Isolation and characterization of Tula virus, a distinct serotype in the

m~u"n~'{ °f.~effe£'ff{ v!£?l?~.~.(!. 99.9.,.,7Z:..3003.~3967.<..P<io!ed.!n,.gr~aLBE~t~!~............................................... S H O R T C O M M U N I C A T I O N
Isolation and characterization of Tula virus, a distinct serotype
in the genus Hontovirus, family Bunyoviridoe
Olli V a p a l a h t i , ~ ~,ke L u n d k v i s t , 2 Sami K. J. K u k k o n e n , 1 Y i n g C h e n g , 1 M a r i Gilljam, 2 Mari K a n e r v a , 1
Tytti M a n n i , 1 M i l a n Pejcoch, 3 J u k k a N i e m i m a a , 4 A s k o K a i k u s a l o , 4 H e i k k i H e n t t o n e n , 4 A n t t i V a h e r P
and A l e x a n d e r P l y u s n i n 1
i Haartman Institute, Department of Virology, POB 2 I, FIN-O001 4 University of Helsinki, Finland
z Swedish Institute for Infectious Disease Control, S-I 05 21 Stockholm, Sweden
3Regional Hygienic Institute of South Moravia, Brno, Czech Republic
4Finnish Forest Research Institute, FIN-01301 Vantaa, Finland
A Vero E6 cell culture isolate of Tula virus (TUL), a
hantavirus first detected in European common voles
(hficrotus arvalis and hr. rossiaemeridionolis) by
RT-PCR was obtained after initial passaging of TULinfected vole lung samples in laboratory-colonized
hl. arvalis. TUL was defined as a classical serotype
by a cross-focus-reduction neutralization test
(FRNT) and was also shown to be distinct from
other hantaviruses by haemagglutination inhibition
assay. The sequences of S, M and partial L genome
segments of the isolate were determined: the S
segment was 9 9 . 9 % identical to the original
rodent-derived sequence. Serological evidence for
a previous TUL infection was obtained from the
serum of a blood donor living near a TUL focus in
Moravia, Czech Republic, showing at least a 16-fold
higher FRNT titre to TUL as compared to Puumala
or other hantaviruses.
Hantaviruses, members of the family Bunyaviridae, are
enveloped negative-stranded RNA viruses with a tripartite
genome. Each hantavirus is carried primarily by a specific
rodent or insectivore host which may transmit the virus to
humans. Hantaan (HTN) (Lee & Lee, I978), Puumala (PUU)
(Brummer-Korvenkontio et al., 1980), Seoul (SEO) and Dobrava
(DOB) viruses, carried by field mice, (Apodemus agrarius), bank
voles (Clethrionomys glareolus), rats (Rattus rattus and R.
norvegicus) and yellow-necked mice (Apodemus flavicollis),
respectively, cause the different forms of haemorrhagic fever
with renal syndrome. In the Americas, Sin Nombre virus (SN)
Author for correspondence:Olli Vapalahti.
Fax + 358 0 434 6491. e-mail [email protected]
The reported TUL isolate sequences are available (EMBL,GenBankand
DDBJ) underaccession numbersZ69991, Z69993 and Z69992.
0001-4188 © 1996 SGM
(Nichol et al., 1993) carried by deer mice (Perornyscus maniculatus), as well as some related viruses carried by Sigmodontinae
rodents, cause hantavirus pulmonary syndrome with 50%
mortality (Khan et al., 1996). Other hantaviruses, such as TUL
and Prospect Hill (PH) (Lee et al., 1982) carried by Microtus
rodents, have not been associated with human disease.
Several hantaviruses have been isolated in cell culture and,
at present, ten have been established as distinct serotypes:
HTN, PUU, SEO, DOB, PH, SN, Khabarovsk (KBR), Black
Creek Canal, Thailand and Thottapalayam (Chu et al., 1994,
1995; Avsic-Zupanc et a]., 1995; Schmaljohn et al., 1995;
H6rling et al., 1996). In addition, some hantaviruses, such as
Bayou, Isla Vista (ILV), E1 Moro Canyon, Rio Segundo, New
York, Rio Mamore and previously TUL, lacking cell culture
isolates have been characterized mainly genetically by
RT-PCR on rodent or patient samples (for reviews see
Lundkvist & Niklasson, i994; Plyusnin et al., 1996b;
Schmaljohn, 1996).
The three RNA segments of hantaviruses code in the viral
complementary sense for the four structural proteins (sizes
given for PUU): the 6"5 kb L segment codes for the
approximately 200 kDa polymerase protein, the 3"7 kb M
segment codes for the two envelope glycoproteins, G1
(68 kDa) and G2 (54 kDa), and the 1"8 kb S segment encodes
the 50 kDa nucleocapsid protein (N) (Antic et al., I992;
Plyusnin et aI., 1996b; Schmaljohn, 1996).
Serologically and genetically, HTN, SEO and DOB carried
by Murinae rodents are relatively closely related to each other,
while PUU, KBR, TUL and PH carried by Arvicdinae rodents,
and SN and other viruses carried by Sigmodontinae rodents
form two more closely related groups (Chu et at., I994;
H6rling et al., 1996; Plyusnin et al, 1996b). The antibody
response to N is strong and highly cross-reactive between the
different serotypes. Antibodies to the envelope glycoproteins
are responsible for the serological responses measured by
neutralization and haemagglutination tests (Chu et al., 1994;
Lundkvist & Niklasson, 1994).
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Wed, 26 Oct 2016 16:29:04
306~
iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii#ii!iiiiiiiiiiiiiiiiii
Tula virus was first detected by RT-PCR in European
common voles (Microtus arvalis and M. rossiaemeridionalis),
originally from the Tula region, Russia (Plyusnin et al., 1994)
and later also from the Czech Republic and Slovakia (Plyusnin
et al., 1995, 1996a; Sibold et al., 1995), and was established as
a distinct genotype, differing 25-27% in the S and M segment
nucleotide sequences from the most closely related PH and ILV
viruses. By using baculovirus-expressed TUL N antigen, we
were able to develop a panel of TUL-specific monoclonal
antibodies (MAbs) of which two reacted exclusively with TUL
(Lundkvist et al., I996 b). While the amino-terminal region of
TUL N was found to be highly antigenic and to contain several
common antigenic sites with PUU (Lundkvist et al., 1996a, b),
several MAb epitopes distinguished TUL N from PUU and PH
proteins.
In order to obtain a cell culture isolate of the agent, five
TUL antigen-positive lung samples from naturally infected M.
arvalis trapped in Tvrdonice, South Moravia, Czech Republic
(Plyusnin et al., 1995) were used for virus isolation attempts in
a laboratory colony of hantavirus-free M. arvalis from Finland.
Twelve animals were injected intraperitoneally in a Biosafety
level 3 laboratory with either 1/10 or 1/1000 dilutions of
homogenized lung suspensions (experiment approved by the
Experimental Animal Committee of the Haartman Institute,
Helsinki University). The rodents were sacrificed 32 days later,
in one case 19 days later. Five animals died of unknown causes
within 10 days. Out of seven animals, only one became
infected (inoculated with the more dilute dose from Moravian
M. arvalis 5302), as judged by antigen positivity in immunoblotting and immunofluorescence assay (IFA) with TULspecific MAbs, and antibody positivity by IFA (Lundkvist et al.,
1996b).
From the infected vole, a lung sample and a pooled sample
of kidney, liver and spleen were frozen and homogenized.
Diluted samples were inoculated to three Vero E6 culture flasks
and the cells were passaged at 27 and 48 days post-infection.
After 4I days, some TUL-positive cells were found in one of
the flasks inoculated with the lung homogenate, and after 55
days 10% of the cells were antigen-positive by IFA with TULspecific MAbs 3D3 and 3F10 (Lundkvist et al., 1996b). A cell
culture supematant which was passaged to new Vero E6 cells
after 48 days was regarded as the first passage of the isolate,
designated Tula/Moravia/Ma5302V/94.
After the first passages the titre of infectious virus in cell
culture supernatants decreased to 1"2 x 104 f.f.u./ml. This
supematant (0"5 ml per 75 cm2 culture flask) infected 80-100 %
of the cells in 6--8 days. During the infection gradual
accumulation of N protein was detected, as first seen by
granular, then filamentous deposits and finally 'crescents'
filling the cytoplasm (not shown). No cytopathic effect was
observed.
The virus was purified, first by centrifugation through a
sucrose pad, and further in a 10--60 % (w/v) sucrose density
gradient. The viral peak was recovered at 42"5% sucrose
~06 z
suggesting a buoyant density of 1"18 g/cm 3. In electron
microscopy (not shown) typical hantavirus particles of
approximately 120 nm with 10 nm long surface projections
could be distinguished. Purified TUL virions were shown to
contain three major protein bands of 68, 53 and 49 kDa, which
were detected in immunoblotting by G1-, G2- and N-specific
antibodies, respectively (Fig. l a, b). The TUL N and G2
proteins migrated slightly faster than the corresponding PUU
proteins.
Viral RNA segments of 1"8, 3"7 and 6"5 kb were found in
purified virions. In infected cells, S RNA dominated and L RNA
was found in relatively small amounts (Fig. lc). The mRNAs
were of roughly similar size to genomic RNA, as no separate
bands could be distinguished from total RNA of infected cells.
Nucleotide sequences of the entire S, M and partial L (nt
1914-2909) genome segments of the isolate were determined.
RT-PCR cloning and sequencing for S and M segments were
performed as described earlier (Plyusnin et al., 1994, 1996a)
(L segment primer sequences available upon request). Similar to
previously reported wild-type TUL genes from Central Europe,
the S and M segments of the TUL/Moravia isolate code for an
N protein of 429 amino acids and a glycoprotein precursor of
1141 amino acids. TUL G1 contained three N-linked glycosylation sites, conserved in all hantaviruses; G2 contained one.
G2 of PUU/Sotkamo had an additional site and was two
amino acids longer, explaining its slower mobility in SDS-PAGE. The S segment of the isolate differed by two nucleotides
from the sequence obtained directly from the original wild
rodent. Both nucleotide changes, which were C to T transitions,
led to an amino acid change, namely Ala to Val (amino acid 28)
and His to Tyr (amino acid 212). The two residues were also
identical in all four rodent-derived TUL sequences from the
same locality (Plyusnin et al., 1995). While the M segment
sequence could not be obtained directly from the same rodent
(5302Ma), the isolate had a nucleotide identity of 99% to
another rodent strain from the trapping area (5286Ma)
(Plyusnin et al., 1996 a). The L segment was the most conserved
of the three genome segments showing 75 %, 76 %, 73 % and
68% nucleotide and 89%, 88%, 84% and 74% amino acid
identity with L segments of PH, PUU, SN and HTN viruses,
respectively.
In order to study the antigenic relationship of TUL virus to
other hantavirus strains, antisera were produced by intranasal
inoculation of New Zealand White rabbits as described
(Niklasson et al., 1991). To achieve adequate neutralization
titres, the rabbits inoculated with TUL, PH and KBR viruses
were boosted by subcutaneous injections of virus (concentrated by ultracentrifugation). Focus-reduction neutralization
tests (FRNTs) for determination of end-point titres of
neutralizing antibodies (Niklasson et al., 1991) and haemagglutination inhibition (HI) (Brummer-Korvenkontio et al., 1986)
were performed as described.
Two rabbits which were immunized intranasally with TUL
developed homologous FRNT titres of 1/320 and 1/80 after
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Wed, 26 Oct 2016 16:29:04
anti-N
6A6
anti-G1
anti-G2
1C8
~6x~ K.6x~
k]
(c)
kDa
i,
i
97 w :
:
66 D
-~-- G1
.,
'
¢
~
~
:
i
-,b-- G2 4 3 w
-.i..- N
I ~ L (6.5kb)
A
aill. ~i r ,
i
4-4 kb
N;;;;
'~:;' :; ~
U (3"7 k b )
) i . ':
2.4 kb
"*" S (1'8 kb)
1
2
3
4
1
2
3
Fig. I. (a) TUL virJon proteins as shown by a standard 10% SDS-PAGE, Coomassie blue stain, in comparison with PUU
proteins. (b) Immunoblot of TUL and PUU proteins, as in (a), with N-specific MAbs (I) 6A6, (2) IC8 (Lundkvist et al., 1996b)
and rabbit antisera raised against recombinant (3) PUU-GI and (4) PUU-G2 GST-fusion proteins (Vapalahti et al., 1995). (c)
Northern blot with S, M and L segment probes of (1) purified TUL virus, (2) non-infected Vero E6 cells and (3) Vero E6 cells
infected for 6 days. Sizes of RNA markers and TUL genome segments are indicated. RNA run in denaturing agarose gel
electrophoresis was transferred to Hybond-N membranes (Amersham) and probed with a mixture of s2p-labelled dCTP probes
representing TUL S (nt I - I 831), M (nt 972-2782) and L (nt 1-946) prepared from 50 ng of each respective insert using a
Ready-To-Go kit (Pharmacia).
Table 1. R e c i p r o c a l cross-FRNT and cross-HI titres with TUL, PH, PUU, KBR and HTN
antigens and rabbit antisera
Titres with the homologous antigen/antiserum are in bold.
Antiserum*
Virus
TULa(MO2V) TULb(MO2V) PH(PH-1) PUU83-L20 KBRMF43 HTN(76-118)
FRNT
TUL (M02V)
PH (PH-I)
PUU (83-L20)
KBR (MF-43)
HTN (76--118)
5120
160
20
80
< 20
80
20
< 20
20
< 20
HI
TUL (MO2V)
PH (PH-I)
PUU (Sotkamo)
KBR (MF-113)
320
40
20
20
ND
NO
ND
ND
160
20
20
80
1280
< 20
< 20
< 20
80
80
< 20
1280
< 20
1280
< 20
< 20
< 20
20
40
4O
40
10
I0
< I0
20
40
2O
< 10
20
160
< 20
160
20
20
160
320
* a and b indicate two different rabbit antisera to TUL.
ND, Not done.
3 months. A cross-FRNT comparison with similarly produced
hantavirus antisera s h o w e d 8-fold or higher titre differences
b e t w e e n h o m o l o g o u s and heterologous antisera for TUL,
PUU, PH, KBR and H T N viruses (Table 1). Thus, TUL was
established, b y FRNT, as a distinct serotype in the genus
Hantavirus. A similar pattern was also obtained in the cross-HI
test with TUL antiserum titres of 1 / 3 2 0 and 1/40, and PH
antiserum titres of 1 / 2 0 and 1 / 1 6 0 , to TUL and PH antigens,
respectively (at least 8-fold higher h o m o l o g o u s to heterologous titres). The a n t i - P U U / 8 3 L 2 0 serum, however, s h o w e d
equal titres ( I / 4 0 ) for HI to P U U / S o t k a m o , PH and TUL
antigens (Table 1).
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Wed, 26 Oct 2016 16:29:04
Table 2. Reciprocal FRNT titres of human sera reactive to
TUL and PUU antigens in EIA and IFA
Mall, serum from a healthy blood donor in Moravia, Czech Republic;
F-Z2655, F-1789, F-1808, representative sera of nephropathia
epidemica (PUU infection) patients from Finland.
Serum
M-11
F-22655
F-1789
F-I808
Virus
TUL
PUU
1280
40
40
160
40
1280
640
2560
Whether TUL could be transmitted to man was unknown
and a virus isolate and FRNT were indispensable tools for
solving this question. A total of 315 sera from healthy blood
donors living near Tvrdonice, Moravia, Czech Republic were
screened in a dilution of 1:20 by PUU-IgG IFA (Vapalahti et al.,
1995) in order to detect PUU/TUL-like antibody responses.
One positive serum was found, of which the TUL or PUU
specificity could not be distinguished by IgG ELISA using
recombinant or native N proteins (data not shown). To
evaluate the antibody response in detail, this serum was
examined by FRNT to TUL, other closely related serotypes of
hantavirus (PH, KBR, PUU), and the more distantly related
HTN, SEO and DOB viruses, At least a 16-fold higher titre to
TUL was found in all cases; TUL (1/1280), PH (1/80), KBR
(1/40), PUU (I/40), DOB ( 1 / ( 4 0 ) , SEO ( I / < 40), H T N
(1/(40).
In contrast, Finnish sera from nephropathia
epidemica patients showed at least 8-fold higher FRNT titres
to PUU than to TUL (Table 2).
This is the first solid evidence indicating that hantaviruses
carried by Microtus rodents can infect man. IFA titres 2-fold
higher to PH than to PUU were reported for sera of four
American mammalogists (Yanagihara et al., 1984), but the
neutralizing titres were measured only for PH and HTN:
evidently, they might also have been infected with some other,
then unknown, American hantavirus.
M . arvalis is a dominant rodent species on large open
agricultural fields in Western, Central and Eastern Europe and
densities can reach hundreds per hectare (Niethammer &
Krapp, 1982). However, even in an area with high TUL antigen
prevalence in rodents, transmission to humans was relatively
rare, with approximately 1/300 seroprevalence. Due to the
high antigenic cross-reactivity of assays based on N antigen, it
was only by using a neutralization assay that reliable
serotyping could be performed, showing 16-fold higher titre
against TUL than against other hantaviruses. As compared to
what has previously been shown for human hantavirus
infections (Chu et al., 1995; Niklasson et al., I99I), our data
~06(
strongly suggest a TUL-like virus as the infectious agent. Thus,
by using the TUL isolate we were able to show that, in addition
to PUU, DOB, SEO and HTN, a fifth hantavirus serotype
infectious to man seems to be circulating in the Old World.
Whether TUL is pathogenic to man or not remains to be
shown. Nevertheless, precise serological typing of human sera
should be performed in a way that makes it possible to
distinguish between PUU and TUL hantavirus infections in
Europe.
O.V. and ,~.L. made an equal contribution to this study. Ms Leena
Kostamovaara, Ms Anja Virtanen and Ms Katarina Brus Sj61ander are
acknowledged for excellent technical assistance. This work was supported
by the Maud Kuistila Memorial Fund, Medical Research Council of the
Academy of Finland, Finnish Culture Foundation, Sigrid Jus61ius
Foundation, Nordic Academy for Advanced Study and the Swedish
Medical Research Council (Project no. 11230).
References
Antic, D., Yong Kang, C., Spik, K., Schmaljohn, C., Vapalahti, O. &
Vaheri, A. (1992). Comparison of the deduced gene products of the L,
M and S genome segments of hantaviruses. Virus Research 24, 35-46.
Avsic-Zupanc, T., Toney, A., Anderson, K., Chu, Y.-K. & Schmaljohn, C.
(1995). Genetic and antigenic properties of Dobrava virus: a unique
member of the Hantavirus genus, family Bunyaviridae. Joy,mat of General
Virology 76, 2801-2808.
Brummer-Korvenkontio, M., Vaheri, A., Hovi, T., Von Bonsdorff, C.-H.,
Vuorimies, J., Manni, T., Penttinen, K., Oker-Blom, N. & L~hdevirta, J.
(1980). Nephropathia epidemica: detection of antigen in bank voles and
serologic diagnosis of human infections. Journal of Infectious Diseases I41,
131-134.
Brummer-Korvenkontio, M., Hanni, T., Ukkonen, S. & Vaheri, A.
(1986). Detection of hemagglutination-inhibiting antibodies in patients
with nephropathia epidemica and Korean hemorrhagic fever by using
Puumala virus cell culture antigen. Journal of Infectious Diseases 153,
997--998.
Chu, Y. K., Rossi, C., Leduc, J.W., Lee, H.W., Schmaljohn, C.S. &
Dalrymple, J. M. (1994). Serological relationships among viruses in the
Hantavirus genus, family Bunyaviridae. Virology 198, 196--204.
Chu, Y. K., lennings, G., Schmaljohn, A., Elgh, F., HjeUe, B., Lee, H. W.,
Jenison, S., Ksiazek, T., Peters, C.J., Rollin, P. & Schmaljohn, C. S.
(1995). Cross-neutralization of hantaviruses with immune sera from
experimentally infected animals and from hemorrhagic fever with renal
syndrome and hantavirus pulmonary syndrome patients. Journal of
Infectious Diseases 172, 1581-1584.
H6rling, J., Chizhikov, V., Lundkvist, ,~., Jonsson, M., Ivanov, L.,
Dekonenko, A., Niklasson, B., Dzagurova, T., Peters, C. J., Tkachenko,
E. & Nichol, $. (1996). Khabarovsk virus: a phylogenetically and
serologically distinct Hantavirus isolated from Microtus fortis trapped in
far-east Russia. Journal of General Virology 77, 687--694.
Khan,A. S., Kslazek, T. G. & Peters, C. J. (1996). Hantavirus pulmonary
syndrome. Lancet 347, 739-741.
Lee, H.W. & Lee, P.W. (1978). Isolation of the etiologic agent of
Korean hemorrhagic fever. Journal of Infectious Diseases 137, 289-308.
Lee, P. W., Am)x, H. L., Gajdusek, D. C., Yanagihara, R. T., Goldgaber,
D. & Gibbs, C. J. I. (198Z). New hemorrhagic fever with renal syndromerelated virus in indigenous wild rodents in the United States. Lancet ii,
1405.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Wed, 26 Oct 2016 16:29:04
Lundlwist,/~. & Niklasson, B. (1994). Haemorrhagic fever with renal
syndrome and other hantavirus infections. Reviews in Medical Virology 4,
177-184.
variation in Tula hantaviruses: sequence analysis of the S and M
segments of strains from Central Europe. Virus Research 39, 237-250.
Lundkvist, ,~., Kallio-Kokko, H., Brus Sj61ander, K., Lankinen, H.,
Niklasson, B., Vaheri, A. & Vapalahti, O. (1996 o). Characterization of
conserved features of Tula hantavirus M gene encoding envelope
glycoproteins G1 and G2. Virus Genes 1Z, 259-266.
Puumala virus nucleocapsid protein: identification of B-cell epitopes and
domains involved in protective immunity. Virology 216, 397-406.
Plyusnin, A., Vapalahti, O. & Vaheri, A. (1996 b). Hantaviruses: genome
structure, expression and evolution. Journal of General Virology 77,
2677-2687.
Lundkvist,/~,., Vapalahti, 0., Plyusnin, A., Brus Sj61ander, K., Niklasson,
Plyusnin, A., Lehv~islaiho, H. & Vaheri, A. (1996o). Unique and
B. & Vaheri, A. (1996b). Tula hantavirus nucleocapsid protein:
characterization of antigenic determinants defined by monoclonal
antibodies raised against baculovirus-expressed protein. Virus Research(in
press).
Schmaljohn, C. S. (1996). Molecular biology of Hantaviruses. In The
Bunyaviridae, pp 63-90. Edited by R. M. Elliott. New York & London:
Plenum Press.
Nichol, S. T., Spiropoulou, C. F., Horzunov, S., Rollin, P. E., Ksiazek,
T. G., Feldmann, H., Sanchez, A., Childs, J., Zaki, S. & Peters, C. J.
Schmaljohn, A., Li, D., Negley, D.L., Bressler, D.S., Turell, M.J.,
Korch, G. W., Ascher, H. S. & Schmaljohn, C. S. (1995). Isolation and
initial characterization of a newfound hantavirus from Califomia. Virology
(1993). Genetic identification of a novel hantavirus associated with an
outbreak of acute respiratory illness in the southwestern United States.
Science 262, 914-917.
Niethammer, J. & Krapp, F. (1982). Microtus arvalis (Pallas, 1779)Feldmaus. In Handbuch der Sfiugetiere Europas, pp. 284-318. Edited by J.
Niethammer & F. Krapp. Wiesbaden: Akademische Verlagsgesellschaft
Niklasson, B., Jonsson, H., Lundkvist,/~., H6rling, J. & Tkachenko, E.
(1991 ). Comparison of European isolates of viruses causing hemorrhagic
fever with renal syndrome by a neutralization test. American Journal of
Tropical Medicine and Hygiene 45, 660--665.
Plyusnin, A., Vapalahti, 0., Lankinen, H., Lehv~islaiho, H., Apekina, N.,
Hyasnikov, Y., Kallio-Kokko, H., Henttonen, H., Lundkvist, A.,
Brummer-Korvenkontio, M., Gavrilovskaya, I. & Vaheri, A. (1994).
Tula virus: a newly detected hantavirus carried by European common
voles. Journal of Virology 68, 7833-7839.
Plyusnin, A., Chen9, Y., Vapalahti,oO. , Pejoch, H., Unar, J., Jelinkova,
Z., Lehv~islaiho, H., Lundkvist, A. & Vaheri, A. (1995). Genetic
206, 963-972.
Sibold, C., Sparr~ S., Schulz, A., Labuda, H., Kozuch, 0., Lysy, J.,
Kriiger, D. H. & Heisel, D. H. (1995). Genetic characterisationof a new
hantavirus detected in Microtus arvalis from Slovakia. Virus Genes 10,
276-281.
Vapalahti, 0., Kallio-Kokko, H, N~irv~nen, A., Julkunen, 1., Lundkvist,
A., Plyusnin, A., Lehv~islaiho, H., Brummer-Korvenkontio, H., Vaheri,
A. & Lankinen, H. (1995). Human B-cell epitopes of Puumala virus
the major antigen in early serological response.
Journal of Medical Virology 46, 293-303.
nucleocapsid protein,
Yanagihara, R., Gajdusek, D.C. & Gibbs, C.J. (1984). Serologic
evidence for infection in mammalogists. New EnglandJournal of Medicine
31(I, 1325-1326.
Received 7 June 1996; Accepted 12 August 1996
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Wed, 26 Oct 2016 16:29:04
~06]