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1 State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 100 Ying Xin St, Beijing 100052, PR China
2 Department of Arbovirology, Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
Guo-dong Liang
gdliang{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU015061–EU015076.
Details of primers used for RT-PCR and sequencing and alignments of GETV and SAGV E2 and 3' UTR sequences are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Alphaviruses are important pathogens of livestock animals and humans worldwide. The alphavirus particle contains a single copy of the virus genome, which is a linear positive-sense single-stranded RNA. The genome length of the alphaviruses that have been sequenced ranges from 11 442 nt for Semliki Forest virus to 11 851 nt for Ross River virus (Faragher et al., 1988; Strauss & Strauss, 1994; Pfeffer et al., 1998). The genome has a 5' terminal cap and the 3' end has a poly(A) tract. The 5' two-thirds of the genome encodes the non-structural proteins nsP1 to nsP4, followed by the 26S junction region, which promotes transcription of the intracellular subgenomic 26S RNA. This latter mRNA contains the structural gene that encodes the C (capsid), E3, E2, 6K and E1 proteins and represents the remaining 3' third of the genome (Pfeffer et al., 1998).
Most alphaviruses are transmitted by haematophagous arthropods and can be grouped into eight antigenic complexes based on their serological cross-reactions. These are the Barmah Forest, Eastern equine encephalitis, Middleburg, Ndumu, Semliki forest, Trocara, Venezuelan equine encephalitis and Western equine encephalitis complexes (Griffin, 2001; Weaver et al., 2005). Antigenic cross-reactivities between alphaviruses reflect sequence conservation in the C protein and E1 glycoprotein, while antibodies directed against the E2 protein are usually virus-specific (Griffin, 2001).
The species Getah virus (GETV) is a member of the Semliki Forest (SFV) complex (Weaver et al., 2005), based on genetic and antigenic properties. GETV was first isolated from Culex gelidus in Malaysia in 1955 (Berge, 1975); the virus was isolated in rubber plantations, hence the name Getah, which means rubber in Malay.
To date seven Alphavirus species, namely GETV, Sindbis virus (SINV), Ross River virus (RRV), Chikungunya virus (CHIKV), Mayaro virus (MAYV) Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), have been isolated from various geographical locations in China (Zhang et al., 1989; Li et al., 1992; Zhao et al., 1997; Liang et al., 2000; Huang et al., 2001; Lv et al., 2001; Wang & Liang, 2003; Wang et al., 2006). Chinese alphaviruses may show significant sequence divergence from their relatives isolated elsewhere; for example, isolate XJ-160 of SINV from China exhibited 18 % overall nucleotide (8.6 % amino acid) variation from the prototype SINV (Liang et al., 2000).
The closest phylogenetic relatives of GETV within the SFV complex are RRV and Sagiyama virus (SAGV) (Calisher et al., 1980; Powers et al., 2001; Attoui et al., 2007). RRV is a known human pathogen and SAGV was isolated from various mammals (Attoui et al., 2007). Previously published data show that the taxonomic status of SAGV should be reassessed (Shirako & Yamaguchi, 2000; Attoui et al., 2007). Sequence analysis suggested that SAGV should be reconsidered as a member of the species GETV.
GETV is widely distributed in the countries of South-east Asia and in northern Australia along the Pacific Ocean (Norder et al., 1996; Turell et al., 2003; Bryant et al., 2005; Wang et al., 2006; Chang et al., 2006). The virus is a known pathogen of horses and pigs. In horses, the disease is characterized by pyrexia, rash, oedema of the hind legs and intumescence of lymph nodes. The infection in pigs results in miscarriage (Kamada et al., 1980; Kono et al., 1980; Kumanomido et al., 1988a, b; Shibata et al., 1991; Brown & Timoney, 1998). Neutralizing antibodies to GETV have been identified in human sera and in birds in Malaysia, northern Australia and Hainan province in China (Kanamitsu et al., 1979; Marchette et al., 1980; Li et al., 1992). Despite these findings, the virus has not been linked to illness in humans.
In 1964, an alphavirus designated M1 was isolated in Hainan province (Li et al., 1992) and was subsequently identified as an isolate of GETV by serological analyses. During investigations of arboviruses in China in recent years, several new GETV isolates have been obtained from mosquitoes. In 2002, 38 years after the first isolation, two more GETV strains were isolated, from wild-caught mosquitoes collected in Hebei province in northern China (Wang et al., 2006). This isolation reflects the first known incursion of GETV into the northern regions of China.
In total, 10 GETV strains have been isolated in China, including the M1 strain from Hainan, two strains from Hebei and seven strains (obtained during this study) from Yunnan, Ganshu and Shanghai (see Fig. 1). The geographical distribution of these isolates ranged between latitudes 1 ° and 4 ° N and longitudes 9 ° and 12 ° E, reflecting the wide distribution of GETV in China. The sequence of the M1 strain has been reported previously (Zhao et al., 2000; Wen et al., 2007). In this study, we report the full-length sequence characterization of various GETV isolates from China, with particular emphasis on the genetic characterization of the envelope genes and 3' untranslated regions (UTRs). The sequence characterization suggests that GETV has spread recently to China and that the virus has been evolving slowly. Arbovirus surveillance studies carried out before 2000 in China failed to identify the circulation of GETV in this country.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), followed by sequencing.
Virus propagation.
Ten viral isolates designated M1 (isolated in 1964 in Hainan province; Li et al., 1992), HB0215-3, HB0234 (isolated in 2002 in Hebei province; Wang et al., 2006), YN0540, YN0542, SH05-6, SH05-15, SH05-16, SH05-17 and GS10-2 (isolated during this study) were propagated in BHK-21 cells at 37 °C or Aedes albopictus C6/36 mosquito cells at 28 °C in minimal essential medium (HyClone) supplemented with 2 % fetal bovine serum (FBS), 2 mM glutamine, 0.12 % NaHCO3, 100 U penicillin ml–1 and 100 U streptomycin ml–1.
Preparation of viral RNA, reverse transcription and PCR amplification of the GETV genome.
Viral RNA was extracted from 140 µl supernatant of virus-infected BHK-21 cell cultures using a viral RNA kit (Qiagen) according to the manufacturer's instruction. cDNA was synthesized using You-Prime-Ready-To-Go beads (Amersham Bioscience) and random hexanucleotide primers. Forty-three primers were designed from the complete sequences of GETV (GenBank accession number AY702913
[GenBank]
; South Korea) and SAGV (AB032553
[GenBank]
; Japan) genomes. These primers were used for full-length amplification and sequencing of Chinese GETV isolates. The list of primers is provided in Supplementary Table S1, available in JGV Online.
The 5' UTR of the viral RNA was amplified using the 5'-Full RACE Core Set (Takara) and the 3' UTR was amplified using primers Ma05F4 (5'-CGGCAATGACATGGGTGCAGC-3', positions 11185–11205), Get3'UR (5'-CTGTAAAATATTAAAAAAACAAATTAGACGC-3', 11670–11698) and M06R1 [5'-CTGTCAGCGAATTCGGTACC(T)18G-3', 11698]. PCR was performed using Ex Taq DNA polymerase (Takara). Amplified products were examined by agarose gel electrophoresis (1 % gel) and purified by QIA quick Gel Extraction kit (Qiagen) and then sequenced directly. At the same time, the amplified products of 5'-RACE and the 3' UTR were cloned into pGEM-T easy vector (Promega) and sequenced using M13 universal primers. Each nucleotide position was sequenced three times. The contig sequences were assembled using ContigExpress Project software (Vector NTI Advance 1001 package; Invitrogen).
Sequence analysis and phylogenetic comparisons.
Nucleotide and amino acid sequence alignments were generated by CLUSTAL_X version 1.8 (Thompson et al., 1997). Analysis of nucleotide and deduced amino acid sequence identities was performed using GeneDoc and DNASTAR (Lasergene). Neighbour-joining phylogenetic trees based on nucleotide sequences were constructed using MEGA version 3.1 (Kumar et al., 2004). The robustness of phylogenetic constructions was evaluated by bootstrapping using 500 replications. Previously published GETV and other alphavirus sequences used in this study include sequences from O'Nyong-nyong virus (ONNV; GenBank accession number M20303
[GenBank]
), Barmah Forest virus (BFV; U73745
[GenBank]
), MAYV (AF237947
[GenBank]
), SFV (X04129
[GenBank]
), RRV (K00046), CHIKV (NC_004162
[GenBank]
), GETV (AF268025
[GenBank]
, AF339484
[GenBank]
, AY702913
[GenBank]
and EF011023
[GenBank]
) and SAGV (AB032553
[GenBank]
and AF242890
[GenBank]
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were also included in this study.
Sequence analysis
Full-length genome sequences were determined for three isolates, M1, HB0234 and YN0540. Details are provided in Table 1. It is noteworthy that the non-structural ORF of strain HB0234 was 3 nucleotides shorter than those of the two other strains, as a result of a deletion of a codon at positions 5473–5475 when compared with SAGV (GenBank accession number AB032533
[GenBank]
). A deletion of 3 nucleotides (positions 99–102) was identified in the capsid gene of strain M1 when compared with SAGV (AB032533
[GenBank]
). Sequence analysis of 3' UTRs of isolates HB0215-3, YN0542, SH05-6 and SH05-17 indicated that their UTRs were 401 nt long, while those of isolates SH05-15, SH05-16 and GS10-2 were respectively 404, 410 and 402 nt long.
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The nucleotide sequence identity between the three Chinese isolates (M1, HB0234 and YN0540) ranged from 98.1 to 99.1 %, while amino acid sequence identity ranged from 98.7 to 99.8 % (Tables 2 and 3). The nucleotide sequence identities between the Chinese and other isolates ranged from 96.9 to 98.8 %, while the amino acid sequence identity ranged from 98.7 to 99.8 % (98.9–99.6 % in the non-structural polyprotein and 98.0–99.8 % in the structural polyprotein).
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Amino acid changes were identified in various genes, as shown in Table 3
. Deletion of a single amino acid was detected in the nsP3 of isolate HB0234. The nsP3–nsP4 junction of the same isolate is not interrupted by the usual leaky opal stop codon (TGA) that is found at position 518 of GETV; rather, this is replaced by a TGG (coding for Trp).
In the capsid protein, seven positions were found to be variable among all analysed GETV isolates. Deletion of a single amino acid was found in the capsid protein of strain M1. Again, the M1 strain showed a single amino acid change in the E3 protein. Seventeen sites showed changes in the E2 protein in the various Chinese isolates. In the 6K protein, only SAGV presented a single change. The results are detailed in Tables 3 and 4.
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. These values ranged between 97.7 and 99.9 % for nucleic acids and 98.3 and 100 % for amino acids among the Chinese isolates and between 94.5 and 95.3 % for nucleic acids and 96.9 to 97.6 % for amino acids when the Chinese isolates were compared with the original Malaysian GETV isolate M2021 (GenBank accession number AF339484
[GenBank]
). Interestingly, the Mongolian isolate (LEIV 17741 MPR; GenBank accession number EF631999
[GenBank]
) showed 100 % amino acid identity with four Chinese isolates (SH05-6, SH05-15, YN0540 and YN0542) isolated in the south of China.
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Analysis of the 3' UTR of the Chinese isolates of GETV
Alphaviruses possess a highly conserved 3' sequence element (3' CSE; approximately 19 nt long) which immediately precedes the poly(A) tail (Pfeffer et al., 1998). Both the poly(A) tail and the 3' CSE are required for virus replication and, more specifically, for efficient minus-strand RNA synthesis (Kuhn et al., 1990, 1992; Raju et al., 1999; Hardy & Rice, 2005). The terminal 19 nt conserved sequence was identical in all GETV isolates, including the M1 isolate that was previously reported to have lost this conserved sequence (Zhao et al., 2000).
Within the 3' UTR, repeated sequence elements (RSE) of 40–60 nt have been described for all alphaviruses investigated so far (Strauss & Strauss, 1994). These were identified as being three repeats of about 54 nt in GETV (Supplementary Fig. S2; underlined) (Pfeffer et al., 1998). An analysis of the 3' UTR of GETV isolates identified considerable variations, as shown in Supplementary Fig. S2. Insertions and/or deletions were found at several positions in the 3' UTR. A deletion was identified in the 3' UTR of a previously published GETV sequence (GenBank accession number AY702913
[GenBank]
) between nucleotides 50 and 153 (positions are relative to the SAGV sequence).
Sequence alignment showed that all isolates including the previously published Taiwan district (GenBank accession number AF242890 [GenBank] ), Russian (EF631998 [GenBank] ) and Mongolian (EF631999 [GenBank] ) isolates show the same deletion (10 nt) at positions 45–54 in the 3' UTR (except YN0540, which showed a deletion of only 9 nt, at positions 46–54).
The Chinese isolates, including the Taiwan district isolate, all showed a T to C transition at position 64 and a C to T transition at positions 43 and 148 (relative to the SAGV sequence, GenBank accession number AB032533 [GenBank] ). These strains were isolated up to 40 years apart (the M1 strain was isolated in 1964). Insertions (or deletions) of adenine residues were identified at several positions (Supplementary Fig. S2; positions 173, 299, 250 and 372). These included an insertion of 10 consecutive adenine residues in the 3' UTR of strain M1 (positions 189–198), which is located towards the end of the second RSE. Insertions of 9 and 3 consecutive adenine residues, respectively, were also identified towards the end of the third RSE in isolates SH05-6 and SH05-15 (Supplementary Fig. S2).
Phylogenetic analysis
The P-distance model and Kimura's two-parameter model were used for tree building. The alpha shape parameter used for the gamma distribution analysis (for the Kimura two-parameter) was calculated using the PAML package (Yang, 1997). The shape parameter measures how variable the rates are among sites. When the value of alpha is >1, most sites have rates around 1 (similar rates). When alpha is 
1 (there is a relatively large amount of rate variation), most sites have very low rates (<1), but there are evolutionary hot spots with higher rates.
A phylogenetic tree (Fig. 2a) was constructed using the complete genome sequences of several alphaviruses including the GETV isolates M1, HB0234 and YN0540 from China and other GETV isolates from South Korea, Russia and Mongolia. Because the sequence of the original Malaysian strain of GETV is incomplete, only the sequence encoding nsp4 and the structural polyprotein (4789 nt) of this particular strain was used in the alignment. The sequence encoding nsP1, nsP2 and nsP3 of this isolate was accounted for as missing information in the MEGA 3.1 package options.
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) was constructed using nucleotide sequences encoding the capsid protein and included, alongside several other alphaviruses, the GETV isolates M1, HB0234 and YN0540 from China, GETV from Malaysia, SAGV from Japan and other GETV isolates from South Korea, Russia and Mongolia.
A third phylogenetic tree (Fig. 2c) was constructed from the E2 protein sequences of all Chinese isolates of GETV alongside the original GETV from Malaysia, SAGV from Japan and other GETV isolates from South Korea, Russia and Mongolia.
Trees constructed for given genes using either the P-distance or Kimura's two-parameter model were found to have identical topologies.
The trees showed that the GETV and SAGV isolates clustered onto a distinct evolutionary branch, rooted by the original GETV from Malaysia. The various trees showed that the most recent isolates of GETV are located at the end branches, rooted by the earlier isolates (the Chinese isolates represent the latest evolutionary events pertaining to GETV). The only noticeable exception is the case of the recent Mongolian and Russian isolates (made in 2000). These two strains do not cluster among those that have been made more recently around the same time. Rather, the Russian isolate made in 2000 roots the M1 Chinese strain (isolated in 1964) which, in turn, roots the Mongolian isolate, also made in 2000. These data suggest that GETV has been evolving slowly (possibly through slow circulation of the virus) and recent years account for the faster evolution of the more recent Chinese isolates. We have calculated the various shape parameters for the available sets of GETV E1, E2 and capsid sequences. Shape parameter values were between 0.21 and 0.23. These low values of the shape parameter are an indication that most of the sites are evolving slowly, but that there are evolutionary hot-spots in these sequences that are driving the evolution of these sequences of the genome.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Differences in antigenicity between GETV and SAGV were only identified by a complement fixation (CF) test, while virus neutralization and haemagglutination assays could not distinguish GETV from SAGV (Kono et al., 1980). Previous sequence analyses showed that GETV and SAGV should be classified as members of the same species within the genus Alphavirus (Shirako & Yamaguchi, 2000; Attoui et al., 2007). In this study, we analysed the sequences of ten GETV strains that were isolated in China during investigations for arboviruses between 1964 and 2006. Seven strains were isolated during the current study in regions within the range of latitude 1 ° (Hainan) and 4 °(Hebei and Ganshu) N and longitude 9 ° (Ganshu and Yunnan) and 12 ° (Shanghai) E. These isolations showed that GETV is distributed widely in China.
Non-plaque-purified virus material was used for the sequencing strategies, which is based on that published by Shirako & Yamaguchi (2000) for the sequence determination of SAGV. The PCR products were not cloned but rather sequenced directly, in order to avoid sequence variations that might result from the presence of quasispecies.
Our analysis of the full-length sequences of the Chinese GETV isolates, South Korean GETV isolate and SAGV (Japanese isolate) showed that these viruses exhibit high identity, therefore confirming previous findings about the relationship of GETV and SAGV. Up to 5.5 % amino acid sequence divergence was calculated between the original Malaysian isolate of GETV and the Chinese isolates.
The E2 protein is responsible for eliciting virus-specific antibodies (Griffin, 2001). We found 17 variable sites among the 14 GETV strains from China, Malaysia, Russia, Mongolia and South Korea (Supplementary Fig. S2). Our results showed that some amino acid changes in E2 are specific to isolates from a particular geographical location. By contrast, the number of variable sites between the E1 proteins of the various Chinese GETV isolates is quite low (only four sites among M1, HB0234 and YN0540), which reflects sequence conservation in the E1 of GETV.
In some arthropod-borne viruses, the nucleocapsid is the antigen responsible for reaction with CF antibodies (Casals & Whitman, 1961; Bose & Sagik, 1970). A CF assay distinguished between SAGV and GETV, while haemagglutination and seroneutralization did not (Kono et al., 1980). Wekesa et al. (2001) analysed the capsid gene of GETV, SAGV isolates in Japan and the Malaysian GETV (M2021) and found that the difference between GETV and SAGV was located at amino acid position 100 (Pro to Leu change). We found that this particular change was also present between the Chinese isolates and SAGV. All of the Chinese isolates have the same amino acid difference (Pro to Leu) from SAGV.
These results show the particularity of the Malaysian isolate and the close relationship between the Chinese GETV isolates and SAGV. In agreement with these results and previous findings (Powers et al., 2001; Attoui et al., 2007); the latter virus should therefore be identified as a member of the species Getah virus and not as a member of the species Ross River virus (according to the current classification; Weaver et al., 2005).
Read-through of leaky opal (TGA) stop codons found upstream of the nsP3/nsP4 cleavage site in most alphaviruses generates the non-structural polyprotein nsP1234 (Kim et al., 2004; Lavergne et al., 2006). The read-through efficiency in alphaviruses was estimated to be around 5–20 % (Strauss & Strauss, 1994), and a TGA to TGG mutation is supposed to increase the expression of nsP1234, which might relate to the efficiency of virus replication. However, in both SFV and SINV (Shirako & Strauss, 1994; Kim et al., 2004), this has not been the case, because free nsP4 was found to be degraded rapidly by the N-end-rule pathway (Takkinen et al., 1991; Ferreira et al., 2003).
Like a few other alphaviruses, strain HB0234 did not possess a stop codon between nsp3 and nsp4, but rather this was replaced by a sense codon. This suggests that strain HB0234 would express the non-structural polyprotein nsP1234 without being dependent on read-through. Our results are in agreement with the previous finding concerning SFV and SINV, as we did not observe any significant differences in proliferation between HB0234 and those Chinese GETV strains that possess the leaky stop codon upstream of the nsP3/nsP4 cleavage site (data not shown).
The 19 nt CSE of the 3' UTR is identical in the various GETV isolates, which suggests an important role of the 3' UTR in viral replication in the host (Hajjou et al., 1996). Mutations were previously reported in the 3' UTR among different alphaviruses, including high rates of mutations in RRV and Venezuelan equine encephalitis virus (Pfeffer et al., 1998). We found a poly(A) insertion in the 3' UTR which is located at the end of the second and/or the third RSE in GETV. The number of poly(A) residues in the insertion that we identified in strain M1 was different from that reported by Zhao et al. (2000) (four adenines; GenBank accession number EF011023
[GenBank]
), which suggest that this might be related to the passage history of the virus.
Several arbovirus investigations were conducted between the 1970s and 1990s in provinces of China (Zhang et al., 1989; Chen & Tao, 1996; Liang et al., 2000; Huang et al., 2001; Lv et al., 2001; Wang & Liang., 2003). None of these investigations reported any isolation of GETV. It has only been since 2000 that GETV strains have been isolated from wild-caught mosquitoes.
The area of GETV dissemination in Russia and Mongolia includes steppe, mixed forest, northern taiga and forest-tundra zones, reaching the tundra zone in the north (L'vov et al., 2000). GETV is the only alphavirus that occurs under such severe climatic conditions in Russia (L'vov et al., 2000). A plausible explanation for the position of the Russian and Mongolian isolates in the various trees would be that these particular strains have not been circulating extensively (and hence have not accumulated as many mutations as the more recent Chinese isolates), given the extreme climatic conditions in the regions where they have been isolated. Various mosquitoes and birds have been found to be infected by GETV (Takashima & Hashimoto, 1985; Kono, 1988) and the virus is supposed not to induce persistent infection in mammals (Wekesa et al., 2001; Chang et al., 2006). Persistent infection in naturally infected mammals as a possible mechanism of overwintering would be ideal to explain the clustering of the Russian and Mongolian isolates of GETV (made in 2000) nearer to the roots than to the exterior branches of the various phylogenetic trees (as is the case of the recent Chinese isolates, made around similar dates).
Finally, the expansion of GETV in China is a recent phenomenon. Beside the original M1 strain that was isolated in 1964 (almost 40 years before the most recent isolations), previous investigation in Gansu, Xinjiang, Yunnan and Hebei provinces over 10 years ago failed to identify any GETV in China (Zhang et al., 1989; Chen & Tao, 1996; Liang et al., 2000; Huang et al., 2001; Lv et al., 2001; Wang & Liang, 2003). This should prompt further investigations as a follow up for the dissemination of this virus in China. The recent isolations of 15 strains of GETV in Russia and Mongolia (L'vov et al., 2000) and the widespread distribution of this virus in China are indications of a change in the world distribution of the re-emerging GETV. This change and the isolation of GETV from various mosquito species of the genera Culex, Aedes and Armigeres identify this virus as a pathogen that is an increasing threat to animal health, particularly horses and pigs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 21 November 2007;
accepted 21 February 2008.
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