Download INVESTIGATION OF HANTAVIRUS INFECTIONS AMONG CCHFV

Acta Medica Mediterranea, 2014, 30: 855
INVESTIGATION OF HANTAVIRUS INFECTIONS AMONG CCHFV NEGATIVE CASES IN THE
WESTERN BLACK SEA REGION OF TURKEY
YAVUZ UYAR1,2, DILEK YAGCI CAGLAYIK2, GÜLAY KORUKLUOGLU2, AHMET CARHAN3, MUSTAFA ERTEK4, CHRISTEL COCHEZ5,
JENNY VERNER-CARLSSON6, ÅKE LUNDKVIST6-7, PAUL HEYMAN5 1
Istanbul University, Cerrahpasa School of Medicine, Dept. of Medical Microbiology, Istanbul, 2Public Health Institute of Turkey
(PHIT), Dept. of Microbiology Reference Laboratories, Virology Reference and Research Laboratory, Ankara,
3
Yildirim Beyazit University, School of Medicine, Dept. of Medical Biology, Ankara, 4Ankara Oncology Training and Research
Hospital, Dept. of Infectious Diseases, Ankara, Turkey, 5Research Laboratory for Vector-Borne Diseases, Reference Laboratory for
Vector-Borne Diseases, ACOS Well-Being / Health / Epi&Biostat, Bruynstraat, Brussels, Belgium, 6The Public Health Agency of
Sweden and Karolinska Institutet, Stockholm, Sweden - 7Uppsala University, Dept. of Medical Biomedicine and Microbiology, Uppsala,
Sweden
ABSTRACT
Objective: The Hantaviruses and Crimean Congo hemorrhagic fever virus (CCHFV) are members of the Bunyaviridae family.
Hantavirus infections causes two main febrile diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). Hantaviruses are transmitted by rodents. In 2009, a hantavirus outbreak occurred in the Western Black
Sea region of Turkey. For the last 10 years, the prevalence of CCHFV has also been high in this region.
Methods: We screened patients’ samples for the presence of hantavirus IgM and IgG using ELISA on 42 clinically suspect
CCHF cases which were CCHFV real-time-RT-PCR and anti-CCHFV IgM negative without a tick-bite history. The current study was
carried out on samples sent between March and September of 2008. SELISA-seropositivity was further evaluated using immunofluorescence assay, immunblotting, and FRNT (focus reduction neutralization test). Real-time-PCR was performed to genotype the seropositive cases.
Results: Anti-Hantavirus IgM and IgG positivity were detected in 3 samples (7.1%) by ELISA and immunoblotting tests. Two
had a 1:640 titer against Puumala virus (PUUV) strain by FRNT, while all three samples were found negative for Dobrova virus
(DOBV). Thus, hantavirus-specific antibodies could be confirmed detected in 2 out of 42 sera (4.8%) by FRNT.
Conclusions: TThe authors postulate that Hantavirus infection should be taken into consideration in patients with clinically
suspected CCHFV infection. This study also shows that Hantavirus was circulating in the area before the 2009 hantavirus outbreak
in the Western Black Sea region of Turkey.
Key words: Hantavirus, CCHFV, ELISA, real time-RT-PCR, FRNT, Turkey.
Received February 18, 2014; Accepted May 19, 2014
Introduction
Hantaviruses belong to the Bunyaviridae family, which includes five genera: Bunyavirus,
Phlebovirus, Nairovirus, Tospovirus, and
Hantavirus. The Bunyavirus family comprises a
number of arthropod-borne viruses (arboviruses,
while the Tospoviruses are plant viruses, and the
Hantaviruses rodent-borne(1). The viruses within the
genus Hantavirus causes various febrile diseases:
hemorrhagic fever with renal syndrome (HFRS) in
Asia and Europe, and hantavirus cardiopulmonary
syndrome (HCPS) in the Americas(2). Hantaviruses
are transmitted by rodents and insectivores. The
infection in humans is transmitted via aerosols of
infected rodents’ saliva, feces and urine(1,2). Recently
published data have revealed that also bats are possible hantavirus vectors(3).
Hantaviruses are negative-sense, singlestranded enveloped RNA viruses with a genome of
three single-stranded RNA segments designated S
(small), M (medium) and L (large)(1). Clinically,
hantavirus infections range from subclinical to fatal
and can be separated into five main phases: febrile,
856
hypotensive, oliguric, diuretic and convalescent(4).
The febrile phase of hantavirus infection lasts for 37 days, accompanied by headache and abdominal
pain(5).
Hantavirus infection is a global public health
problem with approximately 50,000 to 150,000
annual cases reported worldwide. Most cases (about
50,000) are caused by Hantaan virus (HTNV) and
Seoul virus (SEOV) in Asia. In addition, approximately 200 cases are diagnosed annually as HCPS
in the United States(6). Puumala virus (PUUV) and
Dobrava virus (DOBV) have caused the majority of
the HFRS cases in Europe(4).
Each Hantavirus is transmitted predominantly
by its own natural host, whether rodent or insectivore(7,8). Of the approximately 2,000 existing rodent
species, only about 5% so far been tested for the
presence of Hantaviruses (9). The most common
route of hantavirus infection is via inhalation of
infectious aerosolized saliva or excreta originating
from infected rodents (10). Humans can become
infected by exposure to contaminated dust from
rodent nests or droppings(4.6,10). Human to human
transmission is not generally occuring(11) except for
the Andes virus (ANDV) in Argentina(12).
There are three distinct genetic groups of
rodent hosts: Murinae, Arvicolinae, and
Sigmodontinae rodents. HTNV, SEOV and DOBV
viruses causing more severe forms of HFRS are
carried by Murinae rodents mainly found in Asia
and the Balkans. PUUV, hosted by Arvicolinae
voles in most European countries, causes a milder
form of HFRS. The Hantaviruses in the Americas
are mainly transmitted by rodents of the subfamilies
Sigmodontinae and Neotominae(13). In Europe, three
Hantaviruses (PUUV, DOBV, and Saaremaa SAAV)
are known to cause HFRS. PUUV and SAAV generally cause milder HFRS, but DOBV infections
often also induce hemorrhagic complications (4).
PUUV, DOBV and SAAV viruses are carried by the
bank vole (Myodes glareolus), yellow-necked
mouse (Apodemus flavicollis) and striped field
mouse (Apodemus agrarius), respectively. SEOV is
carried by Rattus norvegicus and R. rattus in
Europe, but only a few cases have been reported so
far.(14,15) Co-evolution has been suggested for the
Hantaviruses and their vectors (7,8). Several hantavirus-carrying rodent species have been identified
in Turkey, which also hosts 45 rodent species and
17 endemic insectivore species(16,17).
In 2009, a hantavirus outbreak occurred in the
Western Black Sea region of Turkey from January
Yavuz Uyar, Dilek Yagci Caglayik et Al
to May(18). In this study, we investigated anti-hantavirus ELISA IgM and IgG positivity in samples
from hospilitized clinically suspected Crimean
Congo hemorrhagic fever (CCHF) cases without a
tick-bite history from the same geographical area as
the earlier Hantavirus outbreak, These patients’ sera
samples had been sent before the outbreak, in 2008,
to the Public Health Institute of Turkey (PHIT),
Department of Microbiology Reference
Laboratories (formerly, the Refik Saydam National
Public Health Agency).
Materials and methods
Samples
After the 2009 hantavirus outbreak on the
Western Black Sea coast (specifically Bartin and
Zonguldak provinces), it was decided to investigate
the presence of hantavirus infections among the
CCHF negative (ELISA IgM, IgG and PCR), but
clinically suspected CCHF samples. According to the
case report forms, there was no evidence of tick-bite
history in the selected cases’ anamnesis. For all samples, an ‘in-house’ one-step real-time PCR for
CCHFV was performed as in Yapar et al.(19). AntiCCHFV IgM and IgG assays were performed
according to the CDC (Centers for Diseases Control
and Prevention, US) protocol(20). 42 serum samples
stored at -800C were selected matching the conditions listed above from the Virology Reference and
Research Laboratory of PHIT. Of the 42 samples, 26
were from Kastamonu, 10 from Karabük, two from
Bartin, two from Bursa, one from Bolu and one from
the Zonguldak provinces (Figure 1).
1=Bursa (n:2), 2= Kastamonu (6), 3= Bartin (n:2), 4=
Karabuk (n:10), 5= Zonguldak (n:1), 6= Bolu (n:1).
Figure 1: Distribution of the 26 serum samples were
provinces of Turkey.
Only seven samples, sent from the Kastamonu
province, had follow-up serum samples taken 10-16
days after the initial sampling. This study includes
the results for these follow-up samples.Only seven
samples, sent from Kastamonu province, had follow-
Investigation of hantavirus among C..
up serum samples taken 10-16 days after the initial
sampling. This study includes the results for these
follow-up samples.
Anti-hantavirus IgM and IgG ELISA
Commercial hantavirus ELISA tests (Focus
Diagnostics, DxSelect, Cypress, CA, USA), which
detect antibodies of the most clinically relevant
pathogenic strains of Hantaviruses: SEOV, HTNV,
PUUV, DOBV, and SNV were used according to
the manufacturer. The results were reported as
index values (I.V.) relative to the cut-off calibrator.
To calculate I.V.s, optical density (O.D.) values of
the specimens are divided by the mean of the cutoff calibrator absorbance values. An I.V. of > 1.10
is indicative of the presence of IgM and IgG hantavirus specific antibodies.
Immunoblotting assay for hantavirus antibodies
An immunoblotting assay was used for confirmation of the ELISA IgG and IgM positive samples. The Hanta Profile 1 EUROLINE (Euroimmun,
Luebeck, Germany) tests were performed according
to the manufacturer’s protocol. The strip provides a
semi-quantitative assay for detection of human antibodies of the IgG class for three different hantavirus serotypes: PUUV, DOBV and HTNV. The
processed strips were visually evaluated from (0) to
(+++). Medium (+) to strongly (++/+++) colored
bands were considered to indicate positivity.
RNA extraction for molecular analysis
RNA was extracted from serum samples using
a commercial extraction kit (QIAamp viral RNA
mini extraction kit, QIAGEN), following the manufacturer’s instructions.
Real-time RT-PCR assay for hantaviruses
The Hantavirus Renal Syndrome General-type
I&II Real Time RT-PCR kit (Shanghai ZJ Bio-Tech
Co., Ltd., China) was used for the detection of the
Hantavirus genome in serum using the ABI Prism
7500 real time PCR system (Applied Biosystems,
CA, USA). The following protocol was used for
real time-PCR: 45 ºC for 10 min., 1 cycle; 95 ºC for
15 min., 95 ºC for 15 sec., 60 ºC for 60 sec., and 40
cycles.
Focus reduction neutralization test (FRNT)
for seropositive samples
As PHIT lacked the technical capability at the
857
time of the study, the three seropositive samples
tested as PUUV serotype by hantavirus
immunoblotting were sent to Sweden for confirmation by FRNT tests. For analysis by FRNT, the
PUUV strain Sotkamo (21) and the DOBV strain
Slovenia(22) were used. The test was performed as
described earlier(23). Briefly, sera were serially diluted and mixed with an equal volume of diluted
virus, containing 30-70 focus-forming units/100 μl.
The mixture was incubated at 37°C for 1 h. and
subsequently inoculated into six-well tissue culture
plates containing confluent Vero E6 cell monolayers. The wells were overlaid with a mixture of
agarose and tissue culture medium and incubated
for 9 (DOBV) or 13 days (PUUV). The agarose was
removed from the wells and the cells fixed in
methanol. Rabbit anti-hantavirus sera, followed by
peroxidase labeled goat antibodies to rabbit IgG
(BioRad Laboratories, Hercules, CA), were added
to indicate virus-infected cells. 3, 3’, 5, 5’-tetramethylbenzidine substrate (Sigma) was used as substrate and foci were enumerated. An 80% reduction
in the number of foci compared to the virus control
was used as the criterion for virus neutralization
titers.
Ethical approval
This investigation was designed and conducted
after gaining the official permission of the RSNPHA
Scientific Board, Ankara and approval from the
Erciyes University Ethical Board, Kayseri, Turkey.
Results
The serum samples were drawn from 22 male
(mean age 51) and 20 female patients (mean age:
54 years). According to the patients’ declaration of
their occupations, there was one housewife with no
link to cattle-breeding, 7 wives of cattle-breeders, 6
retired people, 5 cattle-breeders, 5 housewives with
unspecified additional occupation, 4 farmers of
which 3 were also involved in cattle-breeding, 3
students, 2 merchants, 1 forestry worker, 1 unemployed person, 1 nurse, 1 carpenter and 2 patients
with unspecified occupations.
Thrombocytopenia, fever and hemorrhage
were reported in 88.1%, 66.7%, and 7.4% of the
patients respectively. All the samples from patients
without tick-bite history were selected in order to
exclude a diagnosis of CCHF. The patients’ sera
had been sent to our laboratory between March and
September of 2008 for diagnosis of CCHF.
858
Yavuz Uyar, Dilek Yagci Caglayik et Al
Seven follow-up samples were also sent from
the Kastamonu province’s suspected CCHF
patients.
Anti-Hantavirus IgM and IgG positivity were
detected in three (7.1%) of the samples by ELISA.
Two had a 1:640 titer against PUUV strain while all
three were negative for DOBV.
After screening for antibodies by ELISA and
immunoblotting tests, hantavirus-specific antibodies were confirmed by FRNT in 2 (4.8%) of the 42
samples.
The seropositive patients’ occupations were all
housewife with no link to cattle breeding or farming.
These patients were living in the Karabük province,
which borders both Bartin and Zonguldak, where the
Hantavirus outbreak occurred in 2009 (Figure 1).
According to the case report forms of these confirmed patients, one of them had hemorrhage, one
had only thrombocytopenia, while one had both
fever and thrombocytopenia. Table1 summarizes the
characteristics and laboratory findings of the 2 hantavirus cases.
Characteristics of patient
Age (years)
Gender
Occupation
Urban/Rural Area
1st confirmed case
22
Female
housewife – no cattle
breeding
Urban
Time interval between the day of
onset of symptoms (DOS) and
5
sampling (days)
Onset of infection
April 2008
Laboratory findings
ELISA
Anti-Hantavirus IgM
Positive
Anti-Hantavirus IgG
Positive
Immunoblotting
Anti-Hantavirus IgM
2nd confirmed case
24
Male
Farmer
Urban (but he visited
rural areas 2 weeks
prior to DOS)
5
June 2008
Positive
Positive
Positive (PUUV ++) Positive (PUUV +++)
Anti-Hantavirus IgG
Positive (PUUV +)
Real time RT-PCR for Hantavirus
Negative
FRNT for PUUV
Positive (1:640)
FRNT for DOBV
Negative (< 40)
Positive (PUUV ++)
Negative
Positive (1:640)
Negative (< 40)
Table 1: Laboratory findings in the confirmed hantavirus
cases.
Discussion
Hantaviruses are rodent- or insectivore-borne
emerging viruses. The rodent-borne viruses cause
two important human diseases, HFRS in Asia and
Europe, and HCPS in the Americas(2). HFRS cases
usually occur in rural areas with the condition being
an occupational hazard for forest workers, farmers,
shepherds, military personnel and woodcutters(1,4).
The majority of PUUV-associated cases occur
in the fall, while most DOBV-caused HFRS cases
are registered in late spring and summer(1).
HFRS outbreaks due to PUUV are associated
with an increased population of the bank vole
(Myodes glareolus), the main natural rodent host
for PUUV(1,4). Thus, changes in rodent population
density influence the scale of hantavirus infection
outbreaks in humans(1,24).
At the same time, elevated average temperatures in West-Central Europe have also been associated with more frequent PUUV outbreaks, through
high food production (mast years) and subsequently
higher bank vole densities(2,8,24).
The bank vole, the carrier of PUUV, has a distribution range that includes most of Europe and is
present in Turkey as well(16). Human disease incidence and epidemiological patterns vary greatly
across the continent(7).
In Turkey, PUUV infections in rodents was
demonstrated for the first time using immunofluorescence assays in Microtus voles in two regions.
Of 330 rodents captured in Izmir, Trabzon and Rize
by Laakkonen et al in 2004(25), PUUV antibodies
were found in four out of 65 Microtus voles. One of
them (Microtus guentheri lydius) originated from
Izmir, while three (one Microtus roberti and two
Microtus rossiaemeridionalis) were captured
around Trabzon. Laakkonen et al were not able to
amplify RNA from these samples by RT-PCR.
Kavukcu et al. reported that hantavirus
seropositivity was 7.3% among cases with
nephropathy but 2.6% in cases without nephropathy
or any infectious disease. From this, they concluded
that hantavirus disease was only present in the
Aegean Region and Anatolia(26).
Since the first reported outbreak of hantaviruses in Turkey in 2009(18), sporadic cases have been
detected in several Eastern Black Sea region
provinces, such as Giresun and Ordu, and in rural
areas of Istanbul in the Marmara region(27,28).
In order to investigate further, we planned a
hantavirus study of CCHF suspected but CCHFV
negative sera samples (n: 42) collected in 2008. In
the current study, two samples (4.8%) were found
to be PUUV positive by FRNT. These serological
results show that there were hantavirus cases occurring before the 2009 outbreak. One of the two hantavirus seropositive patients had gross hemorrhages, and one had hematuria in their anamnesis
forms. In cases of PUUV serotype, hemorrhages are
seen in only 2% of samples(4). One of our patients
had only fever and another had both fever and
thrombocytopenia. According to the literature,
HFRS cases usually occur in rural areas and are
Investigation of hantavirus among C..
generally seen in farmers, forest workers and shepherds(4).
In the current study, one of the two seropositive patients was a farmer as expected from the literature. The two patients became ill in late spring or
summer and had not been abroad in their anamnesis
during the preceding 2 months. HFRS cases are
also known to coincide with increased agricultural
activities(1).
After the hantavirus outbreak in 2009, a
National Hantavirus Study Group was formed in
Turkey by the Ministry of Health. In June 2009, a
total of 173 rodents and 2 shrews were collected
from 7 sites in rural areas of Bartin Province, where
suspected human hantavirus cases had been reported. Using RT-qPCR, Oktem et al found 23 DOBV
RNA-positive rodents from the outbreak area,
including 10 A. flavicollis mice, 12 A. uralensis
mice, and 1 Rattus rattus rat(29).
According to the results of the current study,
hantavirus infection should be considered in serological and real time-PCR-negative CCHF suspected patients in the Black Sea region in Turkey. The
current findings also show that there were positive
hantavirus human cases before the 2009 outbreak
emerged. This is the first report in the literature to
use neutralization tests to confirm historical hantavirus human infections in Turkey.
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Acknowledgements
This study was funded by the RSNPHA Scientific
Board (BAP Project). We thank the laboratory personnel of the Virology Laboratory of RSNPHA for
excellent technical assistance, and the Primary
Health Care Directorate of the Turkish Ministry of
Health, Provincial Health Directorates, for their collaboration and for organizing the sample collection.
_________
Corresponding Author
YAVUZ UYAR, Assoc. Prof., MD
Medical Microbiologist & Virologist (PhD)
Istanbul University
Cerrahpasa School of Medicine
Dept. of Medical Microbiology
34098- Fatih/ Istanbul
(Turkey)