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Journal of Clinical Microbiology, October 2004, p. 4610-4614, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4610-4614.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evidence of an Antigenic Shift among Palyam Serogroup Orbiviruses

Seiichi Ohashi,^1* Youichi Matsumori,² Tohru Yanase,¹ Makoto Yamakawa,¹ Tomoko Kato,¹ and Tomoyuki Tsuda¹

Laboratory of Clinical Virology, Kyushu Research Station, National Institute of Animal Health, Chuzan, Kagoshima,¹ Central Livestock Hygiene Service Center, Kaizu-machi, Isahaya, Nagasaki, Japan²

Received 11 April 2004/ Returned for modification 19 May 2004/ Accepted 8 June 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Japanese isolates of Palyam serogroup viruses isolated from 1985 to 2001 were investigated for the genome sequence of segments 2 and 7 and were phylogenetically analyzed in comparison with Australian and African isolates of the same serogroup. The nucleotide sequences of segment 7 were highly conserved within Japanese isolates (95.1 to 100%) and between Japanese and Taiwanese isolates (96.0 to 100%), whereas the identities between Japanese and Taiwanese isolates and Australian and African isolates were fairly conserved (84.2 to 92.0%). Phylogenetic analysis based on segment 7 revealed three clusters according to geographical origin. As a result of the nucleotide sequence analysis of segment 2, which encodes a serotype-specific antigen, Japanese isolates were classified into two groups by genome length and nucleotide identities. Four of the nine Japanese isolates were categorized into the same group as prototype strain K-47 of the Chuzan virus, and the remaining isolates were categorized into the same group as the D'Aguilar virus and Nyabira virus. Phylogenetic analysis based on segment 2 revealed two clusters, the cluster containing Chuzan virus and the cluster containing the D'Aguilar and Nyabira viruses. To examine the antigenic relationship among viruses categorized in different clusters, we conducted a cross-neutralization test. KSB-29/E/01, isolated in 2001 in Japan, was neutralized by antiserum not only to strain B8112 of D'Aguilar virus but also to Chuzan virus. These results indicated that genetically and antigenically unique characteristics of KSB-29/E/01 were attributed to genetic reassortment of segment 2 between Chuzan virus and D'Aguilar virus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Palyam serogroup virus of the genus Orbivirus in the family Reoviridae is known as an arthropod-borne virus and is distributed in many countries of the African, Australian, and Asian continents (7, 8, 18). The D'Aguilar, CSIRO Village, and Marrakai viruses were isolated from Culicoides oxystoma in Australia in the 1970s (1, 15). Nyabira virus (NYAV) was isolated from an aborted fetus in Zimbabwe in 1973 (16). Although 11 serotypes have been identified in the serogroup by a cross-neutralization test, the pathogenesis of these viruses was not well understood. Chuzan virus (CHUV) was first described as a causative agent of bovine disease characterized by congenital abnormalities with hydranencephaly and cerebellar hypoplasia syndrome in Japan at the outbreak of the disease in 1985-1986 (2, 3, 10, 11). The virus was isolated from blood of sentinel cattle and Culicoides biting midges at the summer season prior to the disease outbreak and was reported as a new serotype distinct from other viruses by a cross-neutralization test (8, 9); however, it was reported later that CHUV is cross-reactive with Kasba virus, which was isolated in India in 1957 (5). A disease similar to that caused by CHUV occurred sporadically in the southern part of Japan in 1997 and 2001 to 2002, while the virus was isolated from the blood samples of sentinel cattle and Culicoides biting midges during the period from 1985 to 2001. The isolates in 2001 seemed to be related to the disease by seroprevalence in sentinel cattle in the epidemic area and the presence of antibodies in the sera of affected calves and their dam. The development of the inactivated CHUV vaccine has made it clear that the characterization of the isolates is essential for the development of preventive measures, including an assessment of vaccine efficacy.

The orbivirus genome contains 10 double-stranded RNA (dsRNA) segments in a double-layered capsid. These dsRNA segments encode seven structural viral proteins (VP1 to VP7) and four nonstructural proteins (NS1 to NS3 and NS3A) (20). The outer layer is composed of the two major proteins, VP2 and VP5. The VP2 protein encoded by RNA segment 2 is a major neutralizing antigen and has a serotype-specific determinant (4). The VP7 protein encoded by RNA segment 7 is incorporated in the viral inner layer and is a serogroup-specific antigen (19). The sequence analysis of these two segments may reveal the antigenic variation and evolutionary change of the virus.

In this study, we examined the Japanese isolates of Palyam serogroup viruses to determine the genome sequences of segments 2 and 7 and to compare them phylogenetically with Australian and African isolates of the Palyam viruses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viruses and cells. The Palyam serogroup viruses used in this study are shown in Table 1. These viruses were propagated on established baby hamster kidney (BHK-21) cells. The BHK-21 cells were maintained in Eagle's minimum essential medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 0.295% tryptose phosphate broth (Difco Laboratories, Detroit, Mich.), 0.15% sodium bicarbonate, 2 mM L-glutamine, and 5% calf serum.

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TABLE 1. Details of Palyam serogroup viruses used in this study

Preparation of dsRNAs. Viral dsRNAs were extracted from the infected BHK-21 cells as described previously (14), and the individual RNA segments were separated on a 0.8% agarose gel. The separated viral dsRNAs were excised from the agarose gel and purified with the RNaid kit (Bio 101, La Jolla, Calif.) in accordance with the manufacturer's instructions.

RT-PCR of segment 7 and sequencing. Total RNA was extracted from virus culture fluid using High Pure Viral RNA kit (Roche Diagnostics, Indianapolis, Ind.). A portion of segment 7, corresponding to nucleotide positions 258 to 523 of CHUV segment 7, was amplified via reverse transcription-PCR (RT-PCR) with primers S7-1 (5'-ATCTCAAACCTATAGACCATC-3') and S7-2 (5'-GAACTATTGTTCCTTGCTGGA-3'). RT-PCR was conducted as described previously (21). PCR products were purified with the QIAquick PCR Purification kit (QIAGEN, Valencia, Calif.), and direct sequencing was carried out in both directions with the PCR primers using the ABI PRISM Big Dye Terminator Cycle Sequencing kit and the ABI PRISM 3100 automated sequencer (Applied Biosystems).

Molecular cloning of segment 2 and sequencing. RT-PCR was performed with the Titan One Tube RT-PCR kit (Roche Diagnostics) containing a proofreading polymerase. The RT-PCR mixture consisted of 0.2 mM (each) deoxynucleoside triphosphates, 5 mM dithiothreitol, 1x RT-PCR buffer, 1.5 mM MgCl₂, and an enzyme mixture in a total reaction volume of 50 µl. Briefly, the purified viral RNA segment 2 and both strand primers ChuzanS211F (5'-CGCAATGGATGAATTTTCGCTTTGT-3'; nucleotides 11 to 35) and ChuzanS2R (5'-GTAAGTGTGTCCCGCAACACG-3'; nucleotides 3035 to 3055) for CHUV as well as both strand primers PAL2F (5'-GAATTCGTTAAAATTCCGCAATGGATG-3') and PAL2R (5'-GTAAGTGTGTCCCGCAACACGTAGT-3') for D'Aguilar virus (DAGV) were incubated at 94°C for 5 min and were quenched on ice prior to the addition of the reaction mixture. The RT was conducted at 48°C for 60 min. PCR amplification was achieved with initial denaturation (94°C for 5 min) followed by 35 cycles of denaturation (95°C, 30 s), annealing (30 s at 55°C or 47°C, depending on the primers), and elongation (68°C, 2 min), with a final extension at 68°C for 7 min. The purified PCR products were ligated into pGEM-T Easy Vector systems (Promega, Madison, Wis.). The recombinant plasmids were transfected into competent Escherichia coli JM 109 cells. The positive clones were confirmed by PCR using M13 forward and reverse primers. A plasmid containing the viral genome was obtained with a commercial plasmid extraction kit (Promega) according to the manufacturer's protocol. The 5'- and 3'-terminal ends of DAGV segment 2 nucleotide sequences were determined using the 5' RACE (rapid amplification of cDNA ends) kit (Invitrogen Corp., Carlsbad, Calif.). Briefly, first-strand cDNA was made using SuperScript II RT (Invitrogen) and the primer DAGVSP1 (5'-CGTCATACATTCCCATCTCC-3') for the 5' end and the primer DAGSP1/3Term (5'-AGCTGGATAGGGGCGAATTG-3') for the 3' end. The reaction was carried out for 60 min at 42°C followed by 15 min at 70°C. Purified cDNA was tailed with dCTP and the terminal deoxynucleotidyltransferase (TdT), and then it was amplified by PCR with primers DAGVSP2R (5'-GAATAGGCTTGGGATTATGG-3') and DAGSP2/3TermF (5'-GATGATTCCTATCAAGAGTCC-3'), respectively, and the 5' RACE Abridged Anchor Primer (Invitrogen) under the conditions recommended by the manufacturer. Amplified products were cloned into the pGEM-T Easy Vector. Three individual clones were sequenced in both directions with the Thermo Sequenase Cycle Sequencing kit (U.S. Biochemicals, Cleveland, Ohio) on a model 4200 automated DNA sequencer (Li-Cor Inc., Lincoln, Nebr.). Sequences were determined using universal primers (M13 Forward, 5'-CACGACGTTGTAAAACGAC-3'; M13 Reverse, 5'-GGATAACAATTTCACAGG-3') and the segment 2-specific primers ChuzanS2479F (5'-AGTGGATCCAACGTATAGTG-3', nucleotides 479 to 498), ChuzanS22560R (5'-TCCGTTAGAAGTGTTTTCTCC-3', nucleotides 2541 to 2560), DAGVSP2 (5'-GAATAGGCTTGGGATTATGG-3', nucleotides 1090 to 2010), DAGVS23TERM (5'-CATTTGATGAGACAGAGTGG-3', nucleotides 1686 to 1705), DAGVS2n3Term (5'-CTTCCTCTACCTTAATTCTG-3', nucleotides 1738 to 1757), and DAGVSP2/3Term (5'-GGACTCTTGATAGGAATCATC-3', nucleotides 2686 to 2706).

Sequence and phylogenetic analysis. Nucleotide sequences were assembled and analyzed with the GENETYX software (Software Development, Tokyo, Japan). The nucleotide sequences of the open reading frame (ORF) region were aligned with the CLUSTAL W program (17). The aligned sequences were used to construct the phylogenetic trees by the neighbor-joining method (13), and the distances were corrected by using Kimura's two-parameter method (6). The reliability of the branching orders was estimated by bootstrapping (1,000 samples). The phylogenetic trees were drawn with TreeView software (12).

Production of bovine antisera. Bovine immune sera against isolate 31 of CHUV and strain B8112 of DAGV were obtained from bovine infected with each virus. Each virus was inoculated intravenously, and sera were collected 4 to 6 weeks after inoculation. Convalescent bovine serum was collected from sentinel cattle naturally infected with isolate KSB-29/E/01.

Cross-neutralization test. Antisera were used to investigate the antigenic relationship among CHUV, DAGV, and the most recent isolate, KSB-29/E/01, by a neutralization test. The neutralization test was performed on 96-well microtiter plates on established hamster lung (HmLu-1) cells. Serially twofold-diluted serum was mixed with an equal volume of medium containing 100 50% tissue culture infectious doses of virus. The mixtures were incubated at 37°C for 60 min, and then HmLu-1 cells suspended in serum-free medium (GIT; Wako Pure Chemical Industries, Ltd., Osaka, Japan) were added to each well. After incubation at 37°C for 7 days in a humidified 5% CO₂ atmosphere, the antibody titer was expressed as a reciprocal of the highest dilution of serum that completely inhibited the cytopathic effect.

Nucleotide sequence accession numbers. The sequence data determined in this study were deposited in the DDBJ/EMBL/GenBank database under the accession numbers AB177625 to AB177639, and previously reported nucleotide sequences of CHUV, DAGV, and Australian and Zimbabwean Palyam serogroup virus segments 2 and 7 were retrieved from the database. The GenBank accession numbers are shown in Table 1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Partial sequence and phylogenetic analysis of segment 7. To determine the relationship among the Japanese isolates and other Palyam serogroup viruses, we amplified a portion of segment 7 corresponding to nucleotide positions 258 to 523 by RT-PCR. The PCR product was then sequenced for a portion of 224 bases, except for the primer region, and was compared to the corresponding sequences of Palyam serogroup viruses. Nucleotide identities of segment 7 from 19 isolates or strains ranged from 84.4 to 100%. The identities among the nine Japanese isolates, K-47, 31, KY-115, ON 91-5, ON-1/E/97, ON-3/E/98, ON-1/E/00, NS-1/P/01, and KSB-29/E/01, ranged from 95.1 to 100%. A high degree of sequence identity, from 96.0 to 100%, was obtained between Japanese isolates and Taiwanese isolates CY-6 and CY-8. The Japanese and Taiwanese isolates showed relatively lower identities (84.2 to 92.0%) to Australian isolates B8112, CSIRO58, CSIRO11, CSIRO82, and DPP66 and to African isolates 792/73, 1726/76, and 1070/78.

The deduced amino acid sequences of these 19 isolates showed 100% identity, except for African isolate 1726/76, which showed 98.6% identity to other viruses.

In phylogenetic analysis of the nucleotide sequence of segment 7, these 19 viruses were divided into three clusters, which consisted of Japanese and Taiwanese isolates, Australian isolates, and African isolates, respectively (Fig. 1).

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FIG. 1. Phylogenetic tree based on the nucleotide sequence of 224 bp of segment 7 of the Palyam serogroup viruses from Japan, Taiwan, Australia, and Zimbabwe. Segment 7 of North American Bluetongue virus serotype 17 was used as the outgroup to root the tree. Numbers above the internal nodes indicate the bootstrap values obtained with 1,000 replications.

Sequence and phylogenetic analysis of the full length of segment 2. Because RNA segment 2 encodes the VP2 protein, which is responsible for the serotype-specific antigen, it seems that the full sequence of the genome reflects the antigenic variation of the virus. The cDNA of viral RNA segment 2 was cloned into a plasmid vector and was sequenced. The full-length sequence data of isolates were compared to those of Palyam serogroup viruses. The viruses were divided into two groups in lengths coinciding with that of segment 2, that is, with 3,055 nucleotides and 3,022 nucleotides. Isolates 31, FO88-2, FO90-8, and ON-3/E/98 each have 3,055 nucleotides, including an ORF of 3,006 nucleotides flanked by a 5' noncoding region of 14 bases, a presumed initiation AUG codon, and a 3' noncoding region of 35 bases. The length of the genome of these viruses was the same as that of prototype strain K-47 of CHUV. Isolates KY-115, ON 91-5, ON-1/E/00, and KSB-29/E/01 each have 3,022 nucleotides, including an ORF of 2,976 nucleotides flanked by the 5' noncoding region of 16 bases and the 3' noncoding region of 30 bases. The same length of genome was cloned from the prototype strain B8112 of DAGV and strain 792/73 of NYAV.

A comparison of the nucleotide sequences among the Palyam serogroup viruses (Table 2) isolates 31, FO88-2, FO90-8, and ON-3/E/98 showed 96.2 to 99.8% identity to each other and 96.4 to 99.9% identity to strain K-47 of CHUV, whereas the identities of these viruses with Japanese isolates KY-115, ON 91-5, ON-1/E/00, and KSB-29/E/01 ranged from 52.5 to 53.0%. A low percentage of identity was seen in the comparison of these viruses with the prototype strain B8112 of DAGV, 52.4 to 52.7%, and strain 792/76 of NYAV, 52.2 to 52.9%. In contrast, the identities of isolates KY-115, ON 91-5, ON-1/E/00, and KSB-29/E/01 ranged from 95.0 and 99.5% to each other and from 91.9 to 92.9% to the prototype strain B8112 of DAGV and strain 792/76 of NYAV.

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TABLE 2. Percentage identities of segment 2 of nucleotide and deduced amino acid sequences among Palyam serogroup viruses

The deduced amino acid sequences of VP2 of isolates 31, FO88-2, FO90-8, and ON-3/E/98 showed identities from 97.0 to 99.8% to each other and to K-47 of CHUV, whereas there was less identity to other Japanese isolates as well as to B8112 and 792/76. On the other hand, the deduced amino acid sequences of VP2 of the isolates KY-115, ON 91-5, ON-1/E/00, and KSB-29/E/01 showed high similarity to each other, 94.0 to 98.7%, and to B8112 and 792/76 (Table 2).

The phylogenetic analysis of the ORF of VP2 revealed that these viruses could be divided into two major clusters (Fig. 2). The cluster typified by prototype strain K-47 of CHUV contained isolates 31, FO88-2, FO90-8, and ON-3/E/98. The other isolates, KY-115, ON 91-5, ON-1/E/00, and KSB-29/E/01, were included in a cluster together with DAGV strain B8112 and NYAV strain 792/76, while the cluster was subdivided into three minor groups of Japanese isolates, DAGV, and NYAV, respectively.

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FIG. 2. Phylogenetic tree based on the nucleotide sequence of the segment 2 ORF regions of the Palyam serogroup viruses from Japan, Australia, and Zimbabwe. Segment 2 of North American Bluetongue virus serotype 11 was used as the outgroup to root the tree. Numbers above the internal nodes indicate the bootstrap values obtained with 1,000 replications.

Cross-neutralization test. To define the antigenic relationship of viruses classified in different clusters by phylogenetic analysis of segment 2, we performed a cross-neutralization test for three viruses, 31, KSB-29/E/01, and B8112, of which isolate 31 was antigenically identical to strain K-47 of CHUV and strain B8112 was the prototype strain of DAGV (Table 3). The antiserum to isolate 31 neutralized homologous virus at a titer of 2,048; however, the antiserum titers to B8112 and KSB-29/E/01 were lower, by 32- to 256-fold. On the contrary, antisera to B8112 and KSB-29/E/01 neutralized homologous viruses and each other at a titer of 512, while the neutralizing titer of these sera to isolate 31 was 16.

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TABLE 3. Antigenic comparison among CHUV and DAGV strains by cross-neutralization test

Top Abstract Introduction Materials and Methods Results Discussion References

 
CHUV was first demonstrated to be the causative agent of bovine disease by Palyam serogroup viruses. Vital infection of a fetus in the uterus results in hydranencephaly and cerebellar hypoplasia syndrome of the newborn calf. The disease was reported in the southern part of Japan as an epidemic in 1985-1986 and as a sporadic outbreak in 1997 and 2001-2002 (2, 3). In the most recent outbreak, in 2002, the Palyam serogroup virus isolated from sentinel cattle during the previous year seemed to be related to the outbreak. However, it was suggested that the virus was antigenically different from existing isolates of CHUV, because lower antibody titers to CHUV were detected in affected calves in the later outbreak compared to those in the previous outbreak. The phylogenetic analysis of the genome encoding group-specific and serotype-specific antigens was conducted to define the relationship among the viruses isolated during the period from 1985 to 2001 in Japan and with prototype strains of Palyam serogroup viruses.

The overall identities of nucleotide sequences of segment 7 among Palyam serogroup viruses were greater than 85%, and the deduced amino acid sequence was very similar or identical in all the viruses examined. The phylogenetic analysis of viral RNA segment 7 revealed that the viruses were divided in different clusters according to the region where the viruses were isolated. The Zimbabwean isolates, 792/73, 1726/76, and 1070/78, which were classified to distinct serotypes Nyabira, Gweru, and Marondera, respectively, were categorized in the same cluster. Similarly, the Australian isolates, B8812 of DAGV, CSIRO 11 of CSIRO Village virus, CSIRO 82 of Marrakai virus, and CSIRO 58 of Bunyip Creek virus, were included in the same cluster together with another isolate, DPP 66. All the viruses isolated in Japan were categorized in the same cluster with two Taiwanese isolates. Interestingly, the nucleotide sequences of Japanese isolates taken over 15 years were highly conserved, more than 95%, and the deduced amino acid sequences were identical in all the isolates. These results suggested that the nucleotide sequence of segment 7 of Palyam serogroup viruses were conserved among the viruses in the epidemic area even if the serotypes were different, and they exhibited the topotype of viruses. Furthermore, Japanese and Taiwanese isolates of Palyam serogroup viruses seemed to be derived from a common genetic pool.

Segment 2 of viral RNA encodes VP2 viral protein, which is a serotype-specific antigen (4). Therefore, cDNA of the full genome of segment 2 was cloned and sequenced to investigate its genetic variation among the Japanese isolates and its genetic relationship with other Palyam group viruses. In a comparison of nucleotide identities among the isolates, nine isolates were divided into two distinct groups by the length of genome and by the nucleotide identities. Phylogenetic analysis revealed that the Japanese isolates were divided into two clusters, of which four isolates and prototype strain K-47 of CHUV were contained in one cluster and four isolates were contained in another cluster with DAGV and NYAV. The identities of deduced amino acid sequences between the two groups of viruses differed by more than 59%, while identity was greater than 94% among the viruses in the same group.

The differences in amino acid sequence between the two groups indicated that the viruses might exhibit a new antigenic character in a serum neutralization test. The recent isolate KSB-29/E/01 was selected to compare its antigenicity with those of CHUV and DAGV by a cross-neutralization test. The results of the cross-neutralization test demonstrated that KSB-29/E/01 was antigenically close to DAGV. However, the virus was neutralized by antiserum to CHUV. This antigenic change of recent isolates of Palyam serogroup viruses might result from a reassortment event in segment 2 between viruses belonging to CHUV and DAGV. Furthermore, the fact that the viruses were isolated in the southern part of Japan indicates that the viruses might invade Japan from the area where both virus groups are epidemic.

In the period from the first isolation of CHUV in 1985 until 2001, a total of 10 Palyam serogroup viruses were isolated in Japan. During this time, the viruses had not been isolated continuously for 2 years until 2001, and isolation and seroconversion of Palyam serogroup viruses had not been observed between 1992 and 1996. These viruses are regarded as exotic in Japan. It is possible that several different serotypes of Palyam serogroup viruses have been cocirculated around East Asia and that some of these viruses have repeatedly made incursions into Japan. The coexistence of several viruses in an area might generate reassortant viruses. KSB-29/E/01, which showed unique antigenicity and was considered to be reassortant, might have emerged under such conditions. Although segment 2 of KSB-29/E/01 had high identity with DAGV and less identity with CHUV, a partial cross-neutralization reaction was observed between KSB-29/E/01 and CHUV. Segment 6, encoding VP5, may be involved in the unique antigenicity of KSB-29/E/01, because segment 6 as well as segment 2 is correlated with serotype specificity of orbiviruses.

In conclusion, our data indicated that an antigenic shift occurred in Palyam serogroup orbiviruses as a result of reassortment of segment 2. Genetic reassortment appears to have been the main cause of genetic and antigenic diversity of Palyam serogroup orbiviruses. The reassortment event may have contributed to the alteration of viral pathogenicity as well as antigenicity. It is important to understand genetic and antigenic variation of the viruses circulating in Japan and neighboring areas of East Asia and to develop molecular diagnostic tools and a more effective vaccine for the monitoring and prevention of Palyam serogroup orbivirus infection.

 
This work was supported by grants received from the Ministry of Agriculture, Forestry, and Fishery of Japan.

 
* Corresponding author. Mailing address: Laboratory of Diagnosis, Department of Exotic Diseases, National Institute of Animal Health, 6-20-1, Josui-honcho, Kodaira, Tokyo, 187-0022, Japan. Phone: 81-42-321-1441. Fax: 81-42-325-5122. E-mail: ohashis{at}affrc.go.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, October 2004, p. 4610-4614, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4610-4614.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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