5%; however, comparing to subgroup 1.1 (ALD/64/Jap) and 2.1 (TD/96/TWN) only showed about 72.5-80.8%, respectively. Phylogenetic analysis based on positioning from 11,157 to 11,565 nt (NS5B region) revealed that CSFVs were divided into three major lineages and their sublineages. Strain 94.4/IL/94/TWN is the first completely genomic sequence of subgroup 3.4 viruses.     

Phylogenetic characterization of classical swine fever viruses isolated in Korea between 1988 and 2003

Twenty-four isolates of classical swine fever (CSF) virus which were obtained from CSF outbreaks during 1988 and 2003 in the Republic of Korea were genetically characterized for partial E2 gene (190 nucleotides) and compared with CSF viruses reported by other countries. Phylogenetic analyses classified Korean field isolates between1988 and 1999 into subgroup 3.2, forming an independent clade distinct from CSF viruses identified in other countries. In contrast, the viruses isolated during 2002-2003 CSF epidemics were classified into a different subgroup (2.1). The 2.1 viruses showed a close genetic relationship (92.1-100% nucleotide similarity) with CSF viruses reported from China and Taiwan in 1998-2001. As no evidence of CSF virus infection was detected in the wild boar (Sus scrofa coreanus) population that inhabits Korea, the results of molecular characterization strongly suggest that CSF epidemic outbreaks in Korean swine populations during 2002-2003 were attributed to the introduction of a new strain or strains, likely from neighboring countries.     

Mutational analysis of the GDD sequence motif of classical swine fever virus RNA-dependent RNA polymerases

To define the function of the GDD motif of the RNA-dependent RNA polymerase (RdRp) of classical swine fever virus (CSFV), single amino acid substitutions were introduced into the CSFV NS5B. All substitutions within the GDD motif were detrimental to the polymerase activity, the binding activity and the terminal nucleotidyl transferase activity of the NS5B protein. It was also found that the wild-type NS5B had higher RdRp activity with Mg(+2) than with Mn(+2) whereas some mutants worked better with Mn(+2) than with Mg(+2), suggesting that substitutions within the GDD motif modified the enzyme cation preferences and the GDD sequence of CSFV NS5B might be involved in polymerase-metal interaction. Therefore, the GDD amino acid sequence is important for the function of CSFV RdRp.     

Propagation of classical swine fever virus in vitro circumventing heparan sulfate-adaptation

Amplification of natural virus isolates in permanent cell lines can result in adaptation, in particular enhanced binding to heparan sulfate (HS)-containing glycosaminoglycans present on most vertebrate cells. This has been reported for several viruses, including the pestivirus classical swine fever virus (CSFV), the causative agent of a highly contagious hemorrhagic disease in pigs. Propagation of CSFV in cell culture is essential in virus diagnostics and research. Adaptation of CSFV to HS-binding has been related to amino acid changes in the viral E(rns) glycoprotein, resulting in viruses with altered replication characteristics in vitro and in vivo. Consequently, a compound blocking the HS-containing structures on cell surfaces was employed to monitor conversion from HS-independency to HS-dependency. It was shown that the porcine PEDSV.15 cell line permitted propagation of CSFV within a limited number of passages without adaptation to HS-binding. The selection of HS-dependent CSFV mutants was also prevented by propagation of the virus in the presence of DSTP 27. The importance of these findings can be seen from the altered ratio of cell-associated to secreted virus upon acquisition of enhanced HS-binding affinity, a phenotype proposed previously to be related to virulence in the natural host.     

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.     

Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine

Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics in primary porcine macrophage cell cultures, the major target cell of CSFV in vivo, it was markedly attenuated in swine. All RB-C22v-infected pigs survived infection remaining clinically normal in contrast to the 100% mortality observed for BICv-infected animals. Comparative pathogenesis studies demonstrated a delay in RB-C22v spread to, and decreased replication in the tonsils, a 10(2) to 10(7) log10 reduction in virus titers in lymphoid tissues and blood, and an overall delay in generalization of infection relative to BICv. Notably, RB-C22v-infected animals were protected from clinical disease when challenged with pathogenic BICv at 3, 5, 7, and 21 days post-RB-C22v inoculation. Viremia, viral replication in tissues, and oronasal shedding were reduced in animals challenged at 7 and 21 DPI. Notably BICv-specific RNA was not detected in tonsils of challenged animals. These results indicate that a carboxyl-terminal domain of E1 glycoprotein affects virulence of CSFV in swine, and they demonstrate that mutation of this domain provides the basis for a rationally designed and efficacious live-attenuated CSF vaccine.     

Phylogenetic analysis of previously nontypeable hepatitis C virus isolates from Argentina

Phylogenetic analysis of hepatitis C virus isolates from Argentina that were previously nontypeable by restriction fragment length polymorphism (RFLP) analysis revealed that they belong to genotype 1a. A substitution at position 107 (G-->A), which is the landmark of these strains, was shown to be distributed among isolates worldwide. The RFLP patterns obtained for these isolates should be added to the ones reported for genotype 1 isolates.     

Molecular chaperone Jiv promotes the RNA replication of classical swine fever virus

The nonstructural protein 2 (NS2) of classical swine fever virus (CSFV) is a self-splicing ribozyme wherein the precursor protein NS2-3 is cleaved, and the cleavage efficiency of NS2-3 is crucial to the replication of viral RNA. However, the proteolytic activity of NS2 autoprotease may be achieved through a cellular chaperone called J-domain protein interacting with viral protein (Jiv) or its fragment Jiv90, as evidence suggests that Jiv is required for the proper functioning of the NS2 protein of bovine viral diarrhea virus. Hence, the expression of Jiv may be correlated with the replication efficiency of CSFV RNA. We investigated the expression levels of Jiv and viral RNA in CSFV-infected cells and tissues using Real-time RT-PCR or Western blot analysis. The obtained results show that Jiv90 possibly plays an important role in the lifecycle of CSFV because the distribution of Jiv90 protein shows a positive correlation with the viral load of CSFV. Furthermore, the overexpression or knockdown of Jiv90 in swine cells can also significantly promote or decrease the viral load, respectively. The detection of Flow cytometry shows that the overexpression of Jiv90 prolongs the G1 phase of cell cycles but has no effect on apoptosis. These findings are likely to be of benefit in clarifying the pathogenesis of the CSFV.