Wu.s Research, 26 (1992) 1-14 0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1702/92/$05.00
VIRUS 00823
Typing of Hantavimses from five continents by polymerase chain reaction Pilaipan
Puthavathana
a, Ho Wang Lee b and
C.Yong Kang a
a Department
of Microbiology and Immunology, University of Ottawa, Faculty of Medicine, Ottawa, Ont., Canada and ’ The Institute for viral Diseases, Korea University, College of Medicine, Seoul, South Korea
(Received 6 May 1992; revision received and accepted 19 June 1992)
Summary Huntuvirus, a genus in the family Bunyaviridae, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. The present communication reports the use of polymerase chain reaction (PCR) for typing 27 independently isolated Hanfavimes from 5 different continents. Total cellular RNA was extracted from virus-infected Vero E6 cell monolayers by the acid guanidium thiocyanate-phenol-chloroform method. We have utilized 5 different sets of oligonucleotide primers ranging from 18 to 22 nucleotides in length; one set was specific for a conserved region of the S genomic segment and used as genus-specific primers, the other 4 sets of primers were designed from unique sequences of the M genomic segment such that each primer set was specific to only one serological type of Huntavims. The PCR products were analyzed by restriction endonuclease digestion for further confirmation. We typed 10, 12, 3 and 1 isolates into Hantaan, Seoul, Puumala and Prospect Hill respectively. The results of PCR were 100% agreeable with that of serological typing, and thus, PCR can be used as an adjunct test with serological method(s) or an independent test for diagnosis and for typing of new isolates of Huntaviruses. Huntavim;
Polymerase chain reaction; Restriction
endonuclease
digestion
to: C.Y. Kang, Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ont. KlH 8M5, Canada. Fax: (1) (613) 738-5379.
Correspondence
2
Introduction Hantauims, a genus belonging to the family Bunyaviridae, possesses a negative sense RNA genome in tripartite segments designated as S, M and L. The S genomic segment encodes the nucleocapsid (N) protein, the M genomic segment encodes two envelope glycoproteins (Gl and G2) and the L genomic segment is presumed to encode the viral RNA polymerase (L) protein (Antic et al., 1992). Based on the known nucleotide sequences, members of the genus share some degree of homology (Antic et al., 1992) and they also cross-react serologically (Lee et al., 1985; Sugiyama et al., 1987). It has been generally accepted that the genus Hantuuirus is comprised of at least four distinct serotypes: Hantaan (HTN), Seoul (SEO), Puumala (PUU) and Prospect Hill (PH) viruses. Their major reservoir hosts are Apodemus (mouse), Rattus (rat), Clethrionomys (bank vole) and Microtus (meadow vole) respectively (Lee et al., 1985; Lee and Van der Groen, 1989; Sugiyama et al., 1987). Hantaan virus causes the most severe form of hemorrhagic fever with renal syndrome (HFRS). While Seoul virus causes less serious disease, Puumala virus causes only a mild form of HFRS designated nephropathia epidemica, and Prospect Hill virus (PHV) has not been associated with any human disease. In addition to the four well-characterized serotypes of Hantauiruses, new serotypes have been proposed by a few laboratories (Back et al., 1988; Lee and Van der Groen, 1989). Based on serological information, Leakey virus, an isolate from Mus musculus in the USA (Baek et al., 19881, and Maaji, an isolate from Apodemus in Korea (Lee and Van der Groen, 1989) have been proposed as two new serotypes. Recently, an isolate from Bandicota in&x (rat) in Thailand, and another isolate from Suncus murinus (shrew) in India have been quoted as the candidates for the fifth and sixth types, but no solid data have been shown (Antic et al., 1992; Xiao et al., 1992). In our study we designed 5 sets of PCR oligonucleotide primers based on sequences of the four accepted serotypes. One set of primers was designed from the conserved region of S genomic segments of various Hantuuiruses, which has been designated the genus-specific primers. The other 4 sets of primers were designed from the unique sequence of the M genomic segments of each serotype, such that each set was specific either to Hantaan, Seoul, Puumala or Prospect Hill serotype only; thus, they were designated as serotype-specific primers. These PCR primer sets were used to type 27 Huntavirus isolates from 5 different continents including one of the viruses of unclear serotype (Leakey). Our clear results should encourage the application of PCR for typing the Huntavirus strains worldwide.
Materials
and Methods
Cell culture Vero E6 cell line (ATCC 1008, CRL 1586) was used for virus propagation. The cells were grown in minimum essential medium supplemented with 10% fetal calf
3 TABLE
1
Hantacitus isolates
and their serotypes
Virus Isolate Asia A9 JTRN/82/17 i/RN/82/3 JinHae 87/526 Maaji ROK 79/90 SNUS/Hamster 85/4 US 84/2 76/118 80/39 83/14 83/138 Thailand no. 605 Thailand no. 749 Europe GBB NE 672 Yanagihara Cg 3883 Hallnas Bl Fojnica North America Baltimore rat Houston rat no. 4 Lcakey Prospect Hill virus I Tchoupitoulas no. 401613 South America Brazil 2-4 Africa Egypt R/12915
Country
Host of Origin
Serotype
China Japan Korea Korea Korea Korea Korea Korea Korea Korea Korea Korea Thailand Thailand
Apodemur agrarius Rattus norcegicus Rattus norvegicus Apodemus agrarius Apodemur agrarius Human Syrian hamster Human Apodemus agrarius Rattus norcegicus Apodemus agrarius Apodemus agrarius Rattus nomegicus Bandicota indica
HTN SE0 SE0 HTN HTN HTN SE0 HTN HTN SE0 HTN HTN SE0 SE0
England Finland Finland Russia
Laboratory rat Clethrionomys glareolus Clethrionomys glareolus Clethrionomys glareolus
SE0 PUU PUU HTN
Sweden Yugoslavia
Clethrionomys glareolus Apodemus flacicollis
PUU HTN
USA USA USA USA USA
Rattus norvegicus Rattus norvegicus Mus musculus Microtus pennsyluanicus Rattus norcegicus
SE0 SE0 ? PH SE0
Brazil
Rattus nomegicus
SE0
Egypt
Rattus norvegicus
SE0
serum (Grand Island Biological Company (GIBCO), New York, USA), 100 Upg/ml penicillin-streptomycin, 100 pg/ml kanamycin and 2 mM/ml glutamine (GIBCO). Viruses
A total of 27 Huntmims isolates from 5 continents were studied. These viruses represent the major isolates of Huntuuiruses. Detailed information on these viruses are shown in Table 1. The viruses were allowed to grow in Vero E6 cell monolayers at 35°C for 9-30 days, then the infected cells were harvested for immunofluorescence study and RNA extraction. This study also included Indiana serotype of vesicular stomatitis virus (VSV) as the negative control.
4
Serological typing
Serological typing of the Hantauirzu isolates was performed by plaque reduction neutralization test at the WHO Collaborating Centre for Virus Reference and Research (Hemorrhagic fever with renal syndrome), Institute for Viral Diseases, Korea University, Korea. Immunofluorescence
test
Indirect immunofluorescence test was used to confirm the viral infection in Vero E6 cell cultures. Briefly, the infected cells were smeared on a glass slide, air-dried and fixed in pre-cooled acetone at 4°C for 10 min. The fixed slides were stained with rat antisera to Hantaan virus strain 76-118 for 30 min at 37°C before rinsing and soaking in PBS. Fluorescein isothiocyanate-conjugated goat anti-rat IgG (Sigma, St. Louis, MO, USA) was then applied, and the staining process was as described for the first antibody. The slides were observed under fluorescence microscope on which the virus-infected cells showed fluorescent antigen localizing in the cytoplasm. RNA extraction
Total cellular RNA was extracted from the infected Vero E6 cell monolayer by using the acid guanidium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). The infected cells were lysed by the buffer containing 4 M guanidium thiocyanate; 25 mM sodium citrate, pH 7; 0.5% sarcosyl; 0.1 M 2-mercaptoethanol. Sequentially, 0.1 Vol. of 2 M sodium acetate pH 4, 1 Vol. of water saturated phenol, and 0.2 Vol. of chloroform-isoamyl alcohol mixture was added. The final suspension was shaken vigorously, cooled on ice for 15 minutes and then centrifuged at 10,000 x g for 20 min at 4°C. The aqueous phase was collected and mixed with 1 volume of isopropanol, then kept at -20°C for at least one hour to precipitate RNA. The RNA was pelleted by centrifugation at 10,000 x g for 20 min at 4°C. The pellet was then dissolved in the buffer as described above; the RNA solution was again reprecipitated. The final RNA pellet was washed with 70% ethanol, air-dried and dissolved in diethylpyrocarbonate (DEPC)-treated water. The average RNA yield ranged from 80 to 100 pg for one T25 flask of the infected cells solubilized with 1 ml of the buffer. Primers
This study employed 5 sets of oligonucleotide primers. The first set was designated genus-specific and was expected to be reactive to all members of the genus. This set consisted of one pair of primers and was designed from the conserved region of the S genomic segments of 76-118, SR-11, HHllnas Bl and PHV-I (Schmaljohn et al., 1986; Arikawa et al., 1990; Stohwasser et al., 1990; Parrington and Kang, 1990). Details of this primer pair are shown in Fig. 1.
5 Serotype
GenusSpecific
376393 12341253
SE0
382-399
(SR-11) 1240-1259
382-399
Bl) 1252-1271
PH (Prospect Hill-I)
Sequence
Sense
382-399
Function
Products @PS)
=‘GGCCAGACAGCAGATTGG”-“CTACTGTACCTiGGACTCGA5’ %ATGAcATGGATCCTGAGCF
HTN (Hantaan 76-118)
PUU (HPllnls
Primer
Nucleotide Position
+ -
cDNA synthesis and Downstream primer Upstream primer
S’-- A__ ____ _----C---3’878
j 5’___-T______________~~ ~~_A________T_-----3’5’--____--------A I/
g_--_--_----s-_-m-
_____
3’
878
_-__-__r-
_________
890 ------_3’
5’___-__-----~_____-3’_ 890
1252_~27~j~~----~------__~_____3’1
Fig. 1. Genus-specific oligonucleotide primers for Huntavimses as designed from different S genomic sequences. Arrows show direction of DNA synthesis, boxed regions represent the upstream primer binding site; and dashed lines represent homologous nucleotide sequences to the genus-specific primers. Mismatches are shown by bases in the dashed lines.
The other 4 sets of oligonucleotide primers were designated serotype-specific primers which were designed from the unique sequence of the M genomic segments of 76-118, 80-39, HglllnHs Bl and PHV-I (Schmaljohn et al. 1987; Yoo and Kang, 1987; Arikawa et al., 1990; Giebel et al., 1989; Parrington and Kang, 1990). Each set consisted of 3 primers: one primer for the reverse transcriptase reaction and two primers for DNA amplification. We have used two downstream primers, one for the reverse transcription and another internal downstream primer for DNA amplification in order to avoid non-specific amplifications. This approach increased specificity and generated clean PCR products. One virus isolate was expected to react with one primer set only. Detailed information of these primer sets has been shown in Fig. 2.
cDNA synthesis
The method for cDNA synthesis was modified from Frohman et al. (1988). Approximately 5-10 pg of total cellular RNA and 25 pmol of each template primer were used. The RNA was suspended in DEPC-treated water and heated to 65°C for 3 min before quenching on ice. The reaction tube was then brought up to a final volume of 20 ~1 which contained 1 X reverse transcription buffer (50 mM Tris-HCI pH 8.15 at 41°C; 6 mM MgCI,; 40 mM KCI; 1 mM dithiothreitol); 1.5
6
Serotype
Sense
Primer Sequence
Nucleotide Position
Function
Product @PS)
HTN (Hantaan 76-118)
79-97 270-291 711-730
5’ATGGCCTGT’ITTGACACTGSYATACCCAAGTAAG’lTGGAGAGG3~ ,3’lTGTCCAA’fTCTTTAGGAAAs’
+ + -
cDNA synthesis Downstream primer Upstream primer
461
SE0 (Seoul 80-39)
79-97 270-291 71 l-730
~TGGCCA4GGCTTTGCA’ITA~sTAACCAAGGTCATATGGCGGA43~ SGACGGTAGTGTAGCCGTTAC”
+ + -
cDNA synthesis Downstream primer Upstream primer
461
PUU (HQlln& Bl)
79-97 294315 735-754
5’CCAGGGTCTATTACTATGTY5’GGACATGGGAMl-MAAGGTGA3~ ~~‘TcTAAC~TTTCTTGAAACTC~
+ + -
cDNA synthesis Downstream primer Upstream primer
461
PH (Prospect HiIt-I)
83.101 256-277 970-989
S’GGAGTACTACTACTGCAGG3’YGCATACAAGCTCAGCCACACAG3~ ~CCGTGGTAGGTGACTCAGTAY
+ + -
cDNA synthesis Downstream primer Upstream primer
734
Fig.
2. Serotype-specific
oligonucleotide represent
primers
as designed from M genomic of DNA synthesis.
sequence.
Arrows
the direction
mM of each dNTP (Pharmacia, Uppsala, Sweden); 10 units of RNasin (Promega Biotec, Madison, WI, USA); and 10 units of avian myeloblastosis virus reverse transcriptase (Pharmacia), and incubated at 41°C for 2 h. The reaction mixture containing cDNA was added to 480 ~1 of 10% TE (1 mM Tris-HCl pH 7.5; 0.1 mM EDTA pH 8) and kept at -20°C until used. Each RNA sample was used for 5 template primers in 5 different reactions of cDNA synthesis.
Polymerase chain reaction
We have followed the PCR protocol of Frohman et al. (1988). Briefly, a 50 ~1 total volume of the PCR tube contained 2.5-5 ~1 of cDNA, 25 pmol of each primer, 100 PM of each dNTP and 2.5 units of Taq DNA polymerase (Pharmacia, or GIBCO BRL, New York, USA) in the reaction buffer containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl, and 0.01% (w/v> gelatin, and was overlaid with 50 ~1 of mineral oil. The reaction was carried out in a DNA thermal cycler (Perkin-Elmer-Cetus) for 36 cycles as follows: the first cycle was 95°C for 2 min, 48-57°C for 2 min and 72°C for 40 min; the second to the thirty-sixth cycles were 95°C for 40 s, 48-57°C for 2 min and 72°C for 3 min followed by 12 min at 72°C for final extension. Usually, the annealing temperature for PCR was carried out at 55°C except 50°C for that of A9, JinHae 87/526, Brazil 2-4 and Hall& Bl, and 48°C for those of Thailand nos. 605 and 749 in the serotype-specific amplification. The amplified product was visualized by electrophoresis using 5-10% of total DNA in 1.5% agarose gel with 0.5 X Tris-Borate-EDTA buffer and followed by staining in ethidium bromide.
7 TABLE Restriction Enzyme
2 endonuclease
sites on the amplified
Restriction
DNA products
sites on the products
HTN (NT Pos. *)
amplified
by primers
of
(NT ~0s.)
PUU (NT Pas.)
:lz
SE0
Pas.)
BstYl
636 (94/367)
None
None
870
Rmal
312
654
758
Nsp 1
366,492
431 (162/299) 488,492,640
(q1;4,307j
W’Ml
None
None
None
447, 477, 516,618 637 (352/382)
* NT Pos. = Nucleotide position. Fragment length after digestion is given in parentheses.
Restriction endonuclease digestion
The amplified DNA was purified by standard phenol-chloroform extraction (Sambrook et al., 1989) before digestion with restriction endonuclease. The product amplified by Hantaan, Seoul and PH-specific primers was digested with enzymes BstYl (New England Biolabs Inc. NEB, MA, USA), Real (NEB), and PflMl (NEB) respectively, while those amplified by Puumala primers were digested with either Nspl (Boehringer Mannheim, Germany) or Bell (Pharmacia). 2.5 to 4 units of the enzymes were used for cleaving S-15% of the PCR product in 3-4 h under the reaction condition described by the company. The digested product was detected by electrophoresis in a 2% agarose gel and followed by staining with ethidium bromide. The expected sizes of the digested products as shown in Table 2 were calculated from the cleavage site of that enzyme on the M genomic segment of Hantaan 76-118, Seoul 80-39, Hillnh Bl and PHV-I (Yoo and Kang, 1987; Antic et al., 1991; Giebel et al., 1989; Parrington et al., 1991).
Results Polymerase chain reaction A. Genus-specific primers
Fig. 3 shows that the genus-specific primers could amplify sequences from all 27 Vero E6 cells. The lengths of the amplified fragments were expected to range from 878 to 890 nucleotides (Fig. 1) which is consistent with the results shown here. However, differences in fragment sizes between the isolates could not be clearly seen. Hantauirus isolates but not from the VSV or uninfected
GENUS primers Fig. 3. PCR
products
of 27 Huntavirus isolates using digested 4X-174 RF-DNA
genus-specific oligonucleotide is used as markers.
B. Serotype-specific primers The 4 sets of serotype-specific primers (Hantaan, Seoul, primers) were used to amplify sequences from 27 Huntavinu
HTN primers
PUU primers
primers.
Puumala isolates,
Hue111
and PH VSV and
SE0 primers
PH primers
Fig. 4. PCR products of 27 Huntuuirus isolates using four different sets of serotype-specific HaeIII-digested 4X-174 RF-DNA is used as markers. o indicates positive PCR.
primers.
9 TABLE
3
Comparison
on typing of Hantauirus isolates
by PCR and serological typing by PRNT
methods PCR typing
Virus strain
Serological
A9 JinHae 87/526 Maaji ROK 79/90 US 84/2 76/118 83/14 83/138 Cg 3883 Fojnica JTRN/82/17 i/RN/82/3 SNUS/Hamster 85/4 80/39 Thailand no. 605 Thailand no. 749 GBB Baltimore rat Houston rat no. 4 Tchoupitoulas no. 401613 Brazil 2-4 Egypt R/12915 NE 672
Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul
Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul
Seoul
Seoul
Seoul
Puumala
Seoul Puumala
Hallnis Bl Yanagihara Prospect Hill I Leakey
Puumala Puumala Prospect ?
Puumala Puumala Prospect Hill Seoul-Puumala
Hill
uninfected Vero cells. We found that each of 26 isolates reacted with only one of the 4 sets of primers (Fig. 4). Our results demonstrated that 10, 12, 3 and 1 isolates belong to Hantaan, Seoul, Puumala and Prospect Hill types respectively. One isolate named “Leakey” reacted with both Seoul and Puumala specific primers; thus, it was untypable. VSV and uninfected Vero E6 cells were not reactive with any of the primer sets. Our results of typing Hantavimses by PCR was compared to that of serological typing and in all instances the results agreed (Table 3). Restriction endonuclease cleavage
The PCR products were confirmed for their specific amplification by restriction endonuclease digestion. Restriction sites for each enzyme were based on the known sequence of representative strains of the serotype, i.e. 76-118 for Hantaan, 80-39 for Seoul, Hallnas Bl for Puumala and PHV-I for Prospect Hill as has been shown in Table 2.
10
All 10 DNA products amplified by Hantaan specific primers were cleaved by enzyme BstYl and gave rise to two fragments of size 94, and 367 base pairs. All 13 products (including Leakey) amplified by Seoul primers were cleaved by Real and gave rise to two fragments of size 162 and 299 base pairs. PHV-I, the only isolate amplified by PH primers was digested by PfEMl and yielded two fragments sized 352 and 382 base pairs, as expected. Restriction endonuclease sites common to isolates in the Puumala type have not been found yet. Digestion of 4 PCR products (including Leakey) amplified by Puumala primers with enzyme Nspl yielded 3 cleavage patterns. The product amplified from NE672 was cleaved and yielded 2 nucleotide fragments of size 154 -and 307 base pairs which was expected from published sequence of HBlllnas Bl (Giebel et al., 1989). In contrast, the product from our Hallnh Bl isolate was not cleaved at that expected site; it yielded 2 visible bands which were smaller in size. Products from Yanagihara and Leakey viruses showed the same cleavage pattern; the amplified product seemingly was cut at the middle and yielded only one thick visible band (Fig. 5).
Discussion
PCR has been introduced as a method to study the genetic variation and relatedness among Huntuvirus isolates during recent years. Tang et al. (1990) were successful in an attempt to differentiate Hantaan- and Seoul-related strains by using 2 pairs of primers specific for either Hantaan or Seoul M genomic segments. PCR has been used to amplify Puumala-related viruses and subsequently the genomic variation between strains was analysed by nucleotide sequencing of the amplified DNA products (Giebel et al., 1990). Xiao et al. (1992) designed a genus common primer pair from M genomic segments to amplify 41 Huntuvirus isolates, and then analyzed the amplified products by using a panel of restriction endonucleases. One isolate failed to be amplified and the cleavage patterns of the digested PCR products were very diverse even among the strains of the same serotypes. These results strongly indicated genetic diversity among Huntuuiruses and also implied that this was not an ideal approach for typing new virus isolates. We demonstrate here that serotype-specific primers must be used in order to type Huntuvirus isolates. Comparison of nucleotide sequences of Huntuvirus genomes shows that S genomic sequences are more conserved than M genomic sequences (Antic et al., 1992). In this study, we designed the genus-specific primers from the consensus sequence of S genomic segments. The primers were capable of amplifying all 27 isolates studied (Fig. 5). Conversely, serotype-specific primers were designed from the unique sequences of M genomic sequences. Our results showed that the PCR technique could group 26 of 27 Huntuvirus isolates into 4 different types: Hantaan, Seoul, Puumala and Prospect Hill. Only Leakey virus was untypable because it was amplified by both Seoul and Puumala primers. In addition, its patterns of restriction endonuclease cleavage were the same as other isolates within both the Seoul
11
and Puumala serotypes (Fig. 5). There are three possible explanations for this: (1) Leakey virus may be a mixture of Seoul and Puumala viruses due to accidental contamination in the laboratory during the process of virus propagation; (2) Leakey virus was isolated from the animal which had dual infections between the two viruses or (3) Leakey virus is a recombinant virus between Seoul and Puumala serotypes. Nucleotide sequencing of the plaque-purified virus might help to clarify this point.
-461
461. 367-
-299 -162
94-
HTN
-302 -352
-230
PUU
PH
Fig. 5. Endonuclease cleavage patterns of the amplified DNA products. Lanes l-3 are products of Hantaan 76-l 18, Maaji and cg 3883 as amplified by Hantaan-specific primers. Lane lE-3E are Hantaan products digested by BsfYl. Lanes 4-6 are products of Seoul 80-39, Egypt R/12915 and Leakey as amplified by Seoul-specific primers. Lanes 4E-6E are Seoul products digested by Real. Lanes 7-10 are products of Hallnls Bl, NE672, Yanagihara and Leakey as amplified by Puumala-specific primers. Lanes 7E-10E are Puumala products digested by Nspl. Lane 11 is PHV-I product as amplified by PHV-specific primer and lane 11E is PHV product digested by PfIMl. M is HaelI1 digested 4X-174 RF-DNA.
12
When we digested amplified DNA products with restriction endonucleases, we found that all products from viruses within the Hantaan serotype gave rise to only one cleavage pattern when digested with BstYl, as do products from viruses of the Seoul serotype digested with Real (Fig. 5). However, enzyme(s) which should cut all members of Puumala serotype within the amplified region have not been found yet. Moreover, the cleavage pattern of our HBllnEs Bl product did not correspond to the pattern expected from the published sequence of the Hahn% Bl (Giebel et al., 1989). Our amplified products were not cleaved by Bcfl (unpublished data), even though a site should exist according to sequence information. Amplified products were cleaved by Nspl but not at the expected position (Table 2 and Fig. 5). Our finding is consistent with previous reports (Antic et al., 1992; Xiao et al., 1992) and suggests that the published sequence of Hallnas Bl (Giebel et al., 1989) may be incorrect. Among 27 Huntavirus isolates investigated, some were amplified only by lowering the annealing temperature to between 48 and 50°C. These isolates were A9, JinHae X7/526, Thailand no. 605, Thailand no. 749, Brazil 2-4 and Hahn& Bl. This may reflect higher genomic heterogeneity from the prototype strains. Since these isolates were reactive with certain serotype-specific primers and their PCR products (except Hallnas Bl) yielded cleavage patterns corresponding to the prototype virus, they may be classified subtypes, but not as distinct serotypes. Results of serological typing of these isolates strongly supported our PCR typing (Table 3). Nucleotide sequencing of the PCR products should reveal a degree of homology among different strains belonging to the same type. Grankvist et al. (1992) have successfully amplified Puumala virus sequences by RT/PCR using RNAs extracted from the urinary tract cells, the respiratory tract cells and from peripheral blood mononuclear cells of patients with nephropathia epidemica. This result provides a new possibility that RT/PCR can be utilized as a direct diagnostic method for Hantuvirus infection using clinical specimens without in vitro amplification of viruses in cultured cells. Huntuuirus RNA was also detected in tissues of experimentally infected mice by RT/PCR and the sensitivity of this detection method was equal to the cell culture method (Xiao et al., 1991). These results argue the previous belief that viruses from clinical specimens must be amplified to be detected by RT/PCR, but suggest that RT/PCR can be used to detect Huntuuirus genomic sequences in clinical specimens and post-mortem tissues from patients with HFRS. In conclusion, the successful result in typing Huntuvirus isolates by PCR should encourage its application as an adjunct test with serological method(s) or as an independent test for typing of the new Huntuvirus isolates or for diagnostic purpose.
Acknowledgements
We thank Drs. K.E. Wright and E. Brown for their critical and constructive review of this manuscript. We thank M. Dobbs for his excellent help in computer
I3
analyses and N. Delcellier and D. McLean for their technical assistance. P.P. from Mahidol University, Bangkok, Thailand was supported by the Canadian International Development Agency. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. References Antic, D., Kang, C.Y., Spik, K., Schmaljohn. C., Vapalahti, 0. and Vaheri, A. (1992) Comparison of the deduced gene products of the L, M and S genome segments of Huntaciruses. Virus Res., in press. Antic, D., Lim, B.U. and Kang, C.Y. (1991) Molecular characterization of the M genomic segment of the Seoul 80-39 virus, nucleotide and amino acid sequence comparisons with other Hunta~iruses reveal the evolutionary pathway. Virus Res. 19, 47-X Arikawa, J., LaPenotiere, H.F., Iacono-Connors, L., Wang, M. and Schmaljohn, C.S. (1990) Coding properties of the S and the M genome segments of Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal syndrome. Virology 176, 114-125. Baek, L.J., Yanagihara, R., Gibbs Jr., C.J., Miyazaki, M. and Gajdusek, D.C. (1988) Leakey virus: a new Huntuvirus isolated from Mus musculus in the United States. J. Gen. Virol. 69, 3129-3132. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 1566159. Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002. Giebel, L.B., Stohwasser, R., Zoller, L., Bautz, E.K.F. and Darai, G. (1989) Determination of the coding capacity of the M genome segment of Nephropathia epidemica virus strain Hahn% Bl by molecular cloning and nucleotide sequence analysis. Virology 172, 498-505. Giebel, L.B., Zoller, L., Bautz, E.K.F. and Darai, G. (1990) Rapid detection of genomic variations in different strains of HuntuL~iruses by polymerase chain reaction techniques and nucleotide sequence analysis. Virus Res. 16, 127-136. Grankvist, O., Juto, P., Settergren, B., Ahlm, C., Bjermer, L., Linderholm, M., Tarnvik, A. and Wadell, G. (1992) Detection of nephropathia epidemica virus RNA in patient samples using a nested primer-based polymerase chain reaction. J. Inf. Dis. 165, 934-937. Lee, H.W. and Van der Groen, G. (1989) Hemorrhagic fever with renal syndrome. Prog. Med. Virol. 36, 62- 102. Lee, P.-W., Gibbs Jr., C.J., Gajdusek, D.C. and Yanagihara, R. (1985) Serotypic classification of Huntol;iruses by indirect immunofluorescent antibody and plaque reduction neutralization tests. J. Clin. Microbial. 22, 940-944. Parrington, M.A. and Kang, C.Y. (1990) Nucleotide sequence analysis of the S genomic segment of Prospect Hill virus: comparison with the prototype HuntucGw. Virology 175, 167-175. Parrington, M.A., Lee, P.-W. and Kang, C.Y. (1991) Molecular characterization of the Prospect Hill virus M RNA segment: a comparison with the M RNA segments of other Hunturirusev. J. Gen. Viral. 72, 1845-1854. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edit. Cold Spring Harbor Laboratory Press, N.Y. Schmaljohn, C.S., Jennings, G., Hay, J. and Dahymple, 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 157, 31-39. Stohwasser, R., Giebel, L.B., Zoller, L., Bautz, E.K.F. and Darai, G. (1990) Molecular characterization of the RNA S segment of nephropathia epidemica virus strain Hahnas Bl. Virology 174, 79-86. Sugiyama, K., Morikawa, S., Matsuura, Y., Tkachenko, E.A., Morita, C., Komatsu, T., Akao, Y. and Kitamura, T. (1987) Four serotypes of haemorrhagic fever with renal syndrome viruses identified by polyclonal and monoclonal antibodies. J. Gen. Viral. 68, 979-987.
14 Tang, Y.W., Ruo, S.L., Sanchez, A., Fisher-Hoch, S.P., McCormick, J.B. and Xu, Z.Y. (1990) Huntuuirus strains isolated from rodentia and insectivora in rural China differentiated by polymerase chain reaction assay. Arch. Virol. 115, 37-46. Xiao, S.-Y., Yanagihara, R., Godec, M.S., Eldadah, Z.A., Johnson, B.K., Gajdusek, D.C. and Asher, D.M. (1991) Detection of Huntaoirus RNA in tissues of experimentally infected mice using reverse transcriptase-directed polymerase chain reaction. J. Med. Virol. 33, 277-282. Xiao, S.-Y., Chu, Y.-K., Knauert, F.K., Lofts, R., Dahymple, J.M. and LeDuc, J.W. (1992) Comparison of Hantacirus isolates using a genus-reactive primer pair polymerase chain reaction. J. Gen. Virol. 73, 567-573. Yoo, D. and Kang, C.Y. (1987) Nucleotide sequence of the M segment of the genomic RNA of Hantaan virus 76-l 18. Nucleic Acids Res. 15, 6299-6300.
VIRUS 00823
Typing of Hantavimses from five continents by polymerase chain reaction Pilaipan
Puthavathana
a, Ho Wang Lee b and
C.Yong Kang a
a Department
of Microbiology and Immunology, University of Ottawa, Faculty of Medicine, Ottawa, Ont., Canada and ’ The Institute for viral Diseases, Korea University, College of Medicine, Seoul, South Korea
(Received 6 May 1992; revision received and accepted 19 June 1992)
Summary Huntuvirus, a genus in the family Bunyaviridae, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. The present communication reports the use of polymerase chain reaction (PCR) for typing 27 independently isolated Hanfavimes from 5 different continents. Total cellular RNA was extracted from virus-infected Vero E6 cell monolayers by the acid guanidium thiocyanate-phenol-chloroform method. We have utilized 5 different sets of oligonucleotide primers ranging from 18 to 22 nucleotides in length; one set was specific for a conserved region of the S genomic segment and used as genus-specific primers, the other 4 sets of primers were designed from unique sequences of the M genomic segment such that each primer set was specific to only one serological type of Huntavims. The PCR products were analyzed by restriction endonuclease digestion for further confirmation. We typed 10, 12, 3 and 1 isolates into Hantaan, Seoul, Puumala and Prospect Hill respectively. The results of PCR were 100% agreeable with that of serological typing, and thus, PCR can be used as an adjunct test with serological method(s) or an independent test for diagnosis and for typing of new isolates of Huntaviruses. Huntavim;
Polymerase chain reaction; Restriction
endonuclease
digestion
to: C.Y. Kang, Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ont. KlH 8M5, Canada. Fax: (1) (613) 738-5379.
Correspondence
2
Introduction Hantauims, a genus belonging to the family Bunyaviridae, possesses a negative sense RNA genome in tripartite segments designated as S, M and L. The S genomic segment encodes the nucleocapsid (N) protein, the M genomic segment encodes two envelope glycoproteins (Gl and G2) and the L genomic segment is presumed to encode the viral RNA polymerase (L) protein (Antic et al., 1992). Based on the known nucleotide sequences, members of the genus share some degree of homology (Antic et al., 1992) and they also cross-react serologically (Lee et al., 1985; Sugiyama et al., 1987). It has been generally accepted that the genus Hantuuirus is comprised of at least four distinct serotypes: Hantaan (HTN), Seoul (SEO), Puumala (PUU) and Prospect Hill (PH) viruses. Their major reservoir hosts are Apodemus (mouse), Rattus (rat), Clethrionomys (bank vole) and Microtus (meadow vole) respectively (Lee et al., 1985; Lee and Van der Groen, 1989; Sugiyama et al., 1987). Hantaan virus causes the most severe form of hemorrhagic fever with renal syndrome (HFRS). While Seoul virus causes less serious disease, Puumala virus causes only a mild form of HFRS designated nephropathia epidemica, and Prospect Hill virus (PHV) has not been associated with any human disease. In addition to the four well-characterized serotypes of Hantauiruses, new serotypes have been proposed by a few laboratories (Back et al., 1988; Lee and Van der Groen, 1989). Based on serological information, Leakey virus, an isolate from Mus musculus in the USA (Baek et al., 19881, and Maaji, an isolate from Apodemus in Korea (Lee and Van der Groen, 1989) have been proposed as two new serotypes. Recently, an isolate from Bandicota in&x (rat) in Thailand, and another isolate from Suncus murinus (shrew) in India have been quoted as the candidates for the fifth and sixth types, but no solid data have been shown (Antic et al., 1992; Xiao et al., 1992). In our study we designed 5 sets of PCR oligonucleotide primers based on sequences of the four accepted serotypes. One set of primers was designed from the conserved region of S genomic segments of various Hantuuiruses, which has been designated the genus-specific primers. The other 4 sets of primers were designed from the unique sequence of the M genomic segments of each serotype, such that each set was specific either to Hantaan, Seoul, Puumala or Prospect Hill serotype only; thus, they were designated as serotype-specific primers. These PCR primer sets were used to type 27 Huntavirus isolates from 5 different continents including one of the viruses of unclear serotype (Leakey). Our clear results should encourage the application of PCR for typing the Huntavirus strains worldwide.
Materials
and Methods
Cell culture Vero E6 cell line (ATCC 1008, CRL 1586) was used for virus propagation. The cells were grown in minimum essential medium supplemented with 10% fetal calf
3 TABLE
1
Hantacitus isolates
and their serotypes
Virus Isolate Asia A9 JTRN/82/17 i/RN/82/3 JinHae 87/526 Maaji ROK 79/90 SNUS/Hamster 85/4 US 84/2 76/118 80/39 83/14 83/138 Thailand no. 605 Thailand no. 749 Europe GBB NE 672 Yanagihara Cg 3883 Hallnas Bl Fojnica North America Baltimore rat Houston rat no. 4 Lcakey Prospect Hill virus I Tchoupitoulas no. 401613 South America Brazil 2-4 Africa Egypt R/12915
Country
Host of Origin
Serotype
China Japan Korea Korea Korea Korea Korea Korea Korea Korea Korea Korea Thailand Thailand
Apodemur agrarius Rattus norcegicus Rattus norvegicus Apodemus agrarius Apodemur agrarius Human Syrian hamster Human Apodemus agrarius Rattus norcegicus Apodemus agrarius Apodemus agrarius Rattus nomegicus Bandicota indica
HTN SE0 SE0 HTN HTN HTN SE0 HTN HTN SE0 HTN HTN SE0 SE0
England Finland Finland Russia
Laboratory rat Clethrionomys glareolus Clethrionomys glareolus Clethrionomys glareolus
SE0 PUU PUU HTN
Sweden Yugoslavia
Clethrionomys glareolus Apodemus flacicollis
PUU HTN
USA USA USA USA USA
Rattus norvegicus Rattus norvegicus Mus musculus Microtus pennsyluanicus Rattus norcegicus
SE0 SE0 ? PH SE0
Brazil
Rattus nomegicus
SE0
Egypt
Rattus norvegicus
SE0
serum (Grand Island Biological Company (GIBCO), New York, USA), 100 Upg/ml penicillin-streptomycin, 100 pg/ml kanamycin and 2 mM/ml glutamine (GIBCO). Viruses
A total of 27 Huntmims isolates from 5 continents were studied. These viruses represent the major isolates of Huntuuiruses. Detailed information on these viruses are shown in Table 1. The viruses were allowed to grow in Vero E6 cell monolayers at 35°C for 9-30 days, then the infected cells were harvested for immunofluorescence study and RNA extraction. This study also included Indiana serotype of vesicular stomatitis virus (VSV) as the negative control.
4
Serological typing
Serological typing of the Hantauirzu isolates was performed by plaque reduction neutralization test at the WHO Collaborating Centre for Virus Reference and Research (Hemorrhagic fever with renal syndrome), Institute for Viral Diseases, Korea University, Korea. Immunofluorescence
test
Indirect immunofluorescence test was used to confirm the viral infection in Vero E6 cell cultures. Briefly, the infected cells were smeared on a glass slide, air-dried and fixed in pre-cooled acetone at 4°C for 10 min. The fixed slides were stained with rat antisera to Hantaan virus strain 76-118 for 30 min at 37°C before rinsing and soaking in PBS. Fluorescein isothiocyanate-conjugated goat anti-rat IgG (Sigma, St. Louis, MO, USA) was then applied, and the staining process was as described for the first antibody. The slides were observed under fluorescence microscope on which the virus-infected cells showed fluorescent antigen localizing in the cytoplasm. RNA extraction
Total cellular RNA was extracted from the infected Vero E6 cell monolayer by using the acid guanidium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). The infected cells were lysed by the buffer containing 4 M guanidium thiocyanate; 25 mM sodium citrate, pH 7; 0.5% sarcosyl; 0.1 M 2-mercaptoethanol. Sequentially, 0.1 Vol. of 2 M sodium acetate pH 4, 1 Vol. of water saturated phenol, and 0.2 Vol. of chloroform-isoamyl alcohol mixture was added. The final suspension was shaken vigorously, cooled on ice for 15 minutes and then centrifuged at 10,000 x g for 20 min at 4°C. The aqueous phase was collected and mixed with 1 volume of isopropanol, then kept at -20°C for at least one hour to precipitate RNA. The RNA was pelleted by centrifugation at 10,000 x g for 20 min at 4°C. The pellet was then dissolved in the buffer as described above; the RNA solution was again reprecipitated. The final RNA pellet was washed with 70% ethanol, air-dried and dissolved in diethylpyrocarbonate (DEPC)-treated water. The average RNA yield ranged from 80 to 100 pg for one T25 flask of the infected cells solubilized with 1 ml of the buffer. Primers
This study employed 5 sets of oligonucleotide primers. The first set was designated genus-specific and was expected to be reactive to all members of the genus. This set consisted of one pair of primers and was designed from the conserved region of the S genomic segments of 76-118, SR-11, HHllnas Bl and PHV-I (Schmaljohn et al., 1986; Arikawa et al., 1990; Stohwasser et al., 1990; Parrington and Kang, 1990). Details of this primer pair are shown in Fig. 1.
5 Serotype
GenusSpecific
376393 12341253
SE0
382-399
(SR-11) 1240-1259
382-399
Bl) 1252-1271
PH (Prospect Hill-I)
Sequence
Sense
382-399
Function
Products @PS)
=‘GGCCAGACAGCAGATTGG”-“CTACTGTACCTiGGACTCGA5’ %ATGAcATGGATCCTGAGCF
HTN (Hantaan 76-118)
PUU (HPllnls
Primer
Nucleotide Position
+ -
cDNA synthesis and Downstream primer Upstream primer
S’-- A__ ____ _----C---3’878
j 5’___-T______________~~ ~~_A________T_-----3’5’--____--------A I/
g_--_--_----s-_-m-
_____
3’
878
_-__-__r-
_________
890 ------_3’
5’___-__-----~_____-3’_ 890
1252_~27~j~~----~------__~_____3’1
Fig. 1. Genus-specific oligonucleotide primers for Huntavimses as designed from different S genomic sequences. Arrows show direction of DNA synthesis, boxed regions represent the upstream primer binding site; and dashed lines represent homologous nucleotide sequences to the genus-specific primers. Mismatches are shown by bases in the dashed lines.
The other 4 sets of oligonucleotide primers were designated serotype-specific primers which were designed from the unique sequence of the M genomic segments of 76-118, 80-39, HglllnHs Bl and PHV-I (Schmaljohn et al. 1987; Yoo and Kang, 1987; Arikawa et al., 1990; Giebel et al., 1989; Parrington and Kang, 1990). Each set consisted of 3 primers: one primer for the reverse transcriptase reaction and two primers for DNA amplification. We have used two downstream primers, one for the reverse transcription and another internal downstream primer for DNA amplification in order to avoid non-specific amplifications. This approach increased specificity and generated clean PCR products. One virus isolate was expected to react with one primer set only. Detailed information of these primer sets has been shown in Fig. 2.
cDNA synthesis
The method for cDNA synthesis was modified from Frohman et al. (1988). Approximately 5-10 pg of total cellular RNA and 25 pmol of each template primer were used. The RNA was suspended in DEPC-treated water and heated to 65°C for 3 min before quenching on ice. The reaction tube was then brought up to a final volume of 20 ~1 which contained 1 X reverse transcription buffer (50 mM Tris-HCI pH 8.15 at 41°C; 6 mM MgCI,; 40 mM KCI; 1 mM dithiothreitol); 1.5
6
Serotype
Sense
Primer Sequence
Nucleotide Position
Function
Product @PS)
HTN (Hantaan 76-118)
79-97 270-291 711-730
5’ATGGCCTGT’ITTGACACTGSYATACCCAAGTAAG’lTGGAGAGG3~ ,3’lTGTCCAA’fTCTTTAGGAAAs’
+ + -
cDNA synthesis Downstream primer Upstream primer
461
SE0 (Seoul 80-39)
79-97 270-291 71 l-730
~TGGCCA4GGCTTTGCA’ITA~sTAACCAAGGTCATATGGCGGA43~ SGACGGTAGTGTAGCCGTTAC”
+ + -
cDNA synthesis Downstream primer Upstream primer
461
PUU (HQlln& Bl)
79-97 294315 735-754
5’CCAGGGTCTATTACTATGTY5’GGACATGGGAMl-MAAGGTGA3~ ~~‘TcTAAC~TTTCTTGAAACTC~
+ + -
cDNA synthesis Downstream primer Upstream primer
461
PH (Prospect HiIt-I)
83.101 256-277 970-989
S’GGAGTACTACTACTGCAGG3’YGCATACAAGCTCAGCCACACAG3~ ~CCGTGGTAGGTGACTCAGTAY
+ + -
cDNA synthesis Downstream primer Upstream primer
734
Fig.
2. Serotype-specific
oligonucleotide represent
primers
as designed from M genomic of DNA synthesis.
sequence.
Arrows
the direction
mM of each dNTP (Pharmacia, Uppsala, Sweden); 10 units of RNasin (Promega Biotec, Madison, WI, USA); and 10 units of avian myeloblastosis virus reverse transcriptase (Pharmacia), and incubated at 41°C for 2 h. The reaction mixture containing cDNA was added to 480 ~1 of 10% TE (1 mM Tris-HCl pH 7.5; 0.1 mM EDTA pH 8) and kept at -20°C until used. Each RNA sample was used for 5 template primers in 5 different reactions of cDNA synthesis.
Polymerase chain reaction
We have followed the PCR protocol of Frohman et al. (1988). Briefly, a 50 ~1 total volume of the PCR tube contained 2.5-5 ~1 of cDNA, 25 pmol of each primer, 100 PM of each dNTP and 2.5 units of Taq DNA polymerase (Pharmacia, or GIBCO BRL, New York, USA) in the reaction buffer containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl, and 0.01% (w/v> gelatin, and was overlaid with 50 ~1 of mineral oil. The reaction was carried out in a DNA thermal cycler (Perkin-Elmer-Cetus) for 36 cycles as follows: the first cycle was 95°C for 2 min, 48-57°C for 2 min and 72°C for 40 min; the second to the thirty-sixth cycles were 95°C for 40 s, 48-57°C for 2 min and 72°C for 3 min followed by 12 min at 72°C for final extension. Usually, the annealing temperature for PCR was carried out at 55°C except 50°C for that of A9, JinHae 87/526, Brazil 2-4 and Hall& Bl, and 48°C for those of Thailand nos. 605 and 749 in the serotype-specific amplification. The amplified product was visualized by electrophoresis using 5-10% of total DNA in 1.5% agarose gel with 0.5 X Tris-Borate-EDTA buffer and followed by staining in ethidium bromide.
7 TABLE Restriction Enzyme
2 endonuclease
sites on the amplified
Restriction
DNA products
sites on the products
HTN (NT Pos. *)
amplified
by primers
of
(NT ~0s.)
PUU (NT Pas.)
:lz
SE0
Pas.)
BstYl
636 (94/367)
None
None
870
Rmal
312
654
758
Nsp 1
366,492
431 (162/299) 488,492,640
(q1;4,307j
W’Ml
None
None
None
447, 477, 516,618 637 (352/382)
* NT Pos. = Nucleotide position. Fragment length after digestion is given in parentheses.
Restriction endonuclease digestion
The amplified DNA was purified by standard phenol-chloroform extraction (Sambrook et al., 1989) before digestion with restriction endonuclease. The product amplified by Hantaan, Seoul and PH-specific primers was digested with enzymes BstYl (New England Biolabs Inc. NEB, MA, USA), Real (NEB), and PflMl (NEB) respectively, while those amplified by Puumala primers were digested with either Nspl (Boehringer Mannheim, Germany) or Bell (Pharmacia). 2.5 to 4 units of the enzymes were used for cleaving S-15% of the PCR product in 3-4 h under the reaction condition described by the company. The digested product was detected by electrophoresis in a 2% agarose gel and followed by staining with ethidium bromide. The expected sizes of the digested products as shown in Table 2 were calculated from the cleavage site of that enzyme on the M genomic segment of Hantaan 76-118, Seoul 80-39, Hillnh Bl and PHV-I (Yoo and Kang, 1987; Antic et al., 1991; Giebel et al., 1989; Parrington et al., 1991).
Results Polymerase chain reaction A. Genus-specific primers
Fig. 3 shows that the genus-specific primers could amplify sequences from all 27 Vero E6 cells. The lengths of the amplified fragments were expected to range from 878 to 890 nucleotides (Fig. 1) which is consistent with the results shown here. However, differences in fragment sizes between the isolates could not be clearly seen. Hantauirus isolates but not from the VSV or uninfected
GENUS primers Fig. 3. PCR
products
of 27 Huntavirus isolates using digested 4X-174 RF-DNA
genus-specific oligonucleotide is used as markers.
B. Serotype-specific primers The 4 sets of serotype-specific primers (Hantaan, Seoul, primers) were used to amplify sequences from 27 Huntavinu
HTN primers
PUU primers
primers.
Puumala isolates,
Hue111
and PH VSV and
SE0 primers
PH primers
Fig. 4. PCR products of 27 Huntuuirus isolates using four different sets of serotype-specific HaeIII-digested 4X-174 RF-DNA is used as markers. o indicates positive PCR.
primers.
9 TABLE
3
Comparison
on typing of Hantauirus isolates
by PCR and serological typing by PRNT
methods PCR typing
Virus strain
Serological
A9 JinHae 87/526 Maaji ROK 79/90 US 84/2 76/118 83/14 83/138 Cg 3883 Fojnica JTRN/82/17 i/RN/82/3 SNUS/Hamster 85/4 80/39 Thailand no. 605 Thailand no. 749 GBB Baltimore rat Houston rat no. 4 Tchoupitoulas no. 401613 Brazil 2-4 Egypt R/12915 NE 672
Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul
Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Hantaan Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul Seoul
Seoul
Seoul
Seoul
Puumala
Seoul Puumala
Hallnis Bl Yanagihara Prospect Hill I Leakey
Puumala Puumala Prospect ?
Puumala Puumala Prospect Hill Seoul-Puumala
Hill
uninfected Vero cells. We found that each of 26 isolates reacted with only one of the 4 sets of primers (Fig. 4). Our results demonstrated that 10, 12, 3 and 1 isolates belong to Hantaan, Seoul, Puumala and Prospect Hill types respectively. One isolate named “Leakey” reacted with both Seoul and Puumala specific primers; thus, it was untypable. VSV and uninfected Vero E6 cells were not reactive with any of the primer sets. Our results of typing Hantavimses by PCR was compared to that of serological typing and in all instances the results agreed (Table 3). Restriction endonuclease cleavage
The PCR products were confirmed for their specific amplification by restriction endonuclease digestion. Restriction sites for each enzyme were based on the known sequence of representative strains of the serotype, i.e. 76-118 for Hantaan, 80-39 for Seoul, Hallnas Bl for Puumala and PHV-I for Prospect Hill as has been shown in Table 2.
10
All 10 DNA products amplified by Hantaan specific primers were cleaved by enzyme BstYl and gave rise to two fragments of size 94, and 367 base pairs. All 13 products (including Leakey) amplified by Seoul primers were cleaved by Real and gave rise to two fragments of size 162 and 299 base pairs. PHV-I, the only isolate amplified by PH primers was digested by PfEMl and yielded two fragments sized 352 and 382 base pairs, as expected. Restriction endonuclease sites common to isolates in the Puumala type have not been found yet. Digestion of 4 PCR products (including Leakey) amplified by Puumala primers with enzyme Nspl yielded 3 cleavage patterns. The product amplified from NE672 was cleaved and yielded 2 nucleotide fragments of size 154 -and 307 base pairs which was expected from published sequence of HBlllnas Bl (Giebel et al., 1989). In contrast, the product from our Hallnh Bl isolate was not cleaved at that expected site; it yielded 2 visible bands which were smaller in size. Products from Yanagihara and Leakey viruses showed the same cleavage pattern; the amplified product seemingly was cut at the middle and yielded only one thick visible band (Fig. 5).
Discussion
PCR has been introduced as a method to study the genetic variation and relatedness among Huntuvirus isolates during recent years. Tang et al. (1990) were successful in an attempt to differentiate Hantaan- and Seoul-related strains by using 2 pairs of primers specific for either Hantaan or Seoul M genomic segments. PCR has been used to amplify Puumala-related viruses and subsequently the genomic variation between strains was analysed by nucleotide sequencing of the amplified DNA products (Giebel et al., 1990). Xiao et al. (1992) designed a genus common primer pair from M genomic segments to amplify 41 Huntuvirus isolates, and then analyzed the amplified products by using a panel of restriction endonucleases. One isolate failed to be amplified and the cleavage patterns of the digested PCR products were very diverse even among the strains of the same serotypes. These results strongly indicated genetic diversity among Huntuuiruses and also implied that this was not an ideal approach for typing new virus isolates. We demonstrate here that serotype-specific primers must be used in order to type Huntuvirus isolates. Comparison of nucleotide sequences of Huntuvirus genomes shows that S genomic sequences are more conserved than M genomic sequences (Antic et al., 1992). In this study, we designed the genus-specific primers from the consensus sequence of S genomic segments. The primers were capable of amplifying all 27 isolates studied (Fig. 5). Conversely, serotype-specific primers were designed from the unique sequences of M genomic sequences. Our results showed that the PCR technique could group 26 of 27 Huntuvirus isolates into 4 different types: Hantaan, Seoul, Puumala and Prospect Hill. Only Leakey virus was untypable because it was amplified by both Seoul and Puumala primers. In addition, its patterns of restriction endonuclease cleavage were the same as other isolates within both the Seoul
11
and Puumala serotypes (Fig. 5). There are three possible explanations for this: (1) Leakey virus may be a mixture of Seoul and Puumala viruses due to accidental contamination in the laboratory during the process of virus propagation; (2) Leakey virus was isolated from the animal which had dual infections between the two viruses or (3) Leakey virus is a recombinant virus between Seoul and Puumala serotypes. Nucleotide sequencing of the plaque-purified virus might help to clarify this point.
-461
461. 367-
-299 -162
94-
HTN
-302 -352
-230
PUU
PH
Fig. 5. Endonuclease cleavage patterns of the amplified DNA products. Lanes l-3 are products of Hantaan 76-l 18, Maaji and cg 3883 as amplified by Hantaan-specific primers. Lane lE-3E are Hantaan products digested by BsfYl. Lanes 4-6 are products of Seoul 80-39, Egypt R/12915 and Leakey as amplified by Seoul-specific primers. Lanes 4E-6E are Seoul products digested by Real. Lanes 7-10 are products of Hallnls Bl, NE672, Yanagihara and Leakey as amplified by Puumala-specific primers. Lanes 7E-10E are Puumala products digested by Nspl. Lane 11 is PHV-I product as amplified by PHV-specific primer and lane 11E is PHV product digested by PfIMl. M is HaelI1 digested 4X-174 RF-DNA.
12
When we digested amplified DNA products with restriction endonucleases, we found that all products from viruses within the Hantaan serotype gave rise to only one cleavage pattern when digested with BstYl, as do products from viruses of the Seoul serotype digested with Real (Fig. 5). However, enzyme(s) which should cut all members of Puumala serotype within the amplified region have not been found yet. Moreover, the cleavage pattern of our HBllnEs Bl product did not correspond to the pattern expected from the published sequence of the Hahn% Bl (Giebel et al., 1989). Our amplified products were not cleaved by Bcfl (unpublished data), even though a site should exist according to sequence information. Amplified products were cleaved by Nspl but not at the expected position (Table 2 and Fig. 5). Our finding is consistent with previous reports (Antic et al., 1992; Xiao et al., 1992) and suggests that the published sequence of Hallnas Bl (Giebel et al., 1989) may be incorrect. Among 27 Huntavirus isolates investigated, some were amplified only by lowering the annealing temperature to between 48 and 50°C. These isolates were A9, JinHae X7/526, Thailand no. 605, Thailand no. 749, Brazil 2-4 and Hahn& Bl. This may reflect higher genomic heterogeneity from the prototype strains. Since these isolates were reactive with certain serotype-specific primers and their PCR products (except Hallnas Bl) yielded cleavage patterns corresponding to the prototype virus, they may be classified subtypes, but not as distinct serotypes. Results of serological typing of these isolates strongly supported our PCR typing (Table 3). Nucleotide sequencing of the PCR products should reveal a degree of homology among different strains belonging to the same type. Grankvist et al. (1992) have successfully amplified Puumala virus sequences by RT/PCR using RNAs extracted from the urinary tract cells, the respiratory tract cells and from peripheral blood mononuclear cells of patients with nephropathia epidemica. This result provides a new possibility that RT/PCR can be utilized as a direct diagnostic method for Hantuvirus infection using clinical specimens without in vitro amplification of viruses in cultured cells. Huntuuirus RNA was also detected in tissues of experimentally infected mice by RT/PCR and the sensitivity of this detection method was equal to the cell culture method (Xiao et al., 1991). These results argue the previous belief that viruses from clinical specimens must be amplified to be detected by RT/PCR, but suggest that RT/PCR can be used to detect Huntuuirus genomic sequences in clinical specimens and post-mortem tissues from patients with HFRS. In conclusion, the successful result in typing Huntuvirus isolates by PCR should encourage its application as an adjunct test with serological method(s) or as an independent test for typing of the new Huntuvirus isolates or for diagnostic purpose.
Acknowledgements
We thank Drs. K.E. Wright and E. Brown for their critical and constructive review of this manuscript. We thank M. Dobbs for his excellent help in computer
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