Virus Research, 16 (1990) 127-136 Elsevier
127
VIRUS 00576
Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis Lutz B. Giebel ‘, Lothar Zliller 2, Ekkehard and Gholamreza Darai ’
K.F. Bautz ’
’ Institut ftir Molekulare Genetik der Vniversitiit Heidelberg, Heidelberg F.R. G. and 2 Institut ftir Medizinische Virologie der Universitb? Heidelberg, Heidelberg, F. R. G. (Accepted 12 January 1990)
Summary
The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Huntavirw strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nepbropathia epidemica virus strain Hahn& Bl (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level. Immunoblot analysis; RNA hybridization; cDNA Polymerase chain reaction (PCR); Chain termination sequencing
Hantavirus;
synthesis;
3ci Correspondence to: G. Darai, Institut ftir Medizinische Virologie der Universitslt Heidelberg, Im Neuenheimer Feld 324, 6900 Heidelberg, F.R.G. 0168-1702/90/$03.50
6 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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Introduction Hantaviruses, a separate genus of the Bunyaviridae (Schmaljohn and Dalrymple, 1983), are the etiologic agents of a variety of human diseases, collectively termed “hemorrhagic fever with renal syndrome (HFRS)“. Evidence for Huntuuirus infections has been reported from all parts of the world studied. The natural reservoir appears to be muroid rodents. Five serologically distinguishable groups of hantaviruses have been observed: Hantaan (Apodemus) (Lee et al., 1978), Seoul (Rattus), Puumala (Clethrionomys) (Yanagihara et al., 1984), Prospect Hill (Microtus), and Leaky (Mus). The rodent genus listed after each virus indicates the origin of the isolation. The clinical manifestations in humans are severe for Hantaan virus infections, less severe for Seoul virus infections, and quite mild for Puumala virus and serologically related virus strains causing nephropathia epidemica. No pathogenic effects in humans have been reported for Prospect Hill and Leaky viruses (Lee and Dalrymple, 1989; Baek et al., 1988). Hantaviruses possess a single-stranded, negative-sense, three-segmented RNA genome, enclosed in circular nucleocapsid structures which are surrounded by a lipid envelope containing two virus encoded glycoproteins. The large L RNA segments supposedly encode the viral polymerase (Bishop, 1985) and the medium size M segments code for the envelope glycoproteins Gl (- 64 kDa) and G2 ( - 54 kDa) (Schmaljohn et al., 1987; Yoo and Kang, 1987; Giebel et al., 1989). The short RNA S segments encode the 50 to 52 kDa nucleocapsid proteins (Schmaljohn et al., 1986; ZSller et al., 1989; Stohwasser et al., 1989). The Hantaan and nephropathia epidemica virus strain Hallnas Bl (NEV) (Niklasson and LeDuc, 1984) S RNA segments are 1696 and 1785 nucleotides long with coding capacities of 429 and 433 amino acids, respectively (Schmaljohn et al., 1986; Stohwasser et al., 1989). The M RNA segments of the viruses are 3616 and 3682 nucleotides long and encode precursor proteins of 1135 and 1148 amino acid residues, respectively. The precursors are then processed to yield mature Gl and G2 glycoproteins (Schmaljohn et al., 1987; Yoo and Kang, 1987; Giebel et al., 1989). The 3’ and 5’ termini of the Hantaan and NEV M and S segment RNAs show extensive sequence complementarity, a common feature of Bunyaviridae RNA genome segments. So far no nucleotide sequence data have been available for Huntuuims L segment RNAs. The goal of the work reported here was to develop a procedure for rapid cloning and nucleotide sequencing of different Hantavirus genomes in order to study their genetic variability.
Materials and Methods Cells and viruses VERO E6 cells were obtained from the American Type Culture Collection (ATCC C1008) and grown in basal medium Eagle (BME) supplemented with 10% fetal calf serum, 100 IE/ml penicillin G, and 100 IE/ml streptomycin. Viruses were
129
propagated as described previously (Zoller et al., 1989). Hantaan virus type 76-118, and nephropathia epidemica virus strain Hallnas Bl (NEV) were supplied by Drs H.W. Lee (WHO Collaborating Center for Hantaviruses) and J. Pilasky, respectively. Two Puumala virus strains with different passage histories were obtained from Drs H.W. Lee (Seoul) and B. Niklasson (Stockholm).
RNA extraction synthesis of cDNA Total cellular RNA was extracted by either the hot phenol (Maniatis et al., 1985) or the guanidinium isothiocyanate method (Chirgwin et al., 1979). RNA hybridization was performed as described previously (Giebel et al., 1989). The cDNA synthesis was carried out using random priming as described elsewhere (Giebel et al., 1989).
Polymerase
chain reaction (PCR)
First-strand cDNA/RNA hybrids were denatured at 96°C for 5 min and PCR was performed in 100 ~1 volumes of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl,, 200 PM of each dNTP, 100 pg/ml gelatin, 1 PM of each primer and 2.5 units of Taq DNA polymerase (Perkin-Elmer). Thirty cycles of PCR took place in an automated temperature cycling reactor (ERICOMP Inc., U.S.A.) which provided per cycle, 2 min each of incubation at 95, 50, and 72OC or at 95, 37,72”C (Saiki et al., 1988). The specific sequences of the M and S segments of the Hantauirus strains were amplified using the oligonucleotide primers Pl to P4 as listed and described in Table 1. PCR products were then subcloned into M13mp18 and mp19 vectors for DNA sequencing.
TABLE
1
Properties of the ohgonucleotide primers used for PCR amplification of Hantavirus M and S segment specific nucleotide sequences Label
Primer sequence
Specificity
Mismatches t
Pl
5’-AATGGGTTCAATGGTTTGTG-3’ (168-J-1706 2, 5’-CAGTGAAATGCAGTTTTAAG-3’ (2256-2275 2, 5’-GTAATGGGAGTGATI-GTTTTT-3’ (697-718 ‘) 5’-CCTTAGCTCAGGATCCATGTCATC-3’ (1253-1276 3,
M segment messenger-sense M segment genomic-sense S segment messenger-sense S segment genomic-sense
4
P2 P3 P4
3 4 1
’ The number of mismatches indicate nucleotide differences in the corresponding Hantaan (Schmaljohn
sequences
et al., 1986, 1987).
2*3 The numbers denote the nucleotide positions of the messenger-sense M and S segment nucleotide sequence as published by Giebel et al., 1989, and Stohwasser et al., 1989, respectively.
130
DNA sequence analysis The single-stranded Ml3 template recombinant phages per amplification termination procedure (Sanger et al., (Tabor and Richardson, 1987) and a-[ > 1000 Ci/mmol. Computer
DNA of at least 12 independently derived product was sequenced by the dideoxy chain 1977), using a modified T7 DNA polymerase 35S]dATP (Amersham, Braunschweig, F.R.G.)
assistance
The nucleotide and deduced amino acid sequences were computed and analyzed using the BSA program at the German Research Center (DKFZ), Heidelberg, F.R.G., and the PC Gene Program (University of Geneva, Switzerland).
Results Polymerase
chain reaction (PCR)
A previous nucleotide sequence comparison of the Hantaan and NEV M and S segment RNAs (Giebel et al., 1989; Stohwasser et al., 1989) revealed regions of high homologies clustered throughout the sequences. Oligonucleotides corresponding to these regions were derived from the NEV M and S segments for a PCR cloning approach of the different Hantauirus genomes. For cloning of the M segments an oligonucleotide Pl identical to NEV messenger-sense nucleotide positions 1687 to 1706 and a P2 complementary to positions 2256 to 2275 (Giebel et al., 1989) were used. Compared to the corresponding Hantaan M segment sequence, Pl and P2 contained 4 and 3 mismatches, respectively. To clone the S segments an oligonucleotide P3 identical to NEV messenger-sense nucleotide positions 697-718 and an oligonucleotide P4 complementary to positions 1253-1276 (Stohwasser et al., 1989) were synthesized. P3 contained 4 mismatches and P4 one mismatch compared to the corresponding Hantaan S segment sequence. Table 1 summarizes and describes all oligonucleotides used for PCR. The idea of this strategy (summarized in Fig. 1) was to select regions of high homology between the NEV and Hantaan M and S segment sequences for primer synthesis assuming that these regions would also be conserved in the Puumala genome and to use these primers to amplify the corresponding Puumala sequences. The S segment specific primers selected would cover the regions coding for the middle part of the nucleocapsid proteins and the M segment specific primers that encoding the C-terminus of Gl and the N-terminus of G2. Total cellular RNA from Hantaan virus, NEV, and with the two Puumala virus strains infected VERO E6 cells was used to synthesize first strand cDNA by random priming. The individual cDNAs were subjected to PCR using the NEV M and S segment specific primers. Amplification products were then analyzed by agarose gel electrophoresis and Southern blotting using the NEV and Hantaan M and S segment cDNAs as hybridization probes (data not shown). The PCR reactions of all
131
NW-N (Pl;sense) 5'-IWGGGTTCA~WGGTTTGTG-3'
I
-22% I 5'-CAGTGAAAIGCAGTTTTIWG-3' NEWS (P3;sense) NEWi (PZ; ant isense) 5'-GTAfiTGGGI16TGATTGGTTTT-3'
I
I
5'-CCTTAGCTChGGATCCLTGTCATC-3' NEU-S(P4;antisense) Fig. 1. Cloning strategy to isolate Puumala M and S segment specific cDNA by PCR. Panels A and B indicate the NEV M and S segments, respectively, with their corresponding nucleotide numbers in the messenger sense. The positions and nucleotide sequence of the primers used for PCR are depicted. The arrows indicate the direction of DNA synthesis.
virus strains revealed that the M and S segment were specifically amplified. The PCR M and S specific products of the two Puumala viruses were then subcloned into Ml3 vectors mp18 and mp19 and their nucleotide sequences were determined. Nucleotide
sequence analysis
A comparison of the M and S segments of Puumala and NEV viruses revealed at the nucleotide level 81 and 82% homology, respectively (Fig. 2A, B). A Hantaan and Puumala M and S nucleotide sequence comparison revealed 63% homology for both segments. These data demonstrate that NEV and Puumala are very similar viruses whose genomes differ significantly from that of Hantaan virus. In spite of the fact that the two Puumala strains were obtained from two different laboratories (Dr Lee, Seoul and Dr Niklasson, Stockholm) and consequently were propagated and passaged separately, not a single nucleotide difference was detected in the M and S segment sequences of both Puumala strains analysed. Deduced amino acid sequence analysis The 182 amino acids encoded by the PCR amplified Puumala M segment-specific cDNA sequence were compared to those of the NEV M segment (ORF: nucleotide positions 41-3485; Giebel et al., 1989) and Hantaan virus M segment (ORF: nucleotide positions 41-3346; Schmaljohn et al., 1987). The Puumala polypeptide aligns to NEV M aminoacid positions 557 to 738 and Hantaan M positions 547-728. An amino acid sequence comparison of the Puumala M specific polypeptide to the corresponding sequences of NEV and Hantaan virus revealed 94 and
132
A
N1707 :
.A..G...........A.....G.....A..A..AC.G..G..............T.... 1 AGGTATGTCAGTATGAGTGTGAAACTGCTAAGGAGTTAGAATCACATAGAAAGAGCTGTT 1677 .T..C..CA................CTA...A..A...A.GG....CG.GGTATCA..CC
N P H
1767 61 1737
.C....................T..C..............A...A.A.....C.....G. CAATTGGTTCATGCCCTTATTGCCTTAATCCATCTGAGGCTACACCGTCTGCTCTTCAAG .CCAATC.CA...T.....C..TT...C..AT.G...AC.C...GAAG.A..AT.C....
N P H
1827 121 1797
. . . . . . . . . . . ..G.....G..C.G............-...G.....A.GA......l.. CTCATTTTAAACTTTGTAAACTAACATCACGGTTTCA-GGAAAATTTGAAGAAGTCACTA . . . . ..AC..G..A..CC..G.T..-....A.A....G...TG..C.A.....AA..G.T
N
1606 180 1656
..GG.A........T.....A..G..C..C..G..T.....C..C.....A..TA.G... ACAATGTATGAGCCAATGCAGGGTTGTTATCGAACATTATCTCTATTTAGGTACCGCAGT ..TCCTC.AA.TTTT.CA.CA..A.....C..G...C..AA.T.......A...AAA..C
;
B
N 1946 P 240 H 1916
C....C.....G...C....C.....CG....AT.G...CATC.CT.............. AGGTTTTTTGTAGGTTTAGTGTGGTGTGTATGTTGCTTGTTTTAGAGCTAATTGTATGGGCT . . ..GC.ACA.CTT.AC.A.....ATAT.TC.T.....C.....ATCC..AC.G......
N 2006 P 300 H 1976
..C.....-...A........T........A........A..C..A...........TA. GCTAGTGC-TGAGACACAAAACTTAAATGCTGGTTGGACTGATACTGCACATGGATCAGG ,.A.....A.C...G...CC.-....C.C...TC....A...C.A...C.....GGT...
N 2065 P 359 H 2035
. ..T...........AA.......G...T.A..C.....TC.T.AG.....A..A..... AATAATACCTATGAAGGCTGATCTTGAACTGGATTTCTCATTACCTTCATCTGCTAGCTA TTCTG.T......C.TA.A...T.A..G..T........T...A.A..CAG.T.C.AG..
N 2125 P 419 H 2095
. ..A..TA.G.....GC..........A.....C........G.....C.........C. TACTTACCGAAGACAATTACAAAACCCGGCAAATGAACAAGAAAAAATACCATTTCATTT . ..A.....T..GA.G...AC......ACTTG.G...GC.C..TCC..TGACC.A...A.
N 2185 P 473 H 2155
G...T....C.....A...........A..G..C..G...T....T..T........... ACAGATAAGTAAACAGGTGATTCATGCTGAAATTCAACATCTAGGCCACTGGATGGATGC TG.A...GAAG.....ACA...GG..T...TG.G..TGC......A......T.T....G
N 2245 P 539 H 2215
.._. TACATTTAAT .CGTC....C
2254 548 2224
N P H
716 ,.T.....C..T..A..C...C.A..A.A...T..G.....T..,..G.....A.....T 1 TCATTCTTTGTGAAGGATTGGTCTGAGAGAATCAGAGAGTTCATGGAAAAAGAGTGCCCA 701 .A...GG..T.CT..C...A.-.AA..GACTGG..T..TCGT..C...C..TG..-----
N P Ii
778 61 755
. . . . . . . . . ..A.-...-.T.....T..G.........-..G...G.A..AT.T..--G. TTCATAAAGCCTG-AAG-TAAAACCAGGCACACCAGCA-CAAGAGATTGAGATGTT--AA .AAT.G..C.T..C...C.TCTT....AT...G.....GTT..CCTCCTT.G..G.CCTG
N P H
833 116 815
. . . . . . . ..GAG.T..T..C...ACC...........T..........--.......... AAAGAAATAAGATCTACTTTATGCAGCGCCAGGATGTGCTTGACAAAA--ATCATGTGGC C..C...C.G.GA......ACG......G..A.TG.CAT.A.G...T.TGGAG.CAAA.G
N P Ii
891 174 075
T.....C..T.....G...........T........T.......-....G.....TG... AGACATTGACAAGTTAATTGACTATGCAGCCTCTGGAGACCCTA-CATCACCTGACAACA ..T..AA.G.T.--..CGCC.GC......AAG.A.CT.G.TG..G...G:T...AG.T.
N P Ii
950 233 932
.C..A..T.................A.....T...........T..G.....C....... TTGATTCGCCTAATGCACCATGGGTCTTTTGCATGTGCACCAGACCGATGCCCACCAACAT . . ..G..A..ATCAT..AT......T.....TG.A...........T..T..........
N 1010 P 293 H 332
. . ..T........T........T..AT.A..T..A..C.....C..C..A.....r.... GTATCTATGTTGCAGGGATGGCAGAGCTTGGGGCCTTTTTTTCAATATTGCAGGACATGA ..T.G.T.A.A.....T..T..T...........A........C..CC...........C
N 1070 P 353 H 1052
.G.....C..T...........A.....G..................C.G..A..G GAAACACAATAATGGCATCTAAGACTGTTGGCACAGCAGAAGAGAAATTPAAGAAAAAGT . . ..T.....C............ .A.....A...T.T..G.....GC..CG...G..A.
N 1130 P 413 H 1112
.C.....C..T.....A..TT....C.....A...........G.....A........G. CTTCCTTTTACCAATCTTACCTGCGTCGAACTCAATCAATGGGAATTCAGCTTGATCAAA .A..A.....T..G..C.....CA.AA.G..A...........G..A..A..A.GC..G.
N 1190 P 473 H 1172
.G..A.....AC...AC..............A.G...A.....G.....T....... GAATTATCCTCTTGTTTATGTTGGAATGGGGCAAAGAGATGGTAGATCACTTCCATCTTG . . . . . ..TG.GC.C..C...G.T.CC.....A..G...GCT..G..CA.......CT.A.
N 1250 P 533 H 1232
._ GT .G
133
A
0M
N P H
557 1 547
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..T............R.......R.... VCQYECETAKELESHRKSCSIGSCPYCLNPSEATPSALQAHFKVCKLTSRFQENLKKSLT ..K.....Y...KA.GV..PPSQ....FTHC.P.EA.F...Y...QV.H..RDD...TV.
N P
617 61 607
V ........................... V...HH........................R. MYEPMQGCYRTLSLFRYRSRFFVGLVWCMLLVLELIVWAASAETQNLNAGWTDTAHGSGI PQNFTP......N....K..CYIFTM.IF.....S.L.....SETP.TPV.N.N...V
N P H
677 121 667
.. ..T........Q..........................L ................... IPMKADLELDFSLPSSASYTYRRQLQNPANEQEKIPFHLQISKQVIHAEIQHLGHWMDAT V..HT........T..SK.....K.T..LE.AQS.DL.IE.EE.T.GVDVHA....F.GR
N P H
737 181 727
. FN L.
N
226
. . . . . ..P.K........................V.F....RV...T.............
P H
1 229
SFFVKDWSERIREFMEKECPFIKPEVKPGTPAQEIEMLKRNKIYFMQRQOVLDKNHVADI AKDWS.RI.QWLIEPC.LL.DTAAVSLL.G..-------T.RD.LR...VA.GNMETKES
285 60 281
286 61 282
. . . . . . . . . . . . . . ..D.E......................................... DKLIDYAASGOPTSPDNIDSPNAPWVFACAPDRCPPTCIYVAGMAELGAFFSILQDMRNT KAIRQH.EAAGCSMIED.E..SSI....G.........LFI..I................
345 120 341
346 121 342
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Y.....R......... IMASKTVGTAEEKLKKKSSFYQSYLRRTQSMGIQLDQRIILLFMLEWGKEMVDHFHLG . . . . . . . ..S....R....................G....V...VA....A..N....
H
; H
616 SO 606
.S
676 120 666 736 180 726
403 178 399
Fig. 3. Amino acid sequence comparison of Puumala, Hantaan and NEV M and S segment encoded polypeptides. An alignment of the Puumala M (panel A) and S (panel B) ammo acid sequences deduced from the PCR cDNAs with the corresponding regions of the NEV and Han&an M and S segment encoded polypeptides is shown. The dots denote identical amino acid residues, the dashes residues missing in either one of the sequences. The numbers indicate the ammo acid positions of the M and S segment encoded polypeptides.
53% homology, respectively (Fig. 3A, B). There are almost no conservative amino acid exchanges; only one out of 10 differences for the Puumala and NEV comparison and only two out of 81 differences for the Puumala and Hantaan comparison are conservative exchanges. The 178 amino acids encoded by the Puumala S segment specific cDNA were also compared to those of the NEV S segment (ORF: nucleotide positions 43-1342; Stohwasser et al., 1989) and Hantaan virus S segment (ORF: nucleotide positions 37-1324; Schmaljohn et al., 1986). The Puumala S segment specific polypeptide corresponds to NEV ammo acid positions 226-403 and Hantaan positions 229-399. The amino acid homology between the Puumala S segment specific polypeptide and the corresponding sequences of NEV and Hantaan is 94% and 55% (Fig. 3A, B), respectively. Again there are almost no conservative
Fig. 2. Nucleotide sequence comparison of Puumala, Hantaan, and NEV M and S segment cDNAs. An alignment of the Puumala M (panel A) and S (panel B) nucleotide sequences cloned by PCR with the corresponding regions of NEV and Hantaan M and S segments is shown. N, P, and H denote NEV, Puumala and Hantaan sequences. The dots depict identical nucleotides and the dashes nucleotides missing in either one of the sequences. The numbers indicate the nucleotide positions of the M and S segments.
134
amino acid exchanges: none of the 11 differences in the Puumala and NEV comparison is conservative and only one out of 72 is conservative for the Puumala and Hantaan alignment. These protein data are consistent with the nucleotide sequence data, since they also demonstrate that Puumala is extremely similar to NEV but very different from Hantaan virus. Apparently for many amino acid residues of the hantavirus S and M encoded polypeptides there may be little selective pressure for conservation, which is consistent with the observation that amino acid homologies between Hantaan and NEV or Puumala encoded proteins is lower than the nucleotide homologies of the corresponding genomic segments.
Discussion Evidence for Huntauirus infections has been reported from all parts of the world and about 100 different isolates have been collected so far. To study the genetic variance of this important virus group and to relate this variance to the severity of HFRS caused in human infections it is necessary to molecularly clone their genomes and determine the nucleotide sequences. However, the task of analyzing so many different isolates by conventional cloning procedures is not feasible. Therefore we studied the use of the polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis to rapidly detect genomic variations in different Hantauirus strains. For this study we chose Hantaan virus strain 76-118 and nephropathia epidemica virus strain Hallnb Bl whose M and S genomic RNA segments have been sequenced (Schmaljohn et al., 1986, 1987; Giebel et al., Stohwasser et al., 1989). These were compared with the Puumala virus strains which cause nephropathia epidemica in humans. Using oligonucleotides derived from NEV (HallnHs Bl) M and S segment nucleotide sequences which had been found to be conserved in the genome of the Hantaan virus it was possible to isolate the corresponding sequences by a PCR assay from Hantaan, NEV and Puumala viruses. The successful cloning of the genomic sequences of these hantaviruses by the PCR technique shows that this approach works even if the oligonucleotide used for PCR are not perfectly matched. The cloning of genomic fragments derived from Ruumala virus M and S segments by the PCR approach and the subsequent nucleotides sequence analysis revealed that this virus is very similar to the NEV strain Hlllnas Bl. A comparison of the Puumala and NEV sequences showed over 80% homology at the nucleotide level and 94% homology at the deduced amino acid level. A comparison of the Puumala sequences to those of Hantaan revealed 63% nucleotide homology and 53 and 55% amino acid homology for the corresponding regions of the M and S encoded polypeptides, respectively. The results reported in this paper demonstrate that the polymerase chain reaction technique is a powerful tool to rapidly detect genomic variations in hantaviruses. Furthermore, our results confirm the common antigenic sites of the M and S segment encoded proteins between NEV Hallnls Bl and Puumala virus. This should
135
be considered for the development of specific based on recombinant DNA technology.
diagnostic
and prevention
systems
Acknowledgements This study was supported by the Bundesministerium fir Forschung und Technologie, BMFT grant No 0318973A. We thank Drs H.W. Lee, B. Niklasson and J. Pilaski for providing the Hantaoirus strains and the sera used. We gratefully acknowledge Dr C.S. Schmaljobn for providing the Hantaan M and S segment cDNA clones. Finally the authors thank MS Ulrike Heinz for excellent technical assistance.
References Baek, L.J., Yanagihara, R.Y., Gibbs, C.J.JR., Miyazaki, M. and Gajdusek, D.C. (1988) Leaky virus: a new Hantavirus isolated from Mus muxu1u.r in the United States. J. Gen. Virol. 69, 3129-3132. Bishop, D.H.L. (1985) Replication of Arenaviruses and Bunyaviruses. In: B. Fields, D.M. Knipe, R.M. Chanock, J.L. Melnik, B. Roizman and R.E. Shope (Eds), Virology, pp. 1083-1118. Raven Press, New York. Chirgwin, J.M., Pryzybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5295. Giebel, L.B., Stohwasser, R., ZBller, L., Bautz, E.K.F. and Darai, G., (1989) Determination of coding capacity of the M genome segment of nephropathia epidemica virus strain HBllnas Bl by molecular cloning and nucleotide sequence analysis. Virology 172, 4988505. Lee, H.W., Lee, P.W. and Johnson, K.M. (1987) Isolation of the etiologic agent of korean hemorrhagic fever. J. Infect. Dis. 137, 298-308. Lee, H.W. and Dahymple, J.M. (1989) Manual of hemorrhagic fever with renal syndrome. WHO Collaborating Center for Virus Reference and Research. Institute for Viral Diseases, Korea University. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1985) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Niklasson, B. and LeDuc, J. (1984) Isolation of the nephropathia epidemica agent in Sweden. Lancet 1, 1012-1013. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Schmaljohn, C.S., and Dalrymple, J.M., (1983) Analysis of Hantaan virus RNA: evidence of a new genus of Bunyaviridae. Virology 131, 482-491. Schmaljohn, C.S., Jennings, G.B., Hay, J. and Dalrymple, J.M. (1986) Coding strategy of the S genome segment of Hantaan virus. Virology 155, 633-643. Schmaljohn, C.S., Schmaljohn, A.L. and Dalrymple, J.M. (1987) Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order. Virology 151, 31-39. Stohwasser, R., Giebel, L.B., Zbller, L., Bautz, E.K.F. and Darai, G. (1989) Molecular characterization of the RNA S segment of nephropathia epidemica virus strain HBllnPs Bl. Virology 173, in press. Tabor, S. and Richardson, C.C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767-4771.
136 Yanagihara, R., Goldgaber, D., Lee, P.W., Amyx. H.L., Gjdusek, DC.. Gibbs, C.J.JR. and Svedomyr, A. (1984) Propagation of nephropathia epidemica virus in cell culture. Lancet 1,1013. Yoo, D. and Kang, C.Y. (1987) Nucleotide sequence of the M segment of the genomic RNA of Hantaan virus 76-118. Nucleic Acids Res. 15, 6299-6300. Zoller, L., Scholz, J., Stohwasser, R., Giebel, L.B., Sethi, K.K., Bautz, E.K.F. and Darai, G. (1989) Immunblot analysis of the serological response in Hanta virus infections. J. Med. Viral. 27, 231-237. (Received
13 September
1989; revision
received
12 January
1990)
127
VIRUS 00576
Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis Lutz B. Giebel ‘, Lothar Zliller 2, Ekkehard and Gholamreza Darai ’
K.F. Bautz ’
’ Institut ftir Molekulare Genetik der Vniversitiit Heidelberg, Heidelberg F.R. G. and 2 Institut ftir Medizinische Virologie der Universitb? Heidelberg, Heidelberg, F. R. G. (Accepted 12 January 1990)
Summary
The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Huntavirw strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nepbropathia epidemica virus strain Hahn& Bl (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level. Immunoblot analysis; RNA hybridization; cDNA Polymerase chain reaction (PCR); Chain termination sequencing
Hantavirus;
synthesis;
3ci Correspondence to: G. Darai, Institut ftir Medizinische Virologie der Universitslt Heidelberg, Im Neuenheimer Feld 324, 6900 Heidelberg, F.R.G. 0168-1702/90/$03.50
6 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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Introduction Hantaviruses, a separate genus of the Bunyaviridae (Schmaljohn and Dalrymple, 1983), are the etiologic agents of a variety of human diseases, collectively termed “hemorrhagic fever with renal syndrome (HFRS)“. Evidence for Huntuuirus infections has been reported from all parts of the world studied. The natural reservoir appears to be muroid rodents. Five serologically distinguishable groups of hantaviruses have been observed: Hantaan (Apodemus) (Lee et al., 1978), Seoul (Rattus), Puumala (Clethrionomys) (Yanagihara et al., 1984), Prospect Hill (Microtus), and Leaky (Mus). The rodent genus listed after each virus indicates the origin of the isolation. The clinical manifestations in humans are severe for Hantaan virus infections, less severe for Seoul virus infections, and quite mild for Puumala virus and serologically related virus strains causing nephropathia epidemica. No pathogenic effects in humans have been reported for Prospect Hill and Leaky viruses (Lee and Dalrymple, 1989; Baek et al., 1988). Hantaviruses possess a single-stranded, negative-sense, three-segmented RNA genome, enclosed in circular nucleocapsid structures which are surrounded by a lipid envelope containing two virus encoded glycoproteins. The large L RNA segments supposedly encode the viral polymerase (Bishop, 1985) and the medium size M segments code for the envelope glycoproteins Gl (- 64 kDa) and G2 ( - 54 kDa) (Schmaljohn et al., 1987; Yoo and Kang, 1987; Giebel et al., 1989). The short RNA S segments encode the 50 to 52 kDa nucleocapsid proteins (Schmaljohn et al., 1986; ZSller et al., 1989; Stohwasser et al., 1989). The Hantaan and nephropathia epidemica virus strain Hallnas Bl (NEV) (Niklasson and LeDuc, 1984) S RNA segments are 1696 and 1785 nucleotides long with coding capacities of 429 and 433 amino acids, respectively (Schmaljohn et al., 1986; Stohwasser et al., 1989). The M RNA segments of the viruses are 3616 and 3682 nucleotides long and encode precursor proteins of 1135 and 1148 amino acid residues, respectively. The precursors are then processed to yield mature Gl and G2 glycoproteins (Schmaljohn et al., 1987; Yoo and Kang, 1987; Giebel et al., 1989). The 3’ and 5’ termini of the Hantaan and NEV M and S segment RNAs show extensive sequence complementarity, a common feature of Bunyaviridae RNA genome segments. So far no nucleotide sequence data have been available for Huntuuims L segment RNAs. The goal of the work reported here was to develop a procedure for rapid cloning and nucleotide sequencing of different Hantavirus genomes in order to study their genetic variability.
Materials and Methods Cells and viruses VERO E6 cells were obtained from the American Type Culture Collection (ATCC C1008) and grown in basal medium Eagle (BME) supplemented with 10% fetal calf serum, 100 IE/ml penicillin G, and 100 IE/ml streptomycin. Viruses were
129
propagated as described previously (Zoller et al., 1989). Hantaan virus type 76-118, and nephropathia epidemica virus strain Hallnas Bl (NEV) were supplied by Drs H.W. Lee (WHO Collaborating Center for Hantaviruses) and J. Pilasky, respectively. Two Puumala virus strains with different passage histories were obtained from Drs H.W. Lee (Seoul) and B. Niklasson (Stockholm).
RNA extraction synthesis of cDNA Total cellular RNA was extracted by either the hot phenol (Maniatis et al., 1985) or the guanidinium isothiocyanate method (Chirgwin et al., 1979). RNA hybridization was performed as described previously (Giebel et al., 1989). The cDNA synthesis was carried out using random priming as described elsewhere (Giebel et al., 1989).
Polymerase
chain reaction (PCR)
First-strand cDNA/RNA hybrids were denatured at 96°C for 5 min and PCR was performed in 100 ~1 volumes of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl,, 200 PM of each dNTP, 100 pg/ml gelatin, 1 PM of each primer and 2.5 units of Taq DNA polymerase (Perkin-Elmer). Thirty cycles of PCR took place in an automated temperature cycling reactor (ERICOMP Inc., U.S.A.) which provided per cycle, 2 min each of incubation at 95, 50, and 72OC or at 95, 37,72”C (Saiki et al., 1988). The specific sequences of the M and S segments of the Hantauirus strains were amplified using the oligonucleotide primers Pl to P4 as listed and described in Table 1. PCR products were then subcloned into M13mp18 and mp19 vectors for DNA sequencing.
TABLE
1
Properties of the ohgonucleotide primers used for PCR amplification of Hantavirus M and S segment specific nucleotide sequences Label
Primer sequence
Specificity
Mismatches t
Pl
5’-AATGGGTTCAATGGTTTGTG-3’ (168-J-1706 2, 5’-CAGTGAAATGCAGTTTTAAG-3’ (2256-2275 2, 5’-GTAATGGGAGTGATI-GTTTTT-3’ (697-718 ‘) 5’-CCTTAGCTCAGGATCCATGTCATC-3’ (1253-1276 3,
M segment messenger-sense M segment genomic-sense S segment messenger-sense S segment genomic-sense
4
P2 P3 P4
3 4 1
’ The number of mismatches indicate nucleotide differences in the corresponding Hantaan (Schmaljohn
sequences
et al., 1986, 1987).
2*3 The numbers denote the nucleotide positions of the messenger-sense M and S segment nucleotide sequence as published by Giebel et al., 1989, and Stohwasser et al., 1989, respectively.
130
DNA sequence analysis The single-stranded Ml3 template recombinant phages per amplification termination procedure (Sanger et al., (Tabor and Richardson, 1987) and a-[ > 1000 Ci/mmol. Computer
DNA of at least 12 independently derived product was sequenced by the dideoxy chain 1977), using a modified T7 DNA polymerase 35S]dATP (Amersham, Braunschweig, F.R.G.)
assistance
The nucleotide and deduced amino acid sequences were computed and analyzed using the BSA program at the German Research Center (DKFZ), Heidelberg, F.R.G., and the PC Gene Program (University of Geneva, Switzerland).
Results Polymerase
chain reaction (PCR)
A previous nucleotide sequence comparison of the Hantaan and NEV M and S segment RNAs (Giebel et al., 1989; Stohwasser et al., 1989) revealed regions of high homologies clustered throughout the sequences. Oligonucleotides corresponding to these regions were derived from the NEV M and S segments for a PCR cloning approach of the different Hantauirus genomes. For cloning of the M segments an oligonucleotide Pl identical to NEV messenger-sense nucleotide positions 1687 to 1706 and a P2 complementary to positions 2256 to 2275 (Giebel et al., 1989) were used. Compared to the corresponding Hantaan M segment sequence, Pl and P2 contained 4 and 3 mismatches, respectively. To clone the S segments an oligonucleotide P3 identical to NEV messenger-sense nucleotide positions 697-718 and an oligonucleotide P4 complementary to positions 1253-1276 (Stohwasser et al., 1989) were synthesized. P3 contained 4 mismatches and P4 one mismatch compared to the corresponding Hantaan S segment sequence. Table 1 summarizes and describes all oligonucleotides used for PCR. The idea of this strategy (summarized in Fig. 1) was to select regions of high homology between the NEV and Hantaan M and S segment sequences for primer synthesis assuming that these regions would also be conserved in the Puumala genome and to use these primers to amplify the corresponding Puumala sequences. The S segment specific primers selected would cover the regions coding for the middle part of the nucleocapsid proteins and the M segment specific primers that encoding the C-terminus of Gl and the N-terminus of G2. Total cellular RNA from Hantaan virus, NEV, and with the two Puumala virus strains infected VERO E6 cells was used to synthesize first strand cDNA by random priming. The individual cDNAs were subjected to PCR using the NEV M and S segment specific primers. Amplification products were then analyzed by agarose gel electrophoresis and Southern blotting using the NEV and Hantaan M and S segment cDNAs as hybridization probes (data not shown). The PCR reactions of all
131
NW-N (Pl;sense) 5'-IWGGGTTCA~WGGTTTGTG-3'
I
-22% I 5'-CAGTGAAAIGCAGTTTTIWG-3' NEWS (P3;sense) NEWi (PZ; ant isense) 5'-GTAfiTGGGI16TGATTGGTTTT-3'
I
I
5'-CCTTAGCTChGGATCCLTGTCATC-3' NEU-S(P4;antisense) Fig. 1. Cloning strategy to isolate Puumala M and S segment specific cDNA by PCR. Panels A and B indicate the NEV M and S segments, respectively, with their corresponding nucleotide numbers in the messenger sense. The positions and nucleotide sequence of the primers used for PCR are depicted. The arrows indicate the direction of DNA synthesis.
virus strains revealed that the M and S segment were specifically amplified. The PCR M and S specific products of the two Puumala viruses were then subcloned into Ml3 vectors mp18 and mp19 and their nucleotide sequences were determined. Nucleotide
sequence analysis
A comparison of the M and S segments of Puumala and NEV viruses revealed at the nucleotide level 81 and 82% homology, respectively (Fig. 2A, B). A Hantaan and Puumala M and S nucleotide sequence comparison revealed 63% homology for both segments. These data demonstrate that NEV and Puumala are very similar viruses whose genomes differ significantly from that of Hantaan virus. In spite of the fact that the two Puumala strains were obtained from two different laboratories (Dr Lee, Seoul and Dr Niklasson, Stockholm) and consequently were propagated and passaged separately, not a single nucleotide difference was detected in the M and S segment sequences of both Puumala strains analysed. Deduced amino acid sequence analysis The 182 amino acids encoded by the PCR amplified Puumala M segment-specific cDNA sequence were compared to those of the NEV M segment (ORF: nucleotide positions 41-3485; Giebel et al., 1989) and Hantaan virus M segment (ORF: nucleotide positions 41-3346; Schmaljohn et al., 1987). The Puumala polypeptide aligns to NEV M aminoacid positions 557 to 738 and Hantaan M positions 547-728. An amino acid sequence comparison of the Puumala M specific polypeptide to the corresponding sequences of NEV and Hantaan virus revealed 94 and
132
A
N1707 :
.A..G...........A.....G.....A..A..AC.G..G..............T.... 1 AGGTATGTCAGTATGAGTGTGAAACTGCTAAGGAGTTAGAATCACATAGAAAGAGCTGTT 1677 .T..C..CA................CTA...A..A...A.GG....CG.GGTATCA..CC
N P H
1767 61 1737
.C....................T..C..............A...A.A.....C.....G. CAATTGGTTCATGCCCTTATTGCCTTAATCCATCTGAGGCTACACCGTCTGCTCTTCAAG .CCAATC.CA...T.....C..TT...C..AT.G...AC.C...GAAG.A..AT.C....
N P H
1827 121 1797
. . . . . . . . . . . ..G.....G..C.G............-...G.....A.GA......l.. CTCATTTTAAACTTTGTAAACTAACATCACGGTTTCA-GGAAAATTTGAAGAAGTCACTA . . . . ..AC..G..A..CC..G.T..-....A.A....G...TG..C.A.....AA..G.T
N
1606 180 1656
..GG.A........T.....A..G..C..C..G..T.....C..C.....A..TA.G... ACAATGTATGAGCCAATGCAGGGTTGTTATCGAACATTATCTCTATTTAGGTACCGCAGT ..TCCTC.AA.TTTT.CA.CA..A.....C..G...C..AA.T.......A...AAA..C
;
B
N 1946 P 240 H 1916
C....C.....G...C....C.....CG....AT.G...CATC.CT.............. AGGTTTTTTGTAGGTTTAGTGTGGTGTGTATGTTGCTTGTTTTAGAGCTAATTGTATGGGCT . . ..GC.ACA.CTT.AC.A.....ATAT.TC.T.....C.....ATCC..AC.G......
N 2006 P 300 H 1976
..C.....-...A........T........A........A..C..A...........TA. GCTAGTGC-TGAGACACAAAACTTAAATGCTGGTTGGACTGATACTGCACATGGATCAGG ,.A.....A.C...G...CC.-....C.C...TC....A...C.A...C.....GGT...
N 2065 P 359 H 2035
. ..T...........AA.......G...T.A..C.....TC.T.AG.....A..A..... AATAATACCTATGAAGGCTGATCTTGAACTGGATTTCTCATTACCTTCATCTGCTAGCTA TTCTG.T......C.TA.A...T.A..G..T........T...A.A..CAG.T.C.AG..
N 2125 P 419 H 2095
. ..A..TA.G.....GC..........A.....C........G.....C.........C. TACTTACCGAAGACAATTACAAAACCCGGCAAATGAACAAGAAAAAATACCATTTCATTT . ..A.....T..GA.G...AC......ACTTG.G...GC.C..TCC..TGACC.A...A.
N 2185 P 473 H 2155
G...T....C.....A...........A..G..C..G...T....T..T........... ACAGATAAGTAAACAGGTGATTCATGCTGAAATTCAACATCTAGGCCACTGGATGGATGC TG.A...GAAG.....ACA...GG..T...TG.G..TGC......A......T.T....G
N 2245 P 539 H 2215
.._. TACATTTAAT .CGTC....C
2254 548 2224
N P H
716 ,.T.....C..T..A..C...C.A..A.A...T..G.....T..,..G.....A.....T 1 TCATTCTTTGTGAAGGATTGGTCTGAGAGAATCAGAGAGTTCATGGAAAAAGAGTGCCCA 701 .A...GG..T.CT..C...A.-.AA..GACTGG..T..TCGT..C...C..TG..-----
N P Ii
778 61 755
. . . . . . . . . ..A.-...-.T.....T..G.........-..G...G.A..AT.T..--G. TTCATAAAGCCTG-AAG-TAAAACCAGGCACACCAGCA-CAAGAGATTGAGATGTT--AA .AAT.G..C.T..C...C.TCTT....AT...G.....GTT..CCTCCTT.G..G.CCTG
N P H
833 116 815
. . . . . . . ..GAG.T..T..C...ACC...........T..........--.......... AAAGAAATAAGATCTACTTTATGCAGCGCCAGGATGTGCTTGACAAAA--ATCATGTGGC C..C...C.G.GA......ACG......G..A.TG.CAT.A.G...T.TGGAG.CAAA.G
N P Ii
891 174 075
T.....C..T.....G...........T........T.......-....G.....TG... AGACATTGACAAGTTAATTGACTATGCAGCCTCTGGAGACCCTA-CATCACCTGACAACA ..T..AA.G.T.--..CGCC.GC......AAG.A.CT.G.TG..G...G:T...AG.T.
N P Ii
950 233 932
.C..A..T.................A.....T...........T..G.....C....... TTGATTCGCCTAATGCACCATGGGTCTTTTGCATGTGCACCAGACCGATGCCCACCAACAT . . ..G..A..ATCAT..AT......T.....TG.A...........T..T..........
N 1010 P 293 H 332
. . ..T........T........T..AT.A..T..A..C.....C..C..A.....r.... GTATCTATGTTGCAGGGATGGCAGAGCTTGGGGCCTTTTTTTCAATATTGCAGGACATGA ..T.G.T.A.A.....T..T..T...........A........C..CC...........C
N 1070 P 353 H 1052
.G.....C..T...........A.....G..................C.G..A..G GAAACACAATAATGGCATCTAAGACTGTTGGCACAGCAGAAGAGAAATTPAAGAAAAAGT . . ..T.....C............ .A.....A...T.T..G.....GC..CG...G..A.
N 1130 P 413 H 1112
.C.....C..T.....A..TT....C.....A...........G.....A........G. CTTCCTTTTACCAATCTTACCTGCGTCGAACTCAATCAATGGGAATTCAGCTTGATCAAA .A..A.....T..G..C.....CA.AA.G..A...........G..A..A..A.GC..G.
N 1190 P 473 H 1172
.G..A.....AC...AC..............A.G...A.....G.....T....... GAATTATCCTCTTGTTTATGTTGGAATGGGGCAAAGAGATGGTAGATCACTTCCATCTTG . . . . . ..TG.GC.C..C...G.T.CC.....A..G...GCT..G..CA.......CT.A.
N 1250 P 533 H 1232
._ GT .G
133
A
0M
N P H
557 1 547
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..T............R.......R.... VCQYECETAKELESHRKSCSIGSCPYCLNPSEATPSALQAHFKVCKLTSRFQENLKKSLT ..K.....Y...KA.GV..PPSQ....FTHC.P.EA.F...Y...QV.H..RDD...TV.
N P
617 61 607
V ........................... V...HH........................R. MYEPMQGCYRTLSLFRYRSRFFVGLVWCMLLVLELIVWAASAETQNLNAGWTDTAHGSGI PQNFTP......N....K..CYIFTM.IF.....S.L.....SETP.TPV.N.N...V
N P H
677 121 667
.. ..T........Q..........................L ................... IPMKADLELDFSLPSSASYTYRRQLQNPANEQEKIPFHLQISKQVIHAEIQHLGHWMDAT V..HT........T..SK.....K.T..LE.AQS.DL.IE.EE.T.GVDVHA....F.GR
N P H
737 181 727
. FN L.
N
226
. . . . . ..P.K........................V.F....RV...T.............
P H
1 229
SFFVKDWSERIREFMEKECPFIKPEVKPGTPAQEIEMLKRNKIYFMQRQOVLDKNHVADI AKDWS.RI.QWLIEPC.LL.DTAAVSLL.G..-------T.RD.LR...VA.GNMETKES
285 60 281
286 61 282
. . . . . . . . . . . . . . ..D.E......................................... DKLIDYAASGOPTSPDNIDSPNAPWVFACAPDRCPPTCIYVAGMAELGAFFSILQDMRNT KAIRQH.EAAGCSMIED.E..SSI....G.........LFI..I................
345 120 341
346 121 342
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Y.....R......... IMASKTVGTAEEKLKKKSSFYQSYLRRTQSMGIQLDQRIILLFMLEWGKEMVDHFHLG . . . . . . . ..S....R....................G....V...VA....A..N....
H
; H
616 SO 606
.S
676 120 666 736 180 726
403 178 399
Fig. 3. Amino acid sequence comparison of Puumala, Hantaan and NEV M and S segment encoded polypeptides. An alignment of the Puumala M (panel A) and S (panel B) ammo acid sequences deduced from the PCR cDNAs with the corresponding regions of the NEV and Han&an M and S segment encoded polypeptides is shown. The dots denote identical amino acid residues, the dashes residues missing in either one of the sequences. The numbers indicate the ammo acid positions of the M and S segment encoded polypeptides.
53% homology, respectively (Fig. 3A, B). There are almost no conservative amino acid exchanges; only one out of 10 differences for the Puumala and NEV comparison and only two out of 81 differences for the Puumala and Hantaan comparison are conservative exchanges. The 178 amino acids encoded by the Puumala S segment specific cDNA were also compared to those of the NEV S segment (ORF: nucleotide positions 43-1342; Stohwasser et al., 1989) and Hantaan virus S segment (ORF: nucleotide positions 37-1324; Schmaljohn et al., 1986). The Puumala S segment specific polypeptide corresponds to NEV ammo acid positions 226-403 and Hantaan positions 229-399. The amino acid homology between the Puumala S segment specific polypeptide and the corresponding sequences of NEV and Hantaan is 94% and 55% (Fig. 3A, B), respectively. Again there are almost no conservative
Fig. 2. Nucleotide sequence comparison of Puumala, Hantaan, and NEV M and S segment cDNAs. An alignment of the Puumala M (panel A) and S (panel B) nucleotide sequences cloned by PCR with the corresponding regions of NEV and Hantaan M and S segments is shown. N, P, and H denote NEV, Puumala and Hantaan sequences. The dots depict identical nucleotides and the dashes nucleotides missing in either one of the sequences. The numbers indicate the nucleotide positions of the M and S segments.
134
amino acid exchanges: none of the 11 differences in the Puumala and NEV comparison is conservative and only one out of 72 is conservative for the Puumala and Hantaan alignment. These protein data are consistent with the nucleotide sequence data, since they also demonstrate that Puumala is extremely similar to NEV but very different from Hantaan virus. Apparently for many amino acid residues of the hantavirus S and M encoded polypeptides there may be little selective pressure for conservation, which is consistent with the observation that amino acid homologies between Hantaan and NEV or Puumala encoded proteins is lower than the nucleotide homologies of the corresponding genomic segments.
Discussion Evidence for Huntauirus infections has been reported from all parts of the world and about 100 different isolates have been collected so far. To study the genetic variance of this important virus group and to relate this variance to the severity of HFRS caused in human infections it is necessary to molecularly clone their genomes and determine the nucleotide sequences. However, the task of analyzing so many different isolates by conventional cloning procedures is not feasible. Therefore we studied the use of the polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis to rapidly detect genomic variations in different Hantauirus strains. For this study we chose Hantaan virus strain 76-118 and nephropathia epidemica virus strain Hallnb Bl whose M and S genomic RNA segments have been sequenced (Schmaljohn et al., 1986, 1987; Giebel et al., Stohwasser et al., 1989). These were compared with the Puumala virus strains which cause nephropathia epidemica in humans. Using oligonucleotides derived from NEV (HallnHs Bl) M and S segment nucleotide sequences which had been found to be conserved in the genome of the Hantaan virus it was possible to isolate the corresponding sequences by a PCR assay from Hantaan, NEV and Puumala viruses. The successful cloning of the genomic sequences of these hantaviruses by the PCR technique shows that this approach works even if the oligonucleotide used for PCR are not perfectly matched. The cloning of genomic fragments derived from Ruumala virus M and S segments by the PCR approach and the subsequent nucleotides sequence analysis revealed that this virus is very similar to the NEV strain Hlllnas Bl. A comparison of the Puumala and NEV sequences showed over 80% homology at the nucleotide level and 94% homology at the deduced amino acid level. A comparison of the Puumala sequences to those of Hantaan revealed 63% nucleotide homology and 53 and 55% amino acid homology for the corresponding regions of the M and S encoded polypeptides, respectively. The results reported in this paper demonstrate that the polymerase chain reaction technique is a powerful tool to rapidly detect genomic variations in hantaviruses. Furthermore, our results confirm the common antigenic sites of the M and S segment encoded proteins between NEV Hallnls Bl and Puumala virus. This should
135
be considered for the development of specific based on recombinant DNA technology.
diagnostic
and prevention
systems
Acknowledgements This study was supported by the Bundesministerium fir Forschung und Technologie, BMFT grant No 0318973A. We thank Drs H.W. Lee, B. Niklasson and J. Pilaski for providing the Hantaoirus strains and the sera used. We gratefully acknowledge Dr C.S. Schmaljobn for providing the Hantaan M and S segment cDNA clones. Finally the authors thank MS Ulrike Heinz for excellent technical assistance.
References Baek, L.J., Yanagihara, R.Y., Gibbs, C.J.JR., Miyazaki, M. and Gajdusek, D.C. (1988) Leaky virus: a new Hantavirus isolated from Mus muxu1u.r in the United States. J. Gen. Virol. 69, 3129-3132. Bishop, D.H.L. (1985) Replication of Arenaviruses and Bunyaviruses. In: B. Fields, D.M. Knipe, R.M. Chanock, J.L. Melnik, B. Roizman and R.E. Shope (Eds), Virology, pp. 1083-1118. Raven Press, New York. Chirgwin, J.M., Pryzybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5295. Giebel, L.B., Stohwasser, R., ZBller, L., Bautz, E.K.F. and Darai, G., (1989) Determination of coding capacity of the M genome segment of nephropathia epidemica virus strain HBllnas Bl by molecular cloning and nucleotide sequence analysis. Virology 172, 4988505. Lee, H.W., Lee, P.W. and Johnson, K.M. (1987) Isolation of the etiologic agent of korean hemorrhagic fever. J. Infect. Dis. 137, 298-308. Lee, H.W. and Dahymple, J.M. (1989) Manual of hemorrhagic fever with renal syndrome. WHO Collaborating Center for Virus Reference and Research. Institute for Viral Diseases, Korea University. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1985) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Niklasson, B. and LeDuc, J. (1984) Isolation of the nephropathia epidemica agent in Sweden. Lancet 1, 1012-1013. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Schmaljohn, C.S., and Dalrymple, J.M., (1983) Analysis of Hantaan virus RNA: evidence of a new genus of Bunyaviridae. Virology 131, 482-491. Schmaljohn, C.S., Jennings, G.B., Hay, J. and Dalrymple, J.M. (1986) Coding strategy of the S genome segment of Hantaan virus. Virology 155, 633-643. Schmaljohn, C.S., Schmaljohn, A.L. and Dalrymple, J.M. (1987) Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order. Virology 151, 31-39. Stohwasser, R., Giebel, L.B., Zbller, L., Bautz, E.K.F. and Darai, G. (1989) Molecular characterization of the RNA S segment of nephropathia epidemica virus strain HBllnPs Bl. Virology 173, in press. Tabor, S. and Richardson, C.C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767-4771.
136 Yanagihara, R., Goldgaber, D., Lee, P.W., Amyx. H.L., Gjdusek, DC.. Gibbs, C.J.JR. and Svedomyr, A. (1984) Propagation of nephropathia epidemica virus in cell culture. Lancet 1,1013. Yoo, D. and Kang, C.Y. (1987) Nucleotide sequence of the M segment of the genomic RNA of Hantaan virus 76-118. Nucleic Acids Res. 15, 6299-6300. Zoller, L., Scholz, J., Stohwasser, R., Giebel, L.B., Sethi, K.K., Bautz, E.K.F. and Darai, G. (1989) Immunblot analysis of the serological response in Hanta virus infections. J. Med. Viral. 27, 231-237. (Received
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1989; revision
received
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1990)
