Abstract

Hantaviruses are found in widely scattered areas of the world and are transmitted by inhalation of virus-contaminated aerosols of rodent excreta. The present study was undertaken in Kuwait to investigate the serological evidence for hantavirus infection in rodents and humans. Sera were collected from 283 wild rodents and 183 human subjects (46 Kuwaitis and 137 non-Kuwaitis). The rodent sera were investigated for the presence of antibodies against the Seoul and Puumala strains of the hantaviruses by enzyme-linked immunosorbent assay and immunofluorescence technique using the virus-infected Vero E6 cells. The findings showed the presence of anti-hantavirus antibodies in seven out of the 283 (2.8%) rodents. Antibodies against the Seoul strain were present in six (2.1%) and against the Puumala strain in three (1%) rodents. Further, it was observed that three out of 84 (3.6%) of the Rattus norvegicus and four out of 174 (2.3%) Mus musculus had anti-hantavirus antibodies. Two rodents belonging to species Mus musculus had antibodies against both strains of the hantaviruses. Out of 183 human sera, 13 (7%) were positive for hantavirus antibodies. Among the Kuwaitis 5/46 (11%) and among the non-Kuwaitis 8/137 (6%) were positive for the hantavirus antibodies. Antibodies to both Puumala and Hantaan strains were detected in Kuwaitis as well as in non-Kuwaitis. Although no human case of hantavirus illness has yet been reported in Kuwait, the serological evidence of infection suggests a constant vigil.

Introduction

Hantaviruses are enveloped, trisegmented RNA viruses belonging to the Bunyaviridae family. At least 30 hantaviruses are now recognized throughout the world. They are causative agents of at least two different syndromes: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) [1,2,3]. Hantavirus infections have a worldwide importance. Annual incidence of HFRS alone is about 200,000 cases with an approximate mortality rate of 2–10%. The disease occurs in Europe, Asia and the Americas [4]. During recent years hantaviruses have been spreading from one geographical location to the other. For example, the first hantavirus epidemic (HPS) occurred in southwest USA in 1993 [5] and later it reached to other geographical locations [6]. Hantaviruses are primarily rodent-borne and cause persistent infection in rodents. Though the reservoir varies with location, the hantaviruses are primarily associated with the rodents of the Muridae family. Rodents shed the virus and the main source of infection is aerosolized excreta [7–9]. The rodents of the Muridae family are present in Kuwait, and therefore, hantavirus infections pose a potential hazard to the population. However, no data are available on this viral infection in Kuwait. Moreover, the disease is prevalent in other parts of the world but not reported in Kuwait. The present study was therefore conducted to determine the serological evidence of infection with hantaviruses both in rodents and humans living in Kuwait in close proximity with each other.

Materials and methods

Study population

A total of 283 rodents (Rattus norvegicus, n = 84; Mus musculus, n = 174; Nesokia indica, n = 7; and Tattera indica, n = 18) were trapped in 1997–1998 in different areas of Kuwait by the Insect and Rodent Control Division, Community Health Department, Kuwait. The trapped rodents were examined for species, sex, weight, physical and reproductive conditions. They were anesthetized and exsanguinated by cardiac puncture to collect blood for serum separation. In addition, serum samples were collected from 46 Kuwaiti and 137 non-Kuwaiti residents living in the same areas as the rodents with the possibility of frequent exposures to rodents.

Hantavirus antibodies in rodent sera

The rodents’ sera were tested for detection of antibodies against the Puumala and Seoul strains of hantaviruses using the immunofluorescence antibody (IFA) technique described previously [10]. In brief, rodent sera were diluted 20× in phosphate-buffered saline (PBS, pH 7.4) and applied to the multi-well slides with virus-infected Vero E6 cells (American Type Culture Collection C 1008). The slides were incubated in a humidified container for 45 min and washed with PBS for 30 min. The slides were further incubated with fluorescein isothiocyanate-conjugated anti-rat IgG antibodies for 20 min. The slides were washed with PBS and examined for characteristic cytoplasmic inclusions using a fluorescence microscope. All positive sera were then titrated to determine the antibody titer.

Hantavirus antibodies in human sera

Hantavirus-specific IgG antibodies in human sera were detected to Puumala (European) and Hantaan (Asian) strains by using enzyme-linked immunosorbent assay (ELISA). The ELISA kits were purchased from Progen (Heidelberg, Germany) and used according to the manufacturer's instructions. The sera positive by ELISA were also tested by an IFA kit (Progen) according to the manufacturer's instructions.

Results

Hantavirus-specific antibodies in rodent sera

Hantavirus-specific antibodies were detected in four out of 174 (2.3%) and three out of 84 (3.6%) sera of M. musculus and R. norvegicus, respectively (Table 1). Wild rats belonging to Tattera and Nesokia genera did not have detectable level of antibodies to hantaviruses (Table 1). With respect to the presence of virus strain-specific antibodies in sera of 283 rodents, anti-Seoul and anti-Puumala antibodies were detected in six (2.1%) and three (1.1%) sera, respectively (Table 2). Moreover, sera from two (0.7%) rodents showed the presence of anti-Seoul as well as anti-Puumala antibodies (Table 2). These results suggested mixed infection with both of the virus strains.

Table 1

Distribution of hantavirus antibodies in different species of rodents

Rodent species Number of tested rodents Rodents positive for anti-hantavirus antibodies 
  Number Percent 
R. norvegicus 84 3.6 
M. musculus 174 2.3 
N. indica 
T. indica 18 
Total 283 2.5 
Rodent species Number of tested rodents Rodents positive for anti-hantavirus antibodies 
  Number Percent 
R. norvegicus 84 3.6 
M. musculus 174 2.3 
N. indica 
T. indica 18 
Total 283 2.5 
Table 2

Hantavirus strain-specific antibodies in rodent sera

Virus strain Number of tested rodents Rodents positive for anti-hantavirus antibodies 
  Number Percent 
Seoul 283 2.1 
Puumala 283 1.1 
Seoul+Puumala 283 0.7 
Virus strain Number of tested rodents Rodents positive for anti-hantavirus antibodies 
  Number Percent 
Seoul 283 2.1 
Puumala 283 1.1 
Seoul+Puumala 283 0.7 

Hantavirus-specific antibodies in human sera

The results with human sera for anti-hantavirus antibodies with respect to age and virus strain in Kuwaitis and non-Kuwaitis are summarized in Tables 3 and 4. In addition, anti-hantavirus strain-specific antibodies with respect to the nationalities of the tested subjects are also given (Table 5). The results showed that 5/46 (11%) Kuwaitis and 8/137 (6%) non-Kuwaitis were positive for anti-hantavirus antibodies (Table 3). Among the positive Kuwaitis, one (2%) had antibodies to Puumala and four (9%) to Hantaan virus strains. Among the sera from non-Kuwaitis, four (3%) had antibodies to Puumala and four (3%) to Hantaan virus strains (Table 4). With respect to the nationalities of the tested subjects, the results did not show any significant difference in the positivity of sera to anti-hantavirus antibodies (Table 5)

Table 3

Hantavirus antibodies in human sera from Kuwaitis and non-Kuwaitis

Age group (years) Number positive/tested (% positive) 
 Kuwaitis Non-Kuwaitis Kuwaitis+non-Kuwaitis 
21–30 3/13 (23%) 3/37 (8%) 6/50 (12%) 
31–40 2/15 (13%) 2/50 (4%) 4/65 (6%) 
41–50 0/18 (0%) 3/38 (8%) 3/56 (5%) 
51–60 0/0 (0%) 0/12 (0%) 0/12 (0%) 
Total 5/46 (11%) 8/137 (6%) 13/183 (7%) 
Age group (years) Number positive/tested (% positive) 
 Kuwaitis Non-Kuwaitis Kuwaitis+non-Kuwaitis 
21–30 3/13 (23%) 3/37 (8%) 6/50 (12%) 
31–40 2/15 (13%) 2/50 (4%) 4/65 (6%) 
41–50 0/18 (0%) 3/38 (8%) 3/56 (5%) 
51–60 0/0 (0%) 0/12 (0%) 0/12 (0%) 
Total 5/46 (11%) 8/137 (6%) 13/183 (7%) 
Table 4

Hantavirus strain (Puumala and Hantaan)-specific antibodies in human sera from Kuwaitis and non-Kuwaitis

Age group (years) Number positive/tested Kuwaitis Number positive/tested non-Kuwaitis 
 Puumala Hantaan Puumala Hantaan 
21–30 1/13 2/13 1/37 2/37 
31–40 0/15 2/15 2/50 0/50 
41–50 0/18 0/18 1/38 2/38 
51–60 0/0 0/0 0/12 0/12 
Total 1/46 (2%) 4/46 (9%) 4/137 (3%) 4/137 (3%) 
Age group (years) Number positive/tested Kuwaitis Number positive/tested non-Kuwaitis 
 Puumala Hantaan Puumala Hantaan 
21–30 1/13 2/13 1/37 2/37 
31–40 0/15 2/15 2/50 0/50 
41–50 0/18 0/18 1/38 2/38 
51–60 0/0 0/0 0/12 0/12 
Total 1/46 (2%) 4/46 (9%) 4/137 (3%) 4/137 (3%) 
Table 5

Nationality-wise distribution of hantavirus strain (Puumala and Hantaan)-specific antibodies in the human sera

Nationality Number of sera Number of sera positive for antibodies to 
  Puumala virus Hantaan virus 
Kuwaiti 46 
Egyptian 51 
Afghani 36 
Pakistani 35 
Other nationalities 15 
Nationality Number of sera Number of sera positive for antibodies to 
  Puumala virus Hantaan virus 
Kuwaiti 46 
Egyptian 51 
Afghani 36 
Pakistani 35 
Other nationalities 15 

Other nationalities include Iranians (n=9), Bangladeshis (n=3) and Sri Lankans (n=3).

Discussion

The results of this study show for the first time that rats (R. norvegicus), mice (M. musculus) and humans in Kuwait are infected with hantaviruses. However, the 2–3% antibody prevalence in rodents of Kuwait is lower than that found in Wisconsin and Minnesota [4], where 20% of rats and 8% of mice had hantavirus antibodies, and in Northern Ireland (22% for rats, 29% for mice) [11], but it is close to the seropositivity level of 1.1% in rats of Nagoya, Japan (1.1% for rats) [12] and 2–4% seroprevalence in the rodent population of the Nakhon Pathom and Nakhon Ratchasima provinces of Thailand [13].

Among the hantavirus-infected rodents, the presence of antibodies against the Puumala and Seoul strains in two rodents is interesting because it suggests dual infection. Kuwait has a very active seaport where ships come from all over the world. Therefore, the mixing of rodents from east and west is very likely in Kuwait and might have resulted in cross-infection. This also points to active transmission of hantavirus infection in Kuwait.

Thus, despite the relatively low level of hantavirus activity in rodents in Kuwait, there is a chance for humans to acquire infections. Of the 46 Kuwaiti individuals who were at high risk to contact rodent excreta, five (11%) had antibodies to hantaviruses. This prevalence level is the same as reported in studies conducted in Egypt [14], Sweden [15] and The Netherlands [16]. However, the mere presence of antibodies does not have a direct correlation with the manifestation of the disease in humans. In Wisconsin and Minnesota, antibodies to hantaviruses were detected in humans as early as 1984 [3], but serious diseases (HPS) associated with hantaviruses were reported only in 1993 [5].

We are not aware of any report on human hantavirus-associated disease in Kuwait. However, the diagnostic facilities were not available in the past, and without those it is practically impossible to determine the impact of hantavirus infection in the country. The establishment of serological methods to detect anti-hantavirus antibodies will now facilitate the diagnosis of clinically suspected cases of HFRS and HPS.

Acknowledgments

This study was supported by Kuwait University Research Administration Grants MI108 and MI115.

References

[1]
Lee
H.
van der Groen
G.
(
1989
)
Haemorrhagic fever with renal syndrome
.
Progr. Med. Virol.
 
36
,
62
102
.
[2]
Duchin
J.S.
Koster
F.T.
Peters
C.J.
Simpson
G.L.
Tempest
B.
Zaki
S.R.
et al
. (
1994
)
Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease
.
New Engl. J. Med.
 
330
,
949
955
.
[3]
Burek
K.A.
Rossi
C.A.
Leduc
J.W.
Yuill
T.M.
(
1984
)
Serologic and virologic evidence of a Prospect Hill-like hantavirus in Wisconsin and Minnesota
.
Am. J. Trop. Med. Hyg.
 
51
,
286
294
.
[4]
Schmaljohn
C.
Hjelle
B.
(
1997
)
Hantaviruses: a global disease problem
.
Emerg. Infect. Dis.
 
3
,
95
104
.
[5]
Centers for Disease Control
(
1993
)
Update: outbreak of hantavirus infection — southwestern United States, 1993
.
Morbid. Mortal. Wkly Rep.
 
42
,
1325
1326
.
[6]
Young
J.C.
Mills
J.N.
Enria
D.A.
Dolan
N.E.
Khan
A.S.
Ksiazek
T.G.
(
1998
)
New World hantaviruses
.
Brit. Med. Bull.
 
54
,
659
673
.
[7]
Lee
H.W.
French
G.R.
Lee
P.W.
Baek
L.J.
Tsuchiya
K.
Foulke
R.S.
(
1981
)
Observation on natural and laboratory infection of rodents with the etiologic agent of Korean hemorrhagic fever
.
Am. J. Trop. Med. Hyg.
 
30
,
477
482
.
[8]
Hutchinson
K.L.
Rollin
P.E.
Peters
C.J.
(
1998
)
Black Creek Canal virus in experimentally infected Sigmodon hispidus
.
Am. J. Trop. Med. Hyg.
 
59
,
58
65
.
[9]
Mills
J.N.
Yates
T.L.
Ksiazek
T.G.
Peters
C.J.
Childs
J.E.
(
1999
)
Long term studies of hantavirus reservoir population in the southwestern United States: rationale, potential, and methods
.
Emerg. Infect. Dis.
 
5
,
95
101
.
[10]
Lee
P.W.
Gibbs
C.J.
Jr.
Gajdusek
D.C.
Yanagihara
R.
(
1985
)
Serotypic classification of hantaviruses by indirect immunofluorescent antibody and plaque reduction neutralization tests
.
J. Clin. Microbiol.
 
940
944
.
[11]
McCaughey
C.
Montgomery
W.I.
Twomey
N.
Addley
M.
O'Neill
H.J.
Coyle
P.V.
(
1996
)
Evidence of hantavirus in wild rodents in Northern Ireland
.
Epidemiol. Infect.
 
111
,
361
365
.
[12]
Sugiyama
K.
Takagi
K.
Kinjo
T.
Komatsu
T.
Kitamura
T.
(
1995
)
Isolation and characterisation of a hantavirus from Rattus norvegicus in a residential area of Nagoya City, Japan
.
J. Vet. Med. Sci.
 
57
,
51
54
.
[13]
Nitatpattana
N.
Chauvancy
G.
Dardaine
J.
Poblap
T.
Jumronsawat
K.
Tangkanakul
W.
Poonsuksombat
D.
Yoksan
S.
Gonzalez
J.P.
(
2000
)
Serological study of hantavirus in the rodent population of Nakhon Pathom and Nakhon Ratchasima provinces Thailand
.
Southeast Asian J. Trop. Med. Publ. Health
 
31
,
277
282
.
[14]
Corwin
A.
Habib
M.
Olson
J.
Scott
D.
Ksiazek
T.
Watts
D.M.
(
1992
)
The prevalence of arboviral, rickettsial, and hantan-like viral antibody among schoolchildren in the Nile river delta of Egypt
.
Trans. R. Soc. Trop. Med. Hyg.
 
86
,
677
679
.
[15]
Ahlm
C.
Juto
P.
Stegmayr
B.
Settergren
B.
Wadell
G.
Tarnvik
A.
Elgh
F.
(
1997
)
Prevalence of serum antibodies to hantaviruses in northern Sweden as measured by recombinant nucleocapsid proteins
.
Scand. J. Infect. Dis.
 
29
,
349
354
.
[16]
Groen
J.
Gerding
M.N.
Jordans
J.G.
Clement
J.P.
Nieuwenhuijs
J.H.
Osterhaus
A.D.
(
1995
)
Hantavirus infections in the Netherlands: epidemiology and disease
.
Epidemiol. Infect.
 
114
,
373
383
.

Author notes

1
CSIR Emeritus Scientist, Industrial Toxicology Research Centre, M.G. Road, P.O. Box 80, Lucknow 226001, India.