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Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Short communication

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First molecular analysis of West Nile virus during the 2013 outbreak in Croatia

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Ivan C. Kurolt a,∗ , Vladimir Krajinovic´ b , Antea Topic´ c , Ilija Kuzman c,d , Bruno Barˇsic´ b,d , Alemka Markotic´ a,e a

Research Department, University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, Zagreb, Croatia Clinical Department for Adult Intensive Care and Neuroinfections, University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, Zagreb, Croatia c Clinical Department for Acute Respiratory Infections, University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, Zagreb, Croatia d Department of Infectious Diseases, University of Zagreb School of Medicine, Zagreb, Croatia e Medical Faculty, University of Rijeka, Rijeka, Croatia b

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Article history: Received 3 February 2014 Received in revised form 23 April 2014 Accepted 27 April 2014 Available online xxx

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Keywords: West Nile virus Croatia West Nile neuroinvasive disease Outbreak Phylogeny

This is the second subsequent year of West Nile neuroinvasive disease (WNND) outbreak in Croatia. Between July and October 2013, 22 patients presented with symptoms of WNND: all with meningitis and 18 additionally with encephalitis. In contrast to 2012, where six autochthonous infections were confirmed in eastern Croatia, the majority of this year’s cases occurred in and around the city of Zagreb, where no West Nile virus infections have been observed before. Viral RNA was recovered from two patients and phylogenetic analyses revealed West Nile virus lineage 2. This represents the first molecular characterization and phylogenetic analysis of the circulating West Nile virus strain in Croatia. © 2014 Published by Elsevier B.V.

West Nile virus (WNV), family Flaviviridae, genus Flavivirus is a mosquito-borne virus belonging to the Japanese encephalitis antigenic group (Hubálek and Halouzka, 1999) and sporadic cases of human WNV infections have been observed in Europe since the 1960s (Zeller and Schuffenecker, 2004). The virus is sustained by its vector, mainly Culex spp. and Aedes spp. mosquitoes, in a rural cycle between wild birds, but can also be transmitted in an urban cycle (Hubálek and Halouzka, 1999), observed in some WNV outbreaks. Humans and horses represent dead-end hosts. The majority of human infections remain asymptomatic, whereas 20–40% may develop an influenza like illness, termed West Nile Fever (WNF), and less than 1% West Nile neuroinvasive disease (WNND) with at least one of three major clinical syndromes: meningitis, encephalitis or flaccid paralysis (Kramer et al., 2007). The first large outbreak of WNND on the European continent happened 1996 in Romania (Tsai et al., 1998) and was caused by a WNV lineage 1a strain (Savage et al., 1999). In recent years sporadic cases or clusters of WNV infections have been reported by several

∗ Corresponding author at: University Hospital for Infectious Diseases “Dr. Fran ´ Research Department, Mirogojska 8, 10000 Zagreb, Croatia. Mihaljevic”, Tel.: +385 1 2826 283; fax: +385 1 2826 148. E-mail address: [email protected] (I.C. Kurolt).

Mediterranean Countries including Croatia, Hungary, Bulgaria and Romania as well as outbreaks in Greece and neighboring Serbia (Sambri et al., 2013; West Nile fever maps, n.d.). Serological evidence for WNV circulation in Croatia was first detected in 1980s with seroprevalence ranging from 0.28% to 4.9% in inhabitants of Dalmatia and offshore islands (Vesenjak-Hirjan, 1980; Vesenjak-Hirjan et al., 1980). Current data for the continental part showed similar seroprevalence (0.3%) and exposure of brown bears and horses (Golubic´ and Dobler, 2012; Madic´ et al., 1993, 2007). During September 2012, seven human cases of WNND in the eastern parts of Croatia have been confirmed (Merdic´ et al., 2013; Pem-Novosel et al., 2013). This was expected, as an outbreak of WNV in Serbia and acute infection in horses preceded the human cases (Popovic´ et al., 2013). Here we present the first molecular and phylogenetic analysis of WNV causing an outbreak in Croatia between July and October 2013. By 10th October, 22 WNND patients were admitted at the University Hospital for Infectious Diseases, Zagreb. All patients were confirmed or probable cases of WNND as defined by Croatian Public Health Authorities, which was in accordance with the Definition of the European Union (Case definitions for reporting communicable diseases, n.d.).

http://dx.doi.org/10.1016/j.virusres.2014.04.017 0168-1702/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Kurolt, I.C., et al., First molecular analysis of West Nile virus during the 2013 outbreak in Croatia. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.017

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Age

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Duration of hospitalization

Department

Neuroinvasive presentation

Neurological sequelae at discharge

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ICU

Muscle weakness

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ICU

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ICU ICU

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Encephalitis, tremor Encephalitis, tremor, ataxia Meningitis Encephalitis, tremor Encephalitis Encephalitis Meningitis Encephalitis Encephalitis Meningitis Encephalitis, tremor Encephalitis Encephalitis Encephalitis, tremor Encephalitis, tremor, ataxia Encephalitis Encephalitis Meningitis Encephalitis, tremor Encephalitis, ataxia Encephalitis Encephalitis, tremor

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Days before hospitalizationa

Days of symptoms lasting before hospitalization. n.a., not available.

Mechanical ventilation

WNV RNA detection in blood/CSF/urineb

Case classification

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Yes

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Probable

City of Zagreb

Muscle weakness

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Confirmed

City of Zagreb

– Muscle weakness, tetraparesis – – – – – – –

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n.a./−/n.a. n.a./n.a./n.a.

Probable Probable

Zagreb Zagreb

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+/−/− n.a./n.a./n.a. n.a./n.a./n.a. n.a./−/n.a. n.a./n.a./n.a. −/n.a./− n.a./n.a./n.a.

Confirmed Probable Probable Probable Probable Probable Probable

Zagreb City of Zagreb Zagreb Zagreb Zagreb Zagreb Brod-Posavina

Muscle weakness – Bilateral n.VII palsy

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n.a./−/n.a. −/−/− n.a./n.a./n.a.

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Probable

Zagreb

Muscle weakness –

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n.a./n.a./n.a. −/−/−

Probable Probable

Zagreb Zagreb

Glasgow Coma Score

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Case no.

I.C. Kurolt et al. / Virus Research xxx (2014) xxx–xxx

Please cite this article in press as: Kurolt, I.C., et al., First molecular analysis of West Nile virus during the 2013 outbreak in Croatia. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.017

Table 1 ´ Zagreb (n = 22). Clinical and laboratory data of West Nile neuroinvasive disease cases hospitalized at the University Hospital for Infectious Diseases “Dr. Fran Mihaljevic”,

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KF179640 Austria 2008 HQ537483 Greece 2010 70

HG796244 Croatia 2013

Hu - Gr Cluster

DQ116961 Hungary 2004 KF647251 Italy 2013 91

KF179639 Greece 2012 KC407673 Serbia 2012

Lin 2

93 DQ318019 Senegal 1990

NC_001563 Uganda 1937 HM147824 Democratic Republic of the Congo 1958

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FJ425721 Russia 2007 EF429199 South Africa 2000 DQ318020 Central African Republic 1972 AY688948 Sarafend DQ176636 Madagascar 1978 AF260969 Romania 1996 93

AF317203 Russia 1999a AY277252 Russia 1999b

AF404757 Italy 1998 AY268132 France 2000 JF719069 Spain 2010 AF196835 USA 1999b

Lin 1a

DQ118127 Hungary 2003 84 AF202541 USA 1999a

AF481864 Israel 1998 AY268133 Tunisia 1997 91

AF260968 Egypt 1951 70 AY490240 China 2001

Lin 1b Lin 5

D00246 Kunjin virus DQ256376 India 1980 AY277251 Russia 1998 AY765264 Rabensburg isolate

Lin 4

Lin 3

NC 001437 Japanese encephalitis virus 0.05 Fig. 1. Phylogenetic analysis of the WNV sequence detected in Croatian patients with WNND. Phylogenetic tree from WNV strains and isolates based on a 167 bp fragment of the NS5 gene, corresponding to the nucleotides 9116–9282 of the Greek Nea Santa 2010 strain. This Maximum-Likelihood tree, employing the p-distance substitution model including transitions and transversions, was constructed by bootstrap analysis of 1000 replicates using MEGA5 software and rooted by the Japanese encephalitis reference strain 16681 (accession number: NC001437). Scale bar indicates nucleotide substitutions per site. The sequence obtained from the two autochthonous cases is marked with a triangle. Bold lines and brackets mark the earlier proposed Hungarian-Greek Cluster, whereas the square brackets indicate WNV lineages one through five.

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The majority of patients (95.5%) were exposed to WNV in two counties: City of Zagreb and Zagreb County (Table 1). One patient acquired WNND probably visiting Brod-Posavina County where last year WNND cases occurred (Merdic´ et al., 2013). Even though the cases spread over four months, more than two-third of all patients reported onset of symptoms within weeks 34, 35 and 36. The median age of WNND patients was 62.5 years (range 22–80). Of 22 WNND patients, 19 (86.4%) were male. None of the patients had a history of recent blood transfusion or tissue/organ transplantation. Of all WNND cases, 18 (81.8%) presented with encephalitis and four (18.2%) with meningitis alone. None of the patients had acute flaccid paralysis. Ten (45.5%) patients had tremor and/or ataxia. The mean duration of symptoms before hospitalization was 4.9 days. Underlying chronic medical conditions in WNND patients were as

follows: hypertension (n = 10), diabetes mellitus (n = 9), coronary artery disease (n = 4) and long term immunosuppression (n = 2) after kidney and heart transplant. Ten WNND patients (45.5%) were admitted to the Intensive care unit where three patients were mechanically ventilated due to central respiratory failure. As of 10th October 2013, all patients survived WNND. Muscle weakness was the most common neurologic sequel, presented in six (27.3%) patients (Table 1). Cerebrospinal fluid (CSF) findings were typical for viral infection: pleocytosis (mean CSF white-cell count, 228/mm3 , range, 18–1193) and elevated protein concentration (mean 797.7 mg/dl; range 28–1390). There was a lymphocytic predominance in the CSF except in six (27.3%) patients. Glucose levels were normal in all patients.

Please cite this article in press as: Kurolt, I.C., et al., First molecular analysis of West Nile virus during the 2013 outbreak in Croatia. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.017

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Serum samples were routinely tested with commercially available ELISA tests at The Croatian National Institute for Public Health, Zagreb (CNIPH). All samples showed positive IgM and in nine available paired sera, specific IgG was detected. Cross-reactivity for TBEV was seen in five cases. All molecular diagnostics on usual causes of meningoencephalitis in Croatia (enterovirus, herpes viruses and TBEV) was negative. None of the patients had been vaccinated for yellow fever. Amplification of WNV RNA was unsuccessful in all available (54.5%) CSF samples by commercially procurable real-time RT-PCR at CNIPH. Blood and urine from 12 patients, acute and/or convalescent samples, have been tested with two independent, previously described, RT-PCRs: a WNV Lineage 1 specific RT-PCR targeting the junction of the structural proteins C-prM (Lanciotti et al., 2000) or a nested assay with target sequence within the non-structural NS5 gene (Zaayman et al., 2009) able to amplify all WNV Lineages associated with human disease (Lin 1a, 1b, 2, 5). When acute phase samples were unavailable, urine samples taken several weeks after the acute infections were tested instead, as occurrence of WNV RNA in convalescent urine has been reported (Murray et al., 2010; Tonry et al., 2005). The nested RT-PCR assay was able to amplify a WNV specific sequence from total RNA isolated from the blood of two patients. The sequence of the two obtained amplicons are 100% identical, belong to the WNV lineage 2 and comprise the nucleotides 9116–9282 when compared with the Greek Nea Santa 2010 strain and were submitted to the European Nucleotide Archive (accession number: HG796244, HG796245). These two sequences also Exhibit 100% identity with recent WNV lineage 2 isolates from Austria, Greece or Italy (Fig. 1). Here we reported the 2013 WNV outbreak (July–October) within Zagreb region, manifested as WNND. We showed that WNV lineage 2 was responsible for the outbreak. Phylogenetic analysis confirmed association into the Greek/Hungarian Cluster, which is expected due to the geographical proximity to e.g. neighboring Hungary or Serbia, where WNV lineage 2 strains have been detected for several years. No evidence of WNF cases were documented, probably due to mild presentation. Considering that only 1% of infected persons develop WNND it seems probable that several hundred undocumented infections occurred this summer in the wider Zagreb region. Most of WNND cases clustered from August to the beginning of September. Although only in two patients WNV infection were confirmed, according to the European Commission case definitions for reporting communicable diseases, apart from serology proof clinical characteristics (course of disease and neurological sequele) and epidemiological findings (no data of tick bites but mosquito exposure in patients, no circulation of other meningoencephalitis causing viruses) can rule out another pathogen as a causative agent of this encephalitis outbreak. There is a need for continuous epidemiological studies for WNV in Croatia in humans, horses, birds and mosquitoes to provide a better prediction of potential outbreaks and establishment of appropriate preventive measures. For an accurate assessment of the number of patients, particularly WNF patients, it is necessary to further educate physicians and establish routine diagnosis of WNV, at least during WNV season. Physicians should think of WNV infection in patients with meningoencephalitis of unknown etiology, especially during the late summer and early autumn. Systematic studies of WNV in Croatia would enable an adequate assessment for the need of WNV testing among blood and organ donors.

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Acknowledgements ˇ We thank Tatjana Vilibic´ Cavlek, MD, PhD from the Croatian National Institute of Public Health for performing WNV serology and molecular testing of CSF and Oktavija Ðakovic´ Rode, MD, PhD from the University Hospital for Infectious Diseases, Zagreb for performing serology on tick-borne encephalitis virus as part of the routine differential diagnosis approach in hospitalized patients with CNS infections potentially caused by flaviviruses. References Case definitions for reporting communicable diseases (in Croatian), n.d. Available at: http://hzjz.hr/wp-content/uploads/2013/11/definicije zb 12.pdf (accessed 01.08.14). ´ D., Dobler, G., 2012. Flaviviruses in the north-west Croatia. Infektoloˇski Glas. Golubic, 32, 153–157 (in Croatian). Hubálek, Z., Halouzka, J., 1999. West Nile fever – a reemerging mosquito-borne viral disease in Europe. Emerg. Infect. Dis. 5, 643–650, http://dx.doi.org/10.3201/ eid0505.990505. Kramer, L.D., Li, J., Shi, P.-Y., 2007. West Nile virus. Lancet Neurol. 6, 171–181, http://dx.doi.org/10.1016/S1474-4422(07)70030-3. Lanciotti, R.S., Kerst, A.J., Nasci, R.S., Godsey, M.S., Mitchell, C.J., Savage, H.M., Komar, N., Panella, N.A., Allen, B.C., Volpe, K.E., Davis, B.S., Roehrig, J.T., 2000. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J. Clin. Microbiol. 38, 4066–4071. ´ B., 1993. Serologic survey for selected viral and rick´ J., Huber, D., Lugovic, Madic, ettsial agents of brown bears (Ursus arctos) in Croatia. J. Wildl. Dis. 29, 572–576, http://dx.doi.org/10.7589/0090-3558-29.4.572. ´ J., Savini, G., Di Gennaro, A., Monaco, F., Jukic, ´ B., Kovac, S., Rudan, N., Listes, Madic, E., 2007. Serological evidence for West Nile virus infection in horses in Croatia. Vet. 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Please cite this article in press as: Kurolt, I.C., et al., First molecular analysis of West Nile virus during the 2013 outbreak in Croatia. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.017

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First molecular analysis of West Nile virus during the 2013 outbreak in Croatia.

This is the second subsequent year of West Nile neuroinvasive disease (WNND) outbreak in Croatia. Between July and October 2013, 22 patients presented...
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