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

Virus Research 104 (2004) 87–92

Short communication

Phylogenetic analysis of the E2 gene of classical swine fever viruses from Lao PDR Stuart D. Blacksell a,c,∗ , Syseng Khounsy b , David B. Boyle a , Irene Greiser-Wilke d , Laurence J. Gleeson a , Harvey A. Westbury a , John S. Mackenzie c a

c

Australian Animal Health Laboratory, CSIRO Livestock Industries, Private Bag 24, Geelong, Victoria 3220, Australia b Department of Livestock and Fisheries, Ministry of Agriculture and Forestry, Lao PDR Department of Microbiology and Parasitology, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia d Institute of Virology, School of Veterinary Medicine, Buenteweg 17, 30559 Hannover, Germany Received 27 November 2003; received in revised form 27 February 2004; accepted 27 February 2004 Available online 30 April 2004

Abstract 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. © 2004 Elsevier B.V. All rights reserved. Keywords: Classical swine fever virus; Molecular epidemiology; E2; Asia; Lao PDR

1. Introduction Classical swine fever (CSF), also known as hog cholera, is a highly contagious virus infection of swine caused by classical swine fever virus (CSFV), a member of the genus Pestivirus, family Flaviviridae (Wengler et al., 1995). Taxonomic classification of CSF viruses is based on the comparison of 5 -NCR, E2 and NS5B genomic regions (Lowings et al., 1996; Paton et al., 2000) to sort viruses into three main genogroups and a number of subgroups (Paton et al., 2000). Molecular epidemiology techniques have been used to characterize CSF viruses from different countries and to determine the source of viruses that caused CSF outbreaks. Genetic characterisation of viruses from recent European CSF outbreaks indicated that they exclusively belong to genogroup 2 (Paton et al., 2000). The ∗ Corresponding author. Present address: Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, Thailand, 10400. Tel.: +66-2-354-9172; fax: +66-2-354-9169. E-mail address: [email protected] (S.D. Blacksell).

0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.02.041

Western and Central European CSF viruses that caused outbreaks in the early 1990’s were members of the subgroup 2.3 (Lowings et al., 1996; Stadejek et al., 1997; Paton et al., 2000) and Eastern European CSF viruses from the 1990’s belonged to subgroups 2.2 and 2.3 (Bartak and Greiser-Wilke, 2000). The virus responsible for the major CSF outbreak of 1997/98 in the Netherlands was a member of subgroup 2.1 (Widjojoatmodjo et al., 1999; Greiser-Wilke et al., 2000a). Recent molecular epidemiology studies of Asian CSF virus isolates by Japanese (Sakoda et al., 1999), Thai (Parchariyanon et al., 2000) and Chinese (Tu et al., 2001) researchers have indicated all three genogroups are present in East Asia and within these genogroups there is considerable diversity. Classical swine fever is endemic in Lao People’s Democratic Republic (Lao PDR) as it is in the majority of South–East Asia (Edwards et al., 2000) causing severe economic losses in village (McLaren, 1998; Oparaocha, 1997) and commercial (Parchariyanon et al., 2000) pig production systems. Lao PDR is geographically important as it shares borders with Thailand, Vietnam, China, Cambodia and Myanmar and is a major thoroughfare for uncontrolled

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livestock trade (Perry et al., 2002). As there is no information about the genetic characteristics of CSF virus of Lao origin, the objective of this study was to determine the genetic relatedness of Lao CSF viruses to reference viruses from Europe and other Asian countries and to infer possible geographic relationships.

2. Materials and methods Twenty-one spleen samples collected during 1997 and 1999 from suspected-CSF cases throughout Lao PDR were submitted for diagnostic confirmation to the National Veterinary Diagnostic Laboratory in Vientiane. A summary of the collection details of samples is presented in Table 1 and a map of the collection locations is detailed in Fig. 1. Following the confirmation of the presence of CSF virus antigen using an ELISA (Shannon et al., 1993) the samples were processed for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) amplification. RNA isolation was performed initially by the phenol–chloroform method (Vilcek et al., 1994) although this was later replaced by TRIZOLTM (Gibco-BRL) using the manufacturers methodology. RT-PCR amplification was performed using a single-step PCR ACCESS Kit (Promega, USA) using E2 gene specific primers with minor modifications to the manufacturer’s method to generate a 271-bp DNA product. The primer set was based on those described by Lowings et al. (1996) detailed as follows; forward primer 5 -TCR WCA ACC AAY GAG ATA GGG-3 (positions 2467–2487 in Alfort strain) and reverse primer 5 -CAC AGY CCR AAY CCR AAG TCA TC-3 (positions 2738–2716 in Alfort strain). Briefly, the RT-PCR reaction

contained 4.0 ␮l of RNA, 1.0 ␮l of 10 mM dNTP, 3.0 ␮l of 10 ␮M forward primer, 3.0 ␮l of 10 ␮M reverse primer, 10.0 ␮l of 5X AMV/Tfl buffer, 2.0 ␮l of 25 mM MgSO4 , 26 ␮l of DEPC-treated water, 1.0 ␮l of avian myeloblastosis virus reverse transcriptase (5 U/␮l) and 0.5 ␮l Thermus flavus DNA polymerase (5 U/␮l). Production of cDNA took place at a temperature of 48 ◦ C for 45 min followed by heating to 94 ◦ C for 2 min to inactivate the AMV reverse transcriptase. The PCR immediately followed for 40 cycles of 94 ◦ C for 30 s, 54 ◦ C for 60 s, 68 ◦ C for 120 s followed by a final extension of 68 ◦ C for 7 min. Samples were assessed by electrophoresis in 2% agarose gel containing ethidium bromide (0.5 ␮g/ml) in 40 mM Tris–Acetate, 1 mM EDTA buffer at 100 V for 60 min and visualised UV transillumination. The DNA product was purified by silica binding and ethanol precipitation (Geneclean, Bio101, USA) to remove superfluous primers and extraneous PCR reagents prior to nucleotide sequencing of both strands of DNA using an ABI Prism 377-18 automated DNA sequencer (Applied Biosystems, USA). Using the method for CSF virus phylogenetic comparison described by Paton et al. (2000), a 190-nt portion of the deduced 271-bp sequence for each CSF virus was compared with the sequence for the same region held on the European Union CSF virus reference laboratory sequence database accessed at http://viro08.tiho-hannover.de/eg/csf as detailed in Table 2. Dendrograms were calculated using the methods described previously (Greiser-Wilke et al., 2000b).

3. Results and discussion The resultant phylogenetic dendrogram for the 190-nt portion of the E2 gene is presented in Fig. 2. Following

Table 1 History and geographical description for Lao CSF viruses employed in genetic typing analysis Virus

Month

Year

District

Province

GenBank accession numbers

L12 L36 L47 L61 L65 L67 L71 L80 L119 L123 L168 L175 L202 L214 L225 L274 L280 L306 L319 L403 L404

December January February March March March March March April May June July August August September October November December December May May

1997 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1999 1999

Bachiangchaleunsook Pakse Songhon Hinboon Pakse Pakse Moonlapamok Pakse Boontai Xaythany Phaoudom Khanthabouly Xebangfay Nonghed Mahaxay Hinboon Sukhuma Bolikhanh Pakse Sangthong Sangthong

Champassak Champassak Savannakhet Khammouane Champassak Champassak Champassak Champassak Phongsaly Vientiane municipality Bokeo Savannakhet Khammouane Xieng Khouang Khammouane Khammouane Champassak Bhorikhamxai Champassak Vientiane municipality Vientiane municipality

AY283655 AY283650 AY283659 AY283661 AY283651 AY283663 AY283653 AY283652 AY283667 AY283665 AY283666 AY283658 AY283656 AY283662 AY283660 AY283657 AY283654 AY283664 AY283649 AY283668 AY283669

S.D. Blacksell et al. / Virus Research 104 (2004) 87–92

89

L119

L168

L214

L306 L123 L403 L404 L61 L202 L225 L274 Subgroup 2.1 Subgroup 2.2

L47 L175

L12 L36 L65 L67 L71 L80 L280 L319

Fig. 1. Map of Lao PDR with the location of the sources of the CSF viruses examined in this study.

alignment of the field and reference nucleotide sequences, the transition/transversion ratio was calculated as 6.83. To make the dendrograms comparable to those from other studies, they were out-grouped to the sequence of the Kanagawa isolate (CSF0309) and the nomenclature of the subgroups was as proposed by Paton et al. (2000). The results of the phylogenetic analysis demonstrated that all Lao CSF viruses belonged to genogroup 2. Five viruses belonged to subgroup 2.1 and the remaining 16 viruses belonged to subgroup 2.2. No viruses belonged to subgroup 2.3. All Lao subgroup 2.1 viruses originated from the Northern or Central regions of Lao PDR. Phylogenetic analysis of virus L119 that originated in Phongsaly indicated a close relationship with a group of European reference viruses from the 1997 outbreak although the Lao virus was distinct. Virus L119 also demonstrated relationships with, but was distinct from reference viruses representing the United Kingdom outbreak of 2000 (CSF0708) and a Croatian outbreak of

1997 (CSF0669). The remaining subgroup 2.1 viruses L403, L404, L123 and L168 formed two clusters distinct from the European reference viruses. Subgroup 2.2 viruses were derived exclusively from Southern Lao PDR with the exception of one virus from the Central/Northeast province of Xieng Khouang. There was a clear phylogenetic distinction between the Lao PDR subgroup 2.2 viruses and reference viruses including a Thai virus from 1996. The majority of subgroup 2.2 viruses clustered in highly localized geographic groups. This was demonstrated by the close relationship of seven viruses that originated from Champassak province (L12, L36, L65, L71, L80, L280, and L319) during a 12 month period from December 1997 to the same time the following year. Virus L306 from the Northern part of the Southern geographic zone was distinct suggesting a different origin. Previous genetic analyses of CSF viruses from Asia (Malaysia, Japan, Thailand, Taiwan and China) have concluded that viruses from the region belong to all three

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S.D. Blacksell et al. / Virus Research 104 (2004) 87–92 Outgroup

CSF0309

3

KKN93

978 961

991

942

CSF0905 CSF0913 CSF0902 CSF0911 CSF0919 KPP93 CSF0007 CSF0294 CSF0106 CSF0162 CSF0292 CSF0120 648 CSF0166 CSF0171 CSF0281 CSF0084 CSF0107 CSF0669 633 CSF0708

1.2 1.1

2.3

CSF0150

221

L119 847

CSF0301 CSF0277 CSF0273 CSF0283 CSF0305

590 200

505

136

2.1

CSF0023 CSF0022 CSF0934

767

L403 L404 L123 L168 CSF0062 CSF0014

579

L306 945

CBR96 CSF0421 324

316

CSF0075 CSF0118 L225 L61 L214 L67

346

L47

686

2.2

L175 416

L202 L274 L12 L319 L36 L65 L80 L71 L280

0.1

Fig. 2. Neighbour joining (NJ) phylogenetic tree constructed from a 190 nt fragment of the E2 glycoprotein using the SEQBOOT (1000 replicates), DNADIST, NEIGHBOUR and CONSENSE modules of the PHYLIP package (Felsenstein, 1989). Viruses from Lao PDR are underlined. Alignments were calculated with CLUSTAL X (version 1.8) (Thompson et al., 1994). The transition/transversion rate was calculated using the PUZZLE32, Version 4.0, program (Strimmer and von Haeseler, 1996). TREEVIEW version 1.6.5 (Page, 1996) was used to outgroup the tree to the CSF0309 (Kanagawa) sequence and for the graphic output. The bar below the dendrogram indicates the number of substitutions per site.

recognised CSF virus genogroups (Lowings et al., 1996; Vilcek et al., 1994; Stadejek et al., 1997; Sakoda et al., 1999; Parchariyanon et al., 2000; Paton et al., 2000; Tu et al., 2001). From these studies, CSF viruses from two of the five countries bordering Lao PDR have been genetically

characterised. CSF viruses from Thailand collected during the 1980’s and 1990’s have been reported to belong to all three genogroups (Parchariyanon et al., 2000) although only a small number of subgroup 2.2 viruses and no subgroup 2.1 viruses were reported. Chinese CSF viruses that origi-

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Table 2 Virus source and genogroup designation information for reference CSF viruses used for comparative purposes in this study Identificationa

Year

Isolate

Country

Genogroupb

CSF0902 CSF0913 CSF0911 CSF0919 KPP93 CSF0905 CSF0023 CSF0150 CSF0273 CSF0305 CSF0669 CSF0708 CSF0301 CSF0277 CSF0283 CSF0022 CSF0934 CSF0014 CSF0062 CSF0421 CSF0075 CSF0118 CBR96 CSF0007 CSF0084 CSF0106 CSF0166 CSF0171 CSF0294 CSF0162 CSF0292 CSF0120 CSF0281 CSF0107 KKN93 CSF0309

1987 n/a 1968 n/a 1993 n/a 1989 1993 1997 1986 1997 2000 1997 1997 1997 1989 1989 1989 1990 1997 1990 1992 1996 1984 1992 1994 1996 1996 n/a 1996 n/a 1994 1997 1994 1993 1974

Alfort/187 Riems Vaccine Glentorf ALD Kampang Phet (AF241616)c Brescia 907/3 SP 10549/13 V3/97 VRI 2277 S-168 2000/8 1723-1726 V1240/97 MP104 907/2 Rhoen 89 Sch 180 Vi 633/90 SW97-PM1241, KRN SP 2088/90 SP 10022-1 Chonburi (AF241626)c V774 Rostock II, V119/92 Celle/Han94, V422 EWS 1053/96 NWS 718/96 29/5 34/5 25/5 SP 4165/23 Vi302/97/OL V1166 III/94 Khon Kaen (AF241632)c Kanagawa (Tap3)

France Germany Germany Japan Thailand Italy Germany Austria Germany Malaysia Croatia United Kingdom Germany Germany Netherlands Germany Germany Germany Germany Thailand Austria Austria Thailand Germany Germany Germany Germany Germany Poland Poland Poland Austria Germany Germany Thailand Japan

1.1 1.1 1.1 1.1 1.1 1.2 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.2 2.2 2.2 2.2 2.2 2.2 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 3 3.4

a b c

Refers to the CSF virus and sequence database accessible at http://viro08.tiho-hannover.de/eg/ (Greiser-Wilke et al., 2000). According to Lowings et al. (1996) and Paton et al. (2000). Genbank accession numbers.

nated from 26 provinces belonged to genogroups 1.1 and 2.1, 2.2 and 2.3 (Tu et al., 2001) although unfortunately the nucleotide sequences have not been deposited into reference genetic databases to enable comparative analysis. In China, subgroup 2.1 viruses were detected in Yunnan province, which shares a common border with Northern Lao PDR including Phongsaly province. The results presented here indicate that CSF viruses of Lao origin are regionally conserved, with the subgroup 2.1 viruses confined to the North–Central region and subgroup 2.2 viruses confined largely to the South–Central region. It is possible that the subgroup 2.1 viruses that may have originated from cross-border trade although the lack of genetic information on Chinese reference CSF viruses prevented such an epidemiological linkage. Interestingly, subgroup 2.1 viruses were also found in Vientiane municipality (the capital of Lao PDR) but not in the Southern region of Lao PDR. This observation is consistent with Vientiane, the national

capital, being an important focus for animal movement and trade. The majority of the subgroup 2.2 viruses form a distinct cluster separated from European and Thai reference viruses included in this analysis. As they show a clear geographic clustering, they may have evolved independently and probably circulate in a closed viral ecosystem characterised by the local movement of infected animals and associated products. Whether this ecosystem includes neighbouring Cambodia and Vietnam is not presently known. Alternatively the Lao viruses may have been derived from viruses present in neighbouring countries but that are thus far not included in the database. The results presented in this report add significantly to the repository of genetic information on world-wide CSF virus genogroups, although further studies of archival and contemporary CSF viruses from neighbouring countries are required to complete the regional epidemiological picture.

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Acknowledgements This work was supported the Australian Centre for International Agricultural Research (ACIAR), the Department of Livestock and Fisheries of Lao PDR and CSIRO Livestock Industries. The authors wish to acknowledge Ms. Manivanh Phruaravanh and Mrs. Khonsavanh Douangphachanh for excellent technical support. The authors also wish thank Mr. Tony Pye for performing the nucleotide sequencing.

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