Hantaviruses in Serbia and Montenegro.Hantaviruses are endemic in the Balkan Peninsula Balkan Peninsula, southeasternmost peninsula of Europe, c.200,000 sq mi (518,000 sq km), bounded by the Black Sea, Sea of Marmara, Aegean Sea, Mediterranean Sea, Ionian Sea, and Adriatic Sea. . An outbreak of hemorrhagic fever with renal syndrome hemorrhagic fever with renal syndrome n. See epidemic hemorrhagic fever. occurred in 2002 in Serbia and Montenegro Serbia and Montenegro (sûr`bēə, mŏn'tənē`grō), Serbian Srbija i Crna Gora, former country of SE Europe, in the Balkan Peninsula, a short-lived union (2003–6) of the republics of Serbia and the much . The epidemiologic characteristics and genetic relatedness of Dobrava/Belgrade virus strains responsible for most cases are described. ********** Hantaviruses (Bunyaviridae) are enveloped en·vel·op tr.v. en·vel·oped, en·vel·op·ing, en·vel·ops 1. To enclose or encase completely with or as if with a covering: "Accompanying the darkness, a stillness envelops the city" , single-stranded, negative-sense RNA viruses RNA viruses, n See viruses. with a tripartite genome consisting of a small (S), a medium (M), and a large (L) segment, which encode the nucleocapsid nucleocapsid /nu·cleo·cap·sid/ (noo?kle-o-kap´sid) a unit of viral structure, consisting of a capsid with the enclosed nucleic acid. nu·cle·o·cap·sid n. protein, the glycoprotein glycoprotein (glī'kōprō`tēn), organic compound composed of both a protein and a carbohydrate joined together in covalent chemical linkage. precursor and the putative RNA polymerase RNA polymerase n. A polymerase that catalyzes the synthesis of RNA from a DNA or RNA template. , respectively (1). Hantaviruses are transmitted to humans through aerosols of excreta excreta /ex·cre·ta/ (eks-kret´ah) excretion (2). ex·cre·ta pl.n. Waste matter, such as sweat or feces, discharged from the body. from small mammals, mainly rodents, that have had silent lifelong-infections. More than 30 different hantaviruses have been distinguished so far, at least half are related to disease in humans. These viruses cause hemorrhagic fever with renal syndrome (HFRS HFRS Hemorrhagic Fever With Renal Syndrome HFRS Hampshire Fire and Rescue Service (UK) HFRS Humberside Fire and Rescue Service (UK) HFRS High-Float, Rapid-Setting (emulsion) ) in Asia and Europe and hantavirus pulmonary syndrome hantavirus pulmonary syndrome An often fatal RTI caused by a hantavirus; the first cluster occurred in the Four Corners region of Southwestern US Epidemiology Mean age 32, 61% ♀, 72% Native American Case definition Unexplained bilateral interstitial (HPS See Seer*HPS. ) in America. HFRS is caused by Hantaan (HTNV), Dobrava/Belgrade (DOBV), Seoul (SEOV), and Puumala (PUUV) hantaviruses, while HPS is caused by Sin Nombre (SNV SNV Synovus Financial Corp. (stock symbol) SNV Schweizerische Normenvereinigung (Swiss standards body) SNV Stichting Nederlandse Vrijwilligers (Netherlands Development Organization) ) and related viruses. Each hantavirus hantavirus, any of a genus (Hantavirus) of single-stranded RNA viruses that are carried by rodents and transmitted to humans when they inhale vapors from contaminated rodent urine, saliva, or feces. There are many strains of hantavirus. is associated with a specific primary rodent reservoir of the Muridae family; these relationships have coevolved over a long period, probably >50 million years (1). HFRS is endemic in the Balkan Peninsula, where sporadic cases or outbreaks have been reported. The disease is seen during the summer and affects mainly adults (2,3), although infections in children, some fatal (4), have been reported. Hantaviruses associated with disease in humans in Balkans are DOBV, carried by the yellow-necked mouse (Apodemus flavicollis), which causes severe HFRS with a fatality rate fa·tal·i·ty rate n. See death rate. fatality rate see case fatality rate. up to 10%, and PUUV, carried by the red bank vole (Clethrionomys glareolus). PUUV causes nephropathia epidemica, a milder form of HFRS, with a fatality rate <1% (3,5-8). Recently, A. agrarius was found to be an additional host of DOBV, causing a milder disease than that associated with A. flavicollis (9). Additionally, Tula virus RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was amplified from lung tissues of a European pine vole The European Pine Vole (Microtus subterraneus) is a species of rodent in the Cricetidae family. It is found in Albania, Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Estonia, France, Germany, Greece, Hungary, Italy, Liechtenstein, Luxembourg, FYROM, (Pitymys subterraneus) in Serbia (10). The first probable HFRS case was reported in former Yugoslavia in 1952 (11,12); the first identified epidemic of HFRS occurred in 1961 (13). Some years (namely, 1961, 1967, 1979, 1986, 1989, and 1995 [2]) are characterized by increased HFRS cases. Different factors, such as weather and food abundance, could influence the dynamics of rodent populations. The more recent large epidemic in Serbia and Montenegro occurred in 2002 with 128 laboratory-confirmed cases. The number of confirmed cases was lower in the following years. In 2003, 16 cases occurred in Serbia and 18 in Montenegro (1 fatal). In 2004, 20 cases (1 fatal) occurred in Serbia and 11 in Montenegro. During 2002, a total of 376 serum samples from patients with suspected HFRS cases were tested in Torlak Institute, Belgrade, by indirect immunofluorescent assay Immunofluorescent assay (IFA) A blood test sometimes used to confirm ELISA results instead of using the Western blotting. In an IFA test, HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. (IFA Immunofluorescent assay (IFA) A blood test sometimes used to confirm ELISA results instead of using the Western blotting. In an IFA test, HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. ) for the presence of hantavirus antibodies. IFA was performed on spot slides containing Vero E6 cells infected with HTNV, SEOV, PUUV, and DOBV. For 128 cases (77 from Serbia, 51 from Montenegro), a laboratory diagnosis of HFRS was made. Most patients (77.3%) were infected with DOBV-like viruses; the rest were infected with PUUV-like viruses. Briefly, 53 (69%) of 77 samples from Serbia and 46 (90%) of 51 from Montenegro had higher antibody titers to HTNV and DOBV than to PUUV; the other samples had higher titers to PUUV. Two Serbian patients who lived in Leskovac died. Most DOBV-like infections from Serbia occurred in the south (Leskovac, Vranje, Nis, Surdulica, Vlasina), while the PUUV-like infections occurred in the north (Vojvodina and area near the River Drina) (map of Serbia and Montenegro available from http://www.un.org/Depts/Cartographic/map/profile/ yugoslav.pdf). Thirty-one serum samples from the IFA-positive patients were sent to Aristotle University for additional testing. Samples were taken from 21 HFRS patients with a mean age of 40.3 years (21-68 years); 1 sample was obtained from a 5-month-old male infant, whose mother had HFRS at the time of delivery. Two of 21 patients died. Enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay n. ELISA. Enzyme-linked immunosorbent assay (ELISA) A diagnostic blood test used to screen patients for AIDS or other viruses. (ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent. ELISA n. ) to detect immunoglobulin G immunoglobulin G n. Abbr. IgG The most abundant class of antibodies found in blood serum and lymph and active against bacteria, fungi, viruses, and foreign particles. Immunoglobulin G antibodies trigger action of the complement system. (IgG) and IgM antibodies to HTNV and PUUV was performed with kits by Progen (Biotechnik GmbH, Heidelberg, Germany). IgM antibodies to HTNV were detected in 18 of 21 patients; 9 also carried IgM antibodies to PUUV, although in lower titers than to HTNV (Table 1). IgG antibodies to HTNV were present in 17 of 21 patients; in 3 patients low titers of IgM antibodies to PUUV were also detected. The infant had IgG antibodies to HTNV. In 1 sample (DR) no antibodies to HTNV or PUUV were detected, although it was positive by IFA. ELISA results suggested that all 21 patients had an HTNV-like infection. Viral RNA was extracted from IgM-positive samples (a sample from the neonate neonate /neo·nate/ (ne´o-nat) newborn infant. ne·o·nate n. A neonatal infant. neonate a newborn animal. was also included) by using the viral RNA extraction kit (Qiagen GmbH, Hilden, Germany). Reverse transcription reverse transcription n. The process by which DNA is synthesized from an RNA template. and nested amplification were performed with primers previously designed to detect partial S and M segment sequences from hantaviruses associated with rodents of the Murinae and Arvicolinae subfamilies (14,15). Three samples (M.D., T.V., P.V.) gave a PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) product of the expected size of 599 bp, when primers specific for the S segment of hantaviruses associated with Murinae rodents were used; 1 sample (MD) gave a product of 317 bp with the primers for the M segment of the same hantaviruses. No product was obtained when primers specific for the S segment of hantaviruses associated with Arvicolinae rodents were used. Nucleotide sequences were aligned with respective hantavirus sequences retrieved from GenBank; genetic distances were measured by the neighbor-joining method, and phylogenetic trees were constructed by using PHYLIP PHYLIP Phylogeny Inference Package (genetics software) (Phylogeny Inference Package by J. Felsenstein [http://evolution. genetics.washington.edu/phylip.html]). The nucleotide sequences were assigned the accession numbers DQ305279-DQ305282. Two phylogenetic trees were constructed, one for the S segment (Figure 1) and another for the M segment (Figure 2). In both trees, hantavirus strains from Serbia and Montenegro cluster with other DOBV sequences and were associated with the rodent A. flavicollis. In the S segment tree, sequences of this study comprise the Serbian clade clade Cladus, subtype Genetics A branch of biological taxa or species that share features inherited from a common ancestor; a single phylogenetic group or line. See Inheritance, Species. in the DOBV-A. flavicollis cluster. In the same cluster are the Slovenian, Slovakian, and Greek clades. Sequences of this study differ by 0.3%-2.6% at the nucleotide level, with identical deduced amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. sequences. Genetic distances with other DOBV sequences are seen in Table 2. Concerning the M segment, a fragment of the G1-coding region of patient MD differed by 5.7% at the nucleotide level from the Slovenian DOBV strain isolated from A. flavicollis, with identical deduced amino acid sequences. The differences from DOBV strains from northwestern Greece were 8.5%-9.4% and 1% at nucleotide and amino acid levels, respectively. Patient TV was a 38-year-old woman who lived in Vranje. Patient PV was a 29-year-old woman who lived in Leskovac. Both of these locations are in southeastern Serbia. PV died on day 6 of illness. Patient MD was living in Beograd. However, his sequences were similar to those of patients TV and PV. His travel history showed that 18 days before the onset of illness, he was on vacation in Kolasin Mountain in Montenegro, where he was probably infected. Thus, all sequences of this study were from the southern region of the country and clustered with other DOBV strains associated with A. flavicollis rodents. However, the involvement of other hantaviruses in the outbreak cannot be excluded. Although the number of samples tested was limited, this study gives the first genetic information on DOBV strains circulating in Serbia and Montenegro. Further studies of both patients and small mammals in the region are needed to find out the exact epidemiology of HFRS in the country. Acknowledgments We thank Vassiliki Pavlidou and Panagiota Papadopoulou for excellent technical assistance. Dr Papa is assistant professor of medicine and head of the molecular diagnostics and special pathogens laboratory at the Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki The Aristotle University of Thessaloniki (often referred to in English as Aristotelian University), named after the philosopher Aristotle, is the largest university of Greece. Its campus covers 429 metric acres close to the center of the city of Thessaloniki. , Greece. Her major interest is the molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of hantaviruses and nairoviruses. References (1.) Nichol ST, Ksiazek TG, Rollin PE, Peters CJ. Hantavirus pulmonary syndrome and newly described hantaviruses in the United States. In: Elliott RM, editor. The Bunyaviridae. New York New York, state, United States New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of : Plenum Press; 1996. p. 269-80. (2.) Avsic-Zupanc T. Hantaviruses and hemorrhagic fever with renal syndrome in the Balkans. In: Saluzzo JF, Dodet B, editors. Factors in the emergence and control of rodent-borne viral diseases. Paris: Elsevier SAS (1) (SAS Institute Inc., Cary, NC, www.sas.com) A software company that specializes in data warehousing and decision support software based on the SAS System. Founded in 1976, SAS is one of the world's largest privately held software companies. See SAS System. ; 1999. p. 93-8. (3.) Antoniadis A, Stylianakis A, Papa A, Alexion-Daniel S, Lampropoulos A, Nichol ST, et al. Direct genetic detection of Dobrava virus in Greek and Albanian patients with hemorrhagic fever with renal syndrome. J Infect Dis. 1996;174:407-10. (4.) Peco-Antic A, Popovic-Rolovic M, Gligic A, Popovic D, Jovanovic O, Kostic M. Clinical characteristics of haemorrhagic fever with renal syndrome in children. Pediatr Nephrol. 1992;6:335-8. (5.) Glicic A, Dimkovic N, Xiao SY, Buckle GJ, Jovanovic D, Velimirovic D, et al. Belgrade virus: a new hantavirus causing severe hemorrhagic fever with renal syndrome in Yugoslavia. J Infect Dis. 1992;166:113-20. (6.) Avgic-Zupanc T, Xiao SY, Stojanovic R, Gligic A, van der Groen G, LeDuc JW. Characterization of Dobrava virus: a Hantavirus from Slovenia, Yugoslavia. J Med Virol. 1992;38:132-7. (7.) Lundkvist A, Hukic M, Horling J, Gilljam M, Nichol S, Niklasson B. Puumala and Dobrava viruses cause haemorrhagic fever with renal syndrome (HFRS) in Bosnia-Herzegovina: evidence of highly cross-neutralizing antibody responses in early patient sera. J Med Virol. 1997;53:51-9. (8.) Markotic A, Nichol ST, Kuzman I, Sanchez AJ, Ksiazek TG, Gagro A, et al. Characteristics of Puumala and Dobrava infections in Croatia. J Med Virol. 2002;66:542-51. (9.) Avgic-Zupanc T, Nemirov K, Petrovec M, Trilar T, Poljak M, Vaheri A, et al. Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of two distinct genetic lineages within the same natural focus. J Gen Virol. 2000;81:1747-55. (10.) Song JW, Gligic A, Yanagihara R. Genetic analysis of Tula hantaviral sequences amplified from tissues of Pitymys subterraneus captured in the Cacak region of Serbia-Yugoslavia. Int J Infect Dis. 2002;6:31-6. (11.) Simic M, Miric V. Successful application of peritoneal dialysis peritoneal dialysis n. The removal of soluble substances and water from the body by transfer across the peritoneum, utilizing a solution which is intermittently introduced into and removed from the peritoneal cavity. in a case of renal insufficiency renal insufficiency A defect in renal ability to 'clear' waste products, a sign of inadequate glomerular filtration [in Serbian]. Vojnosanit Pregl. 1952;9:285-90 (12.) Radosevic Z, Mohacek 1. The problem of nephropathia epidemica Myhrman-Zetterholm in relation to acute interstitial nephritis acute interstitial nephritis Acute allergic nephritis Nephrology Renal inflammation characterized by cellular—primarily mononuclear—and fluid exudates, often with epithelial degeneration Types Idiopathic, 2º to drugs or infections . Acta Med Scand. 1954;149:221-8. (13.) Heneberg D, Vuksic L. Epidemic of hemorrhagic fever hemorrhagic fever (hĕm'ərăj`ĭk), any of a group of viral diseases characterized by sudden onset, muscle and joint pain, fever, bleeding, and shock from loss of blood. in certain workplaces in Fruska Gora [in Serbian]. Zbornik Vojnomedicinske Akademije. 1962;4:263-71. (14.) Bowen MD, Gelbmann W, Ksiasek TG, Nichol ST, Nowotny N. Puumala virus and two genetic variants of Tula virus are present in Austrian rodents. J Med Virol. 1997;53:174-81. (15.) Papa A, Johnson AM, Stockton PC, Bowen MD, Spiropoulou CF, Alexiou-Daniel S, et al. Retrospective serological serological pertaining to or emanating from serology. serological test one involving examination of blood serum usually for antibody. and genetic study of the distribution of hantaviruses in Greece. J Med Virol. 1998;55:321-7. Address for correspondence: Anna Papa, A Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki, 54006, Thessaloniki, Greece; email: annap@med.auth.gr All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. Anna Papa, * Bojana Bojovic, ([dagger]) and Antonis Antoniadis * * World Health Organization Collaborating Center for Reference and Research on Arboviruses arboviruses (ar´bōvī´r  sz),n. and Haemorrhagic Fever Viruses at Aristotle University of Thessaloniki, Thessaloniki, Greece; and ([dagger]) Torlak Institute of Immunology and Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression , Belgrade, Serbia and Montenegro
Table 1. ELISA and PCR results from 31 serum samples tested in this
study *
ELISA (indexes)
Collection date
Patient Sex Year of birth (day of illness) IgM HTNV
GD M 1979 Oct 17 2.5
CS M 1972 Oct 17 2.9
PM M 1958 Oct 9 Neg
DZ F 1951 Sep 20 3.2
RD M 1968 Aug 19 3.3
DR M 1958 Aug 9 Cutoff
SS F 1973 Aug 1 2.9
DO M 1956 Jul 26 1.8
RM M UNK Jul 12 2.65
VM M 1962 Jun 14 2.7
MM M 1936 Jun 10 2.7
GM M 1937 May 13 3.2
TV F 1964 Apr 23 (day 11) 1.6
Apr 24 (day 12) 1.7
May 8 (day 26) Neg
IR F 1981 May 17 (day 8) 6.9
May 24 (day 15) 6.6
CJ M 1957 May 24 (day 11) 5.0
May 27 (day 14) 4.2
Oct 23 (day 5) Neg
TD M 1958 Jun 11 (day 12) 4.0
Jun 13 (day 14) 5.0
Jun 28 (day 29) 4.4
GA M 2002 Jun 19 Neg
SM M 1961 Jul 3 (day 7) 3.1
Jul 7 (day 11) 4.6
PV F 1972 Jun 20 (day 5) 4.3
MD M 1981 Sep 18 (day 8) 4.3
Sep 30 (day 20) 4.1
ELISA (indexes) PCR
Patient IgG HTNV IgM PUUV IgG PUUV MS MM-G1 PPT
GD 1.8 Cutoff Cutoff Neg
CS Neg Cutoff Neg
PM 1.8 Neg 1.4
DZ 2.0 Cutoff Neg Neg
RD 2.3 Cutoff Cutoff Neg
DR Neg Cutoff Cutoff
SS 2.1 Neg Neg
DO 2.5 1.5 1.5 Neg Neg
RM 2.13 Cutoff Cutoff
VM Cutoff 1.61 Cutoff Neg Neg
MM 2.4 Neg Neg
GM 2.8 Cutoff Cutoff Neg
TV 3.3 Neg 1.7 Neg
3.5 Neg 2.0 DOBV Neg
4.7 Neg 1.9
IR 2.0 1.6 Cutoff Neg Neg
2.9 1.5 Cutoff
CJ 2.5 1.5 Neg Neg Neg
2.7 1.8 Neg
4.2 Neg Neg
TD 1.7 1.9 2.2 Neg Neg
2.5 1.8 2.2
3.4 2.0 2.2
GA 1.8 Neg Cutoff Neg Neg
SM 1.1 Neg Neg Neg Neg
2.4 Neg Neg
PV 2.0 Neg Neg DOBV Neg
MD 1.2 Neg Neg DOBV DOBV
5.0 Nea Nea Nea
* ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain
reaction; HTNV, Hantaan virus; PUUV, Puumala virus; DOBV,
Dobrava/Belgrade virus; MS, primers for partial N coding regions of
hantaviruses associated with rodents of the Murinae subfamily; MM-G1,
primers for partial G1 coding regions of hantaviruses associated with
rodents of the Murinae subfamily; PPT, primers for partial N coding
regions of hantaviruses associated with rodents of the Arvicofinae
subfamily; Unk, unknown; neg, negative; DOBV, a positive PCR band
yielding Dobrava/Belgrade virus nucleotide sequences.
Table 2. Genetic distances (%) in partial S segment fragment (364-963
nucleotides) of hantaviruses associated with Murinae rodents among
Yugoslavian DOBV strains and representative DOBV strains related with
different Apodemus spp. Rodents *
A. flavicollis related
Yugoslavian DOBV strains
GR-EA GR-PA GR-TD
7937/0 5157/02 9744/02 NW NC NE
Strain 2 (PV) (TV) (MD) Greece Greece Greece
PV -- 0.3 2.6 4.9 4.9 3.0
TV -- 2.2 4.5 4.5 2.8
MD -- 4.5 4.5 2.8
A. flavicollis
related A. agrarius
related A. sylvaticus
AP-Af9 related,
NE DOBV-1 862 East Saarema Krasnodar
Strain Greece Slovenia Slovakia Estonia Russia
PV 3.2 4.0 17.5 15.6 9.5
TV 3.0 3.6 17.0 15.0 9.0
MD 3.4 3.6 16.1 15.3 9.4
* DOBV, Dobrava/Belgrade virus; --, 0.0.
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