Antigenic and molecular characterization of hantavirus isolates from China

Virus Virus Research 31 (1994)219-233

Antigenic and molecular characterization isolates from China

Research

of hantavirus

Mifang Liang a, Dexin Li a, Shu-Yuan Xiao ap1,Changshou Hang a, Cynthia A. Rossi b, Connie S. Schmaljohn a,* ’ Virology Division, United States Army Medical Research Institute of Infectious Dbeases, Fort Detrick, Frederick, MD 21702-5011, USA b Applied Research Division, United States Army Medical Research Institute of Infectious Diseases, Fort Derrick, Frederick, MD 21702-5011, USA

(Received 9 September 1993; revised 25 October 1993; accepted 1 November 1993)

Abstract

Hemorrhagic fever with renal syndrome (HFRS) is caused by certain viruses in the genus family Bunyauirihe, and is a major public health problem in China. By using molecular and serological tests, we characterized 15 hantaviruses isolated either from patients with HFRS or from rodents captured in endemic areas of China. By cross plaque-reduction neutralization tests performed with rabbit immune sera, we identified two serologically distinct groups of viruses, comprised of those related to Hantaan virus, and those related to Seoul virus. To study the genetic relationships among these viruses, we amplified a 330 base pair region of the medium (M) genome segment of each isolate by reverse transcription and polymerase chain reaction (PCR) and compared the nucleotide sequences to those of other, well-characterized hantaviruses. In addition, we PCR-amplified and analyzed the entire coding region of the small (S) genome segment of each isolate by restriction enzyme digestion with a battery of enzymes. The results of our genetic analyses of both the M and S segments of these isolates confirmed our serological data, indicating that Hantaan and Seoul viruses co-circulate in endemic disease regions of China. We constructed a phylogenetic tree based on multiple alignment of the partial M segment sequences. The resulting dendrogram distinguished three genetic subtypes of Hantaan viruses and one type of Seoul virus. Huntuuirzq

author. Fax: + 1 (301) 619-2439. ’ Present address: Laboratory of Infectious Diseases, NIAID, National Institute of Health, Bethesda, MD 20892, USA. * Corresponding

0168-1702/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1702(93)E0089-G

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Key words: drome

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31 (1994) 219-233

Hantaan virus; Hantavirus; Bunyaviridae; Hemorrhagic fever with renal syn-

1. Introduction

Hemorrhagic fever with renal syndrome (HFRS) is an infectious human disease that is distributed widely throughout the world. It is clinically characterized by high fever, various hemorrhagic manifestations and transient renal dysfunction. The causative agents of HFRS are members of the Huntuvirus genus, family Bunyaviridae. Four serologically distinct groups of hantaviruses are represented by Hantaan (HTN), Seoul (SEO), Puumala (PUU) and Prospect Hill (PH) viruses (Lee et al., 1985; Schmaljohn et al., 1985; Sugiyama et al., 1987; Dantas et al., 1987). Major reservoir hosts for these viruses, respectively, are Apodernus (mouse), Rattus (rat), Chethrionomys (bank vole) and Microtus (meadow vole) (Lee et al., 1978, 1982; Brummer-Korvenkontio et al., 1980; Yanagihara et al., 1984). HTN, SE0 and PUU viruses are known to cause HFRS, but no disease has yet been associated with PH virus. HTN-like viruses generally cause the most severe form of HFRS, while PUU-like viruses cause the mildest disease. However, HTN, SE0 and PUU viruses have all been associated with fatal cases of HFRS (Lee, et al. 1990). M segment gene sequence homologies among the prototype strains of viruses have been determined to be approximately 70% for HTN vs SEO, 59% for HTN vs PUU and 63% for HTN vs PH; and for the S segments, 72%, 60%, and 62%, respectively (Antic, et al., 1992). In addition to these viruses, at least three other genetically distinct hantaviruses (Xiao et al., 19931, have also been described: Thailand, Thottapalayam and Dobrava, isolated, respectively, from Bandicota in Thailand (Elwell et al., 1985), Suncus in India (Carey et al., 1971) and Ap0demu.s in Slovenia (Avsic-Zupanc et al., 1992). Thailand and Thottapalayam viruses have not yet been associated with human disease, but Dobrava virus is suspected to be a cause of severe HFRS in the Balkans. Another group of genetically distinct hantaviruses has recently been detected in the United States, and certain of these viruses are responsible for a very severe pneumonia-like illness (Nichol, et al., 1993). The extent of this disease problem in the U.S. is currently unknown. HFRS is endemic in China where approximately 70 - to 130,000 cases occur annually throughout 26 provinces (Lee et al., 1990). In recent years, numerous hantavirus isolates have been obtained from various hosts, including patients with HFRS and a variety of rodents captured in many endemic and non-endemic areas of China (Song, 1991; Chen et al., 1985; Xing et al., 1987; Liang et al., 1992). Preliminary serological characterization of these isolates indicated that both HTN and SE0 viruses may cause HFRS in China. To examine the etiology of viruses causing HFRS in China more closely, we examined the genetic and serological characteristics of 15 hantavirus isolates collected from humans or rodents in various geographic locals. We prepared immune sera to each virus, and used those sera to perform cross plaque-reduction neutralization assays. We examined the

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genetic relationships of the viruses by analyzing the nucleotide sequence of a portion of the M genome segment of each isolate, and by restriction enzyme digestion of the entire coding region of their S genome segments. Our results indicate that at least three subtypes of HTN viruses and one type of SE0 virus are present in China.

2. Material and methods 2.1. Wuses, antiserum and cell culture The origins of the Chinese hantavirus isolates are described in Table 1. Strain 486 was isolated from the lung tissue of an Apodemus mouse in Guizhou province. Strains 84FLi, H3, H5, B6-59, Luyao, Lwcu, HB55, JB2 and JB3 were isolated from

Table 1 Origin of hantavirus isolates used in this study Virus

Source

Country (province)

References

A. A. A. A.

Korea Yugoslavia China (Jiangsu) China (Guizhou) China (Shandong) China (Shandong) China (Shandong) China (Shanxi) China (Anhui) China (Hubei) China (Helongjiang)

(Lee et al., 1978) (Gligic et al., 1989) (Song et al., 1982) (“1 f”)

Korea Egypt Brazil U.S.A. Thailand China (Henan) China (Hubei) China (Jiangxi) China (Henan) China (Jiangsu) China (Jiangsu)

(Lee et al., 1983) (Lee, 1990) (LeDuc et al., 1985) (Childs et al., 1987) (Elwell et al., 1985) (Song et al., 1982) (Lee et al., 1980) (Liu et al., 1984)

C. glareolus

Finland Russia

(Schmaljohn et al., 1985) (Tkachenko et al., 1984)

M. petmylvanica

USA

(Lee et al., 1982)

HTN

HTN (76-118) Fojnica A9 486 B6.59 Luyao Luxu 84FLi Chen H3 H5 SE0 SE0 (HR 80-39) Egypt Brazil Baltimore Thailand 605 R22 Hubei-1 L99 HB55 JB2 JB3 PUU PUU (Sotkamo) CG18-20 PH PH (PHV-1)

agrarius jlauicollis agrarius agrarius

Human Human Human Human Human Human Human R. R. R. R. B. R.

norvegicus norvegicw norvegkus

norvegicus indica norvegicus

Human R. rosea

Human Human Human C. glareolus

a Song, G. and Hang, C.S., Unpublished data.

I:; (“1 (Ni et al., 1983) I:;

I:; (“1

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blood specimens of patients with HFRS in different endemic regions from 1985 1991 (Song, G. and Hang, C.S., unpublished data). The passage history of each virus prior to our studies follows and is listed as (VP#>, indicating number of passes in Vero E6 cells, or (SMP#), indicating number of passes in suckling mouse brain, or as a combination of these: 486 (VP14); 84FLi (VP9); H3 (VP3-SMP3-VPl); H5 (VP4-SMP3-VPl); B659 (VP7); Luyao (VP4-SMP3-VPl); Luxu (VP4-SMP3VPl); HB55 (SMPS-VP6); JB2 (VP3) and JB3 (VP3). The other isolates, are described in references listed in Table 1. All isolates were passed two additional times in Vero E6 cells in our laboratory. Strain L99 (SMP18-VP31 is currently being used to develop an inactivated, cell culture-derived vaccine for HFRS in China (Song et al., 1992). All viruses were propagated in Vero-E6 cells (ATCC, Cl008 CRL 1586) as previously described (Schmaljohn et al., 1983). Cell cultures were maintained in Eagle’s MEM supplemented with 5% fetal bovine serum (FBS) and 1% antibioticantimycotic (GIBCO BRL, Gaithersburg, MD). Hantavirus immune sera in experimentally infected rabbits or rats were prepared as described previously (Schmaljohn et al., 1985; Schmaljohn et al., 1988). Briefly, immune sera to each hantavirus (see Fig. 1) was generated by a single intramuscular inoculation of at least two New Zealand white rabbits or four adult Wistar rats with infectious, cell culture-propagated virus. Rabbit sera were tested at weekly intervals by ELISA for antibody response, and high-titer sera were collected and pooled. Rat sera were collected 28 days after infection and individual serum samples were screened by ELISA to determine the presence of hantavirus-specific antibody. 2.2. Cross plaque-reduction neutralization test (PRNT) PRNT was performed on Vero-E6 cell monolayers, in 6-well cell culture plates, according to methods previously described (Schmaljohn et al., 1985). Briefly, approximately 100 PFU of virus were incubated with two-fold serial dilutions of each immune serum for 1 h at 37°C before inoculation of 6-well plates. Cells were further incubated for 1 h at 37°C before adding an overlay of EMEM containing 0.6% agarose (SeaKern, Rockland ME), 5% FBS and antibiotics. After incubation for 8-12 days postinfection, a second overlay was applied, which was nearly identical to the first, with the addition of 5% neutral red. Neutralization titers were expressed as the reciprocal of the highest dilution of antibody resulting in greater than 80% reduction of approximately 100 plaques. 2.3. Preparation of viral RNA, cDNA synthesis, polymerase chain reaction (PCR) Viral RNA was extracted from hantavirus-infected Vero-E6 cells by using RNAzol B (Cinna/Biotec Lab, INC, TX). Briefly, Vero-E6 cells were infected with different Chinese hantavirus isolates for 8-12 days, after which the infected cells (approximately lo6 cells) were rinsed with cold diethyl pyrocarbonate (DEPC)-treated water, scraped from flasks and pelleted in a microfuge. The

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supernatants were aspirated and 1.5 ml of RNAzol B was added. Cell lysates were extracted with chloroform and centrifuged at 4°C. The aqueous phase was removed and mixed with an equal volume of isopropanol which then was precipitated at - 20°C and RNA was recovered by centrifugation in a microfuge. The RNA pellet was washed with 75% ethanol and resuspended in 50 ~1 DEPC-treated water. Genus-reactive forward and reverse oligonucleotide primers, HG2F and HG2R (forward at base 1970, and reverse at base 2334 with respect to HTN strain 76-118 M segment RNA), were used for cDNA synthesis and PCR amplification of a 365 bp fragment of the M segment (Xiao et al., 1992). The primers SF1 (5’-ATGGCAACTATGGAAGAA-3’) which is forward from the start codon with respect to HTN virus strain 76-118; and SHRl (S’TTAGAGTITGAAAGGCT CAGG-3’) or SSRl (S’TTATAATITCATAGGTTCCTC-3’1, which are reverse from the stop codons with respect to HTN or SE0 virus S segment RNA, respectively (Schmaljohn et al., 1986; Giebel et al., 19911, were used to amplify the coding region of the S segment of each isolate. cDNA was synthesized by using a BRL cDNA synthesis kit according to the manufacturer’s directions, but with the following modifications: The RNA sample (11 ~1) was mixed with primers and heated at 95°C 5 min, then was added to a 9 ~1 reaction mixture containing reverse transcriptase, DTT and reaction buffer and was incubated for 60 min at 42°C. The cDNA was amplified by using the Gene Amp kit (Perkin-Elmer Cetus Norwalk, CT) according to the manufacturer’s directions. Amplification was performed for 30 cycles, with each cycle consisting of melting at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 3 min. 2.4. Cloning, sequencing and computer analysis PCR products were either gel-purified by using Geneclean resin, (Bio 101 Inc., Gaithersburg, MD) or directly cloned into the pCR II vector according to the protocol of TA cloning kit (Invitrogen Corp., San Diego, CA). The ligations were used to transform competent Escherichia coli DHSa (GIBCO BRL), and at least two positive clones containing the insert of each isolate were used for sequence analysis. Nucleotide sequences were determined with the dideoxy chain termination method using a Sequenase version 2.0 sequence kit (U.S. Biochemical) with T7 promoter and Ml3 reverse primers (Invitrogen). Sequence data were entered into and edited in the MacVector program (IBI, New Haven, CT). Initial multiple sequence alignment was performed by using the GCG program (University of Wisconsin; Devereaux et al., 1984), and phylogenetic analysis was performed by using the PAUP 3.0 program (David L. Swofford, Illinois Natural History Survey, Champaign, IL). The flanking primer regions from each sequence were excluded from the comparison to eliminate potential artifacts caused by the consensus primer sequences. For comparison, existing sequence data were either obtained from GenBank DNA database or from our previous studies. The M segment sequences include: HTN virus, strain 76-118, (Schmaljohn et al., 19881, SE0 virus, strains HR80-39, and SR-11 (Arikawa et al., 1990; Antic et al.,

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19911, PUU virus, strain CG18-20 (Stohwasser et al., 1992); and PH virus, strain PHV-I (Parrington et al., 1991). 2.5. Restriction endonuclease digestion The PCR-amplified products representing the coding region of the S segment of each isolate were gel-purified, then recovered with Geneclean resin, and restriction patterns were obtained by individual digestion with 13 restriction endonucleases. Enzymes used were: AvaII, BcEI, BarnHI, DraI, H&II, HindIII, HaeIII, HpaI, MspI, NdeI, NciI, PstI and PuuI (GIBCO, BRL). Ten units of each enzyme, in the optimal buffer described by the manufacturer, were used to digest 1 pg of PCR product at 37°C for at least 1-2 h. The digested products were detected by electrophoresis in 2% agarose gels containing 0.05% ethidium bromide. The size of the digested fragments were estimated by comparing the position of each band with 1 Kb and a 100 bp DNA ladders (GIBCO, BRL).

3. Results 3.1. Comparative plaque-reduction neutralization test (PRiVT)

Cross-PRNT was performed with 15 Chinese hantavirus isolates from HFRS patients or rodents captured in various endemic areas of China by using sera from experimentally infected animals. To compare the isolates to other well-characterized hantaviruses, the immune sera were also tested for neutralizing activity with viruses representing each of the four recognized, serologically distinct groups of hantaviruses: HTN, SEO, PUU and PH. In addition, eight antisera to other hantaviruses from diverse geographic regions were reacted with the Chinese isolates. Our results indicate that both HTN and SE0 viruses cause HFRS in China (Fig. 1). From our data, it appears that both viruses extend over a broad geographic region and may co-circulate in many areas. We did not find any evidence for HFRS caused by PUU virus. 3.2. Genetic relationships based upon a 330 bp region of the M genome segment

We used a pair of universal primers flanking a region from nucleotides 1970 to 2334 of the M segment of HTN virus in reverse transcription and PCR to produce and amplify cDNAs of the M segment of each of the 15 Chinese isolates. We cloned the resultant 0.36 Kb fragments and determined their nucleotide sequences. For comparisons, we ignored the nucleotide sequences of the primer pairs. We found that all 9 of the Chinese isolates, which were determined to be HTN by PRNT, were also closely related to HTN virus by multiple alignment of their nucleotide sequences (Fig. 2A). Similarly, the 6 Chinese isolates, which were found to be SE0 by PRNT, aligned well with prototype SE0 virus sequences, but not with the HTN virus sequences (Fig. 2B). By examining the multiple alignments,

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we discovered greater variations in nucleotide sequences among the HTN isolates than among the SE0 isolates. This finding was also reflected in a phylogenetic tree that we constructed by pairwise comparison of the nucleotide sequences of each isolate (Fig. 3). Among the HTN-like isolates, three branches were apparent: one made up of two human isolates, H3 and H.5; a second comprised of two human isolates, Chen and 84FLi; and a third with three human isolates and two Apodemus isolates (Fig. 3). We determined the percent identities among all viruses by using the GCG GAP program (not shown) and from these estimated the average divergence between each branch. For the HTN viruses, we calculated average differences of 22.5%, 14.9% and 14.1%, respectively, for the H.5, Chen and A9 branches as compared to HTN virus, strain 76-118. Average differences of 21.9%, 21.2% and 17.1%, respectively, were calculated for the HS vs Chen branches, the H5 vs A9 brances and the Chen vs A9 branches. These values are proportional to the relative distance numbers listed above the branches in Fig. 3. Among the SE0 isolates, we observed far less divergence (average differences approximately 6%).

Fig. 2. Multiple alignment of the nucleotide sequences of a portion of the G2 coding region of the M (B), and those genome segment (330 bp) of HTN virus, strain 76-118 (A), or SE0 virus strain HR80-39 of related Chinese isolates. Identical nucleotides are marked with dots.

M. Liang et al. / VCU.YResearch31 (1994) 219-233

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227

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We calculated the average divergence between the HTN and SE0 clusters to be approximately 32%. 3.3. Genetic relationships as determined by restriction endonuclease digestions of the S genome segment of each isolate

We also examined the genetic relationships of the Chinese hantavirus isolates to each other and to prototype hantaviruses by using restriction enzyme mapping of cDNA representing the entire coding region of the S genome segment of each isolate. We compared the restriction patterns obtained to those predicted for prototype HTN virus, strain 76-118, or SE0 virus, strain HR 80-39. We were unable to amplify the S segments of the H3 and H5 isolates, thus those data are not included in this study. We found that none of the Chinese isolates had restriction patterns identical to that deduced for HTN virus (Fig. 4). Of the seven HTN isolates that we examined, two Apodemus (A9 and 486) and two human isolates (Luyao and Luxu) had identical restriction patterns, and another Apodemus isolate had only minor differences (B6.59). The other two human isolates (Chen and 84FLi) had patterns identical to each other, but quite different from all other isolates. In contrast, among the SE0 isolates, we found only minor differences from the restriction patterns generated for prototype SE0 virus, strain HR80-39 (Fig. 4). Thus, our restriction pattern analyses of the S genome segments

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Fig 3. Dendrogram generated by paitwise comparison of the 330 bp sequences described in Fig. 2. The sequences were aligned by using the UWGCG program PILEUP and the phylogenetic tree was created by the PAUP program based on 1000 bootstrap replications. The numbers above each tree branch represent the distance to the nodes, and are proportional to nucleotide sequence differences. Numbers in the parentheses are the bootstrap confidence of each branch (%o).

confirm the data obtained by sequence analyses with the M genome segments of these Chinese hantaviruses.

4. Discussion Hemorrhagic fever with renal syndrome (HFRS) is a serious human infectious disease associated with some viruses in the Huatavirus genus of the family ~~~y~~~~~ffe. China is the most seriously affected country and has the most cases annually in the world, HTN and SE0 viruses were first isolated from Apodemus and Ruttus in China in 1981 to 1982 (Song et al., 1982a,b). Since then, many additional isolates have been obtained from a variety of rodents in numerous geographic regions in China (Song, 1991; Chen et al., 1985; Liang et al., 1992). The goal of our study was to provide info~ation concerning the antigenic and genetic diversity among hantaviruses in endemic regions of China. Toward this goal we

M. Liang et al. / Virus Research 31 (1994) 219-233

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Fig 4. Restriction endonuclease maps of the coding region of the S genome segment of each Chinese hantavirus isolate. Fragment sizes are listed only for the first isolate of a particular digestion pattern. The shadings indicate different restriction patterns. That is, the dark shading for HTN viruses 486, B659, Luyao, and Luxu indicates that the pattern is identical to that of A9. The only two differences observed among these isolates were in B659, as listed. Similarly, for the SE0 viruses, the shading indicates that the pattern is identical to that listed in the column to the far left. For example, for MspI and NciI digests, all patterns are the same as listed for HR80-39 except for those of SR-11. Likewise, for BarnHI digests, HR SO-39 and SR-11 patterns are identical and the rest are the same as Hubei-1; etc. Fragments that could not be visualized, but were calculated based on sequence information are also shaded and shown in italics and a reduced font.

characterized 15 Chinese hantavirus isolates and compared them to prototype hantaviruses by serological and molecular means. The results that we obtained by cross-PRNT and by genetic analyses of a portion of the M genome segments, and of the entire coding region of the S genome segments of these isolates produced concordant findings, in that we found that all isolates were either HTN or SE0 viruses by all three methods used. Our findings with the rodent isolates are consistent with the generally accepted theory that there is a strong association between a particular serological type of hantavirus and its rodent host. That is, usually &&emus isolates are HTN virus, Rattus isolates are SE0 virus and Clethrionomys isolates are PUU virus (Schmaljohn et al., 1985). Thus, although none of our rodent isolates were related to PUU or PH viruses, this finding was not unexpected, because we examined only Apodemus and Rattus isolates. None of the human isolates we studied appeared to be similar to PUU virus either. There are Clethrionomys species in China, thus it

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may be possible that PUU-like viruses are present. To our knowledge, however, there are no reports of PUU-like viruses in China. We recently demonstrated that a phylogenetic tree constructed from pair-wise comparisons of a 330 bp region of the M segment correlated well with a tree established by comparing the amino acid sequences encoded in the entire M segments of hantaviruses (Xiao et al., 1994). The results presented in this study were generated by comparison of this same M segment gene region, thus, we are confident that this method provides an accurate view of the genetic diversity among the Chinese isolates that we examined. By using this method, we were able to discern three genetically separable groups of Chinese HTN viruses. Interestingly, although previous reports describing human isolates have always found a very similar rodent counterpart, two of the human isolates that we examined, Chen and H.5, appeared quite different from any of the rodent isolates. Direct comparison of the 330 bp nucleotide sequences of the M segments of Chen and HS to the Apodemus isolates, A9 and 486, revealed only 83% and 79% homology. In contrast, the B659, Luxu and Luyao isolates displayed 98% to 99% homolo~ to A9 and 486. Similar studies with other hantaviruses have also demonstrated a very close rodent counterpart to each human isolate. For example, comparison of the complete nucleotide sequence of the M genome segment of prototype HTN virus, strain 76-118, to those of two human isolates from Korea, HoJo and Lee, revealed nucleotide sequence homologies of appro~mately 95% (Schmaljohn et al., 1988). Also, the M and the S segment nucleotide sequences of two human PUU virus isolates from Russia, K27 and P360, were reported to be approximately 99% homologous to a Cfethrionomys isolate from the same region (Xiao et al., 1994). Because human to human transmission of HFRS has never been demonstrated, it is likely that a rodent counterpart to these human isolates like Chen and H5 does exist, but has not yet been identified. In contrast to the HTN virus isolates, the SE0 virus isolates that we studied were all very similar. The 330 nucleotides of the M segments of the human isolates HB55, JB2 and JB3 were 97% to 99% homologous with the batter isolates, R22 and L99, and 94% homologous with SE0 virus strains HR 80-39 (isolated in Korea), or SR-11 (isolated in Japan). Human isolate Hubei-1 was 98.8% and 98.2% homologous with strains HR 80-39 and SR-11. These data are consistent with the findings of others, which indicate that SE0 viruses are generally highly conserved at both the antigenic and genetic level (Schmaljohn et al., 1985; Xiao et al., 1992; Puthavathana et al., 1992; Xiao et al., 1994; Chu et al., 1994). Our restriction map studies of the S segments of these viruses, confirmed our M segment findings. The restriction pattern similarities observed among the HTN isolates coincided with the groupings observed on the phylogenetic tree, except for H3 and H5, for which we were unable to obtain PCR products with the S segment primers. This result, in itself, may indicate that H3 and HS are quite different from the other isolates. Similarly, for the SE0 isolates, the homologies of the S segment restriction patterns confirmed the M segment findings, in that there appeared to be less genetic diversity among the SE0 viruses as compared to the HTN viruses that we examined.

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In summary, our study provides a data base for the genetic variation among HTN and SE0 viruses in China. This information may be valuable in determining the appropriate vaccine to combat HFRS in areas where more than one of these viruses co-circulate.

Acknowledgements

We thank Drs. Kevin Anderson and Erik A. Henchal for their excellent help in computer analyses and Kristin W. Spik for her technical assistance. We also acknowldege the NC1 Biomedical Supercomputing Center for their generous allotment of computer time for these studies. M.L., D.L. and C.H. are visiting scientists from the Institute of Virology, Chinese Academy of Preventive Medicine and held Courtesy Associateships from the Centers for Disease Control and Prevention. S.-Y.X. held a Senior Research Associateship from the National Research Council when he worked at USAMRIID.

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