J. Neurovirol. (2014) 20:437–441 DOI 10.1007/s13365-014-0276-0
MINI REVIEW
Review of West Nile virus epidemiology in Italy and report of a case of West Nile virus encephalitis Serena Delbue & Pasquale Ferrante & Sara Mariotto & Gianluigi Zanusso & Antonino Pavone & Mauro Chinaglia & Roberto L’Erario & Salvatore Monaco & Sergio Ferrari
Received: 4 July 2014 / Accepted: 25 July 2014 / Published online: 20 August 2014 # Journal of NeuroVirology, Inc. 2014
Abstract West Nile virus (WNV) is a flavivirus that causes neurological disorders in less than 1 % of infected subjects. Human cases of WNV-associated fever and/or neurological disorders have been reported in Italy since 2008. The first outbreak occurred in the northeastern region of Italy surrounding the Po River and was caused by the Po River lineage 1 strain, and since then, WNV infections have been reported in several regions of central Italy. Although the virus is highly genetically conserved, stochastic mutations in its genome may lead to the emergence of new strains, as was observed in Italy in 2011 with the identification of two new lineage 1 strains, the WNV Piave and WNV Livenza strains. To help further define WNV epidemiology in Italy, we describe a case of an Italian man living in the Po River area who developed fatal encephalitis in 2009 due to infection with the WNV Piave strain. This finding supports the notion that the Piave strain has been circulating in this area of Italy for 2 years longer than was previously believed.
S. Delbue : P. Ferrante (*) Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Pascal 36, 20133 Milan, Italy e-mail: [email protected] P. Ferrante Foundation Ettore Sansavini, Health Science Foundation, Lugo, Italy S. Mariotto : G. Zanusso : S. Monaco : S. Ferrari Department of Neurological and Movement Sciences, University of Verona, P.le L.A. Scuro 10, 37134 Verona, Italy A. Pavone Neurology Unit, Garibaldi Hospital, Via Palermo 636, 95122 Catania, Italy M. Chinaglia : R. L’Erario Department of Neurosciences, Neurology Unit, Hospital of Rovigo, Viale Tre Martiri 89, 45100 Rovigo, Italy
Keywords West Nile virus . Lineage . Viral encephalitis
Introduction West Nile virus (WNV) is an arthropod-borne virus that is transmitted to humans by mosquitos primarily of the genus Culex in a cycle involving birds as amplifying hosts. The WNV virion is an icosahedral particle that is surrounded by an envelope and contains a single-stranded positive-sense RNA genome ranging in size from 10,842 to 11,057 nucleotides that encodes seven non-structural and three structural proteins (Deubel et al. 2001). Based on nucleotide sequence data, WNV strains are phylogenetically classified into five genetic lineages, but only lineages 1 and 2, which have a nucleotide sequence identity of approximately 75 %, have been associated with major epidemics. Lineages 1 and 2 are primarily distributed throughout North Africa, Central/ Southern Europe and North America and Sub-Saharan Africa, and Central/Eastern Europe and Greece, respectively. This virus, which belongs to the Flaviviridae family within the Flavivirus complex, was first isolated from the blood of a febrile patient in 1937 in the West Nile district of Uganda (Smithburn et al. 1940). Since, WNV has spread worldwide and has been responsible for both sporadic cases and outbreaks of symptomatic disease. Initially, WNV-associated fever was considered to be a minor asymptomatic disease but became a public health concern at the end of the 1990s when several outbreaks of infection occurred, causing severe and sometimes fatal neurological disorders (ND) (Pesko and Ebel 2012; Petersen et al. 2013). Pathogenesis of WNV infection WNV is maintained in a bird-mosquito-bird transmission cycle and occasionally infects humans. Although WNV has
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been detected in many different mosquito species, only a few of the genus Culex (i.e., Culex pipiens, Culex quinquefasciatus, and Culex tarsalis) can spread the virus to humans. The predominant and preferred reservoirs of the virus are birds, which develop viremia that is high enough to infect the mosquitos that feed on them. Bites from infected mosquitos account for nearly all infections in non-avian vertebrate hosts, including humans and equines, which are considered to be “dead-end” hosts because they develop low-level viremia that is insufficient for transmission to mosquitos. The incubation period for WNV infection is usually 3– 15 days after a bite from an infected mosquito. However, symptoms of infection, such as fever, headache, generalized weakness, myalgia, anorexia, nausea, and morbilliform or maculopapular rash, occur in approximately 25 % of infected people. The disease may last a few days, generally with complete recovery (Petersen et al. 2013). Approximately 1 % of subjects who are infected via mosquito bites develop neuroinvasive diseases with clinical manifestations consisting of meningitis, encephalitis, and acute flaccid paralysis. In these cases, the illness may last weeks to months and may cause death, especially among elderly individuals (over 70 years old) or immunocompromised patients. The fatality rate among patients with neuroinvasive diseases is approximately 10 %. Treatment of WNV infection is supportive, and thus far, no human vaccine is available. The most efficient preventive measure is the use of mosquito repellents and communitybased mosquito control programs.
Epidemiology of WNV in Italy The presence of WNV in Europe was first revealed in 1958 when specific antibodies were detected in two Albanians (Bárdo et al. 1959). The first cases of WNV fever in Italy were described approximately 10 years later through personal communications (Hubalek and Halouzka 1999); however, the first laboratory-confirmed human case of neuroinvasive infection was reported in 2008 and occurred in a rural area near the Po River (northeastern Italy), which is an area that is heavily infested with both Culex and Aedes albopictus vectors and where an outbreak of WNV among horses was concurrently reported (Rossini et al. 2008). By the end of 2008, WNV infection had caused fever, aseptic encephalitis, and meningitis in seven patients living in the Emilia-Romagna and Veneto regions. Five cases of asymptomatic WNV infection were also reported in the same year (Barzon et al. 2009). In the summer of 2009, 18 cases of neuroinvasive disease due to WNV were again diagnosed in Emilia-Romagna and Veneto in addition to the Lombardia region. During these two outbreaks, genomic sequences of WNV were obtained from magpie isolates that
J. Neurovirol. (2014) 20:437–441
were found to belong to the lineage 1 clade 1a within the European Mediterranean/North African cluster (Schuffenecker et al. 2005; Magurano et al. 2012). Notably, the isolates, which are termed the Po River strains, showed high levels of similarity, and the virus was able to maintain its pathogenic characteristics over the winter season. These observations allowed for the formulation of the hypothesis that between 2008 and 2009, WNV established an endemic cycle in Italy through local birds and vectors (Monaco et al. 2011). During the following 2 years, several human cases of neuroinvasive WNV disease and fever were reported in the areas surrounding the Po River in addition the Friuli-Venezia Giulia region (northeastern Italy) and Sardinia Island (five cases of neuroinvasive WNV disease in 2011 and two cases in 2012) (Magurano et al. 2012). In 2011, two new WNV lineage 1 strains, the Piave and the Livenza strains, spread throughout the Veneto region, whereas the Po River strain simultaneously disappeared (Barzon et al. 2012a, 2013a). Interestingly, the first human cases of autochthonous WNV lineage 2 infection that were related to Hungarian-Greek strains were also reported in Italy in September 2011 in the Marche and Sardinia regions (Central Italy). The largest human outbreak of WNV infection in Italy was reported in 2012 with a total of 42 human cases of neuroinvasive disease and fever, mainly occurring in northeastern Italy (Veneto and Friuli Venezia Giulia regions), which were most likely caused by the hot climate that facilitated the rapid spread of the vectors. These infections were mainly sustained by lineage 1 Livenza (Barzon et al. 2013b). Finally, as of November 2013, the Italian Health Institute (ISS) reported 40 cases of WNV infection due to both lineages 1 and 2, occurring mainly in the Veneto and Emilia-Romagna regions, which were the areas that were affected by the first outbreaks in 2008–2009 (http://www.epicentro.iss.it/ problemi/westnile/bollettino/WN_News_2013_13.pdf). In the period between 2008 and 2011, a case fatality rate of 16 % was reported compared with that of 8 % in Israel and 9 % in the USA; additionally, an association between age and severe disease was observed (Rizzo et al. 2012).
Diagnostic and surveillance issues The diagnosis of WNV infection is primarily based on clinical evidence and laboratory data that are obtained by commercially available serological and/or molecular tests. The detection of a specific IgM immune response in either the cerebrospinal fluid (CSF) or serum remains to be the most widely used and reliable approach for indicating the early stages of an infection. This test, which is known as the IgM antibody capture enzyme immunoassay (MAC-EIA), allows for the identification of the presence of IgM against the virus for up to 47 days post-infection in serum and up to 199 days post-
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infection in CSF (Kapoor et al. 2004). The calculated sensitivity of the test is 91.7 %, and the specificity is 99.2 % (Long et al. 2006). Subsequently, the diagnosis of infection should be confirmed by indirect IgG EIA or by an immunofluorescence assay that is performed on samples that are collected at different stages of the disease and during convalescence. IgG seroconversion should be indicated by at least a fourfold increase in serum antibodies. Because of broad antigenic crossreactivity among flaviviruses and to improve the specificity of these tests, diagnoses should be confirmed using the plaque reduction neutralization test (PRNT). However, the PRNT test requires viable virus isolates and a high biosafety level laboratory, which are not always available for routine use. Additionally, WNV genomes may also be detected in the blood, CSF, or urine of the patients with suspected WNV infections using reverse transcription PCR (RT-PCR), RTnested PCR, or real-time RT-PCR. The sensitivity of the test depends on the specific techniques that are employed and on the target sequence and varies from the equivalent of 0.1 plaque-forming units (PFU) to 0.01 PFU. In cases of WNV neuroinvasive disease, the viral load in clinical specimens should be higher than in asymptomatic infected subjects from day 2 through day 18 post-infection (Sambri et al. 2013). In light of the wide circulation of WNV in several regions of Italy, a national plan for human surveillance has been defined for affected areas where laboratory-confirmed infections in horses and/or humans had been reported in previous years or during the surveillance period (between June 15 and November 15, which was the period with the highest vector activity). Moreover, if human cases of WNV-associated neuroinvasive disease are detected during the same year, immediate nucleic acid amplification test screening of all blood and hematopoietic stem cell donations is implemented. This screening is performed using commercial nucleic acid amplification tests (NATs) that reach high levels of sensitivity (40.3 genome copies/mL) and that are fully automated on high-throughput instruments and allow for the testing of hundreds of samples per day (Rossini et al. 2008). Thus far, two different commercially licensed tests are available, including one that is based on real-time RT-PCR and a second that is based on transcription-mediated amplification technology (TMA) (Pai et al. 2008; Ziermann and SanchezGuerrero 2008). The main limitation of these tests is the genetic variability of the Italian WNV strains, particularly after the recent appearance of lineage 2. At the national level, all blood, tissue, and solid organ donors who have travelled to an affected area are temporarily deferred for 28 days http://www.centronazionalesangue.it/sites/default/files/prot._ n._1172.cns_.2011_ulteriori_indicazioni_wnv.pdf. Additionally, several Italian regions, such as EmiliaRomagna, that are mainly affected by WNV circulation have developed their own laboratory surveillance schemes and protocols (www.saluter.it).
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Case report To obtain further insight into WNV epidemiology in Italy, we describe here a case of fatal and neuroinvasive encephalitis that was caused by the WNV lineage 1a/Piave strain, occurring in 2009 in the Veneto region of Italy. Although this is a single case, it importantly demonstrates the introduction of this particular strain into Italy 2 years earlier than was previously believed. In August 2009, an 82-year-old man living in the Veneto region of northeastern Italy was admitted to the hospital because of altered mental status, disequilibrium, and frequent falls. The patient had hypertension and stable ischemic heart disease and a negative history of travelling outside of his area of residency. A computed tomography (CT) scan of the brain showed a slight bilateral hypodensities in the basal ganglia and white matter that were interpreted as ischemic lesions. An electroencephalogram (EEG) showed diffuse bilateral slowing of cortical background activity. Upon neurological examination, the patient was somnolent with right-sided hemiparesis. Three weeks after admission, the patient developed septic fever. A lumbar puncture yielded clear, colorless CSF showing 2 cells/ mL and protein levels of 74 mg/dL. Antimicrobial drug therapy consisting of quinolones and a combination of piperacillin and tazobactam was administered. A repeat CT scan of the brain showed the presence of a new hypodense lesion in the right caudate nucleus. On the twenty-fifth day, the patient became comatose. Magnetic resonance imaging (MRI) of the brain revealed bilateral hyperintense signal abnormalities in the temporo-parietal regions and corona radiata; basal ganglia, cerebellum, and pons in the T2-weighted; and fluid attenuation recovery (FLAIR) images in addition to hyperintense signals in the white matter surrounding the occipital horns of the lateral ventricles in the diffusion-weighted images (DWI) (Fig. 1). Encephalitis was suspected, but in-house CSF PCR failed to show the presence of the HSV1, HSV2, VZV, enterovirus, or WNV genomes. Serum but not CSF IgM and IgG antibodies against WNV were detected using a commercial enzymelinked immunosorbent assay (ELISA) kit, and a positive plaque reduction neutralization test on the serum substantiated the serological diagnosis. Unfortunately, the results were obtained after death and 1 month after admission to the hospital. A general autopsy showed increased adrenal gland size with the presence of a diffuse large B cell primary bilateral adrenal lymphoma. A neuropathological examination revealed focal lesions of different sizes at the level of the internal capsule, putamen, insula, centrum semiovale, thalamus, and medulla oblongata. A microscopic examination demonstrated that the leptomeninges were infiltrated with lympho-monocytes. A variable degree of lymphocyte and monocyte infiltration was
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Fig. 1 Brain MRI of the patient obtained 25 days after admission shows bilateral hyperintense signals in basal ganglia in T2-weighted sequences (a); in DWI-weighted axial (b), and coronal FLAIR images (c, d); hyperintense signal abnormalities are present in the white matter surrounding the occipital horns. Neuropathological findings in regions indicated by dashed squares in c (parietal cortex, e hematoxylin and eosin stain, 25× original magnification) and d (cerebellum, f, 25×) showing cellular inflammation and necrotic foci. Perivascular mononuclear infiltrates (g, 50×), gliosis (h, 100×), and neuronophagia (i, 100×) were also observed in basal ganglia (50×)
present in all brain areas. Inflammation and liquefactive necrosis prevailed in the cortical gray matter, thalamus, basal ganglia, substantia nigra, cerebral peduncles, inferior olivary
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nuclei, and cerebellar cortex. Glial nodules, perivascular cuffing by mononuclear cells, neuron loss, and neuronophagia were also observed in the involved brain areas (Fig. 1). Total RNA was isolated from tissues that were collected from both the basal ganglia and the cerebellar cortex of the brain using a previously described protocol (Ha et al. 2004). RT-semi-nested PCR against a portion of the NS5 gene sequence of WNV was performed (Scaramozzino et al. 2001). Because nested PCR is prone to contamination, strict precautionary measures were taken to avoid cross-contamination from prior PCRs. The nucleic acids were extracted, amplification mixtures were prepared and nested PCRs were run in separate rooms, and multiple negative controls (which contained water instead of the DNA template) were included with each assay batch. All RNA samples that were extracted from brain tissues revealed the presence of WNV. A sequencing analysis of the amplified region identified a 96 % rate of homology with the previously described strain Italy/2011/Piave (Barzon et al. 2012b) (Fig. 2). In the present case, the combination of fever and mental status changes in addition to MRI signal abnormalities in the basal ganglia, thalamus, and pons in an elderly man suggested viral encephalitis after ruling out ischemic brain disease and other inflammatory and infectious disorders of the CNS. The possibility of WNV neuroinvasive disease was considered due to the season, age of the patient, and the occurrence of two outbreaks of WNV infection affecting humans, horses, and birds in the Po River area in 2008 and 2009 (Barzon et al. 2013b). WNVencephalitis was diagnosed based on positive ELISA and PRNT assays. In contrast, RT-PCR on CSF did not detect the viral genome, which was later amplified from the brain tissues that were obtained during autopsy. The subsequent sequencing analysis of the amplified fragment allowed for the strain to be identified as Italy/2011/Piave. Interestingly, the infection that was caused by this strain was generally not recognized in this area of Italy until 2 years later, but the case described here demonstrates that it was already circulating and causing deaths along the Po River in northeastern Italy in 2009. We hypothesize that the epidemiology of WNV in Italy is more complex than what has been reported thus far
Fig. 2 Schematic representation of the WNV NS5 gene fragment nucleotide sequence. The WNV infecting the brain was identified as strain Italy/2011/ Piave (JQ928175.1) with deletions of three bases at nt 9,227; 9,233; and 9,234 and with a point mutation at nt 9,244
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and that perhaps additional strains could be identified (Barzon et al. 2013a). Because WNV is generally present at low levels in the body fluids of patients with ND, it is difficult to perform biomolecular analyses on the infectious viral strain and to study the phylogeny of WNV. Therefore, the case we report here is of particular interest because it was possible to isolate and sequence the viral strain and obtain rare genetic information by studying the brain tissue of the patient. Funding statement This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sector. Conflict of interest The authors declare that they have no conflict of interest
References Bárdo V, Adamcová J, Dedei S, Gjini N, Rosický B, Imková A (1959) Neutralizing antibodies against some neurotropic viruses determined in human sera in Albania. Journal of Hygiene, Epidemiology. Microbiol Immunol (Prague) 3:277–282 Barzon L, Squarzon L, Cattai M, Franchin E, Pagni S, Cusinato R, Palu G (2009) West Nile virus infection in Veneto region, Italy, 2008–2009. Euro Surveill 6:14(31) Barzon L, Pacenti M, Franchin E, Martello T, Lavezzo E, Squarzon L, Toppo S, Fiorin F, Marchiori G, Scotton GP, Russo F, Cattai M, Cusinato R, Palù G (2012a) Clinical and virological findings in the ongoing outbreak of West Nile virus Livenza strain in northern Italy, July to September 2012. Euro Surveill 17:20260 Barzon L, Pacenti M, Franchin E, Squarzon L, Lavezzo E, Toppo S, Martello T, Cattai M, Cusinato R, Palù G (2012b) Novel West Nile virus lineage 1a full genome sequences from human cases of infection in north-eastern Italy, 2011. Clin Microbiol Infect 18:E541– E544 Barzon L, Pacenti M, Franchin E, Squarzon L, Lavezzo E, Cattai M, Cusinato R, Palù G (2013a) The complex epidemiological scenario of West Nile virus in Italy. Int J Environ Res Public Health 10: 4669–4689 Barzon L, Pacenti M, Franchin E, Pagni S, Lavezzo E, Squarzon L, Martello T, Russo F, Nicoletti L, Rezza G, Castilletti C, Capobianchi MR, Salcuni P, Cattai M, Cusinato R, Palù G (2013b) Large human outbreak of West Nile virus infection in north-eastern Italy in 2012. Viruses 5:2825–2839 Deubel V, Fiette L, Gounon P, Drouet MT, Khun H, Huerre M, Banet C, Malkinson M, Despres P (2001) Variations in biological features of WN viruses. Ann N Y Acad Sci 951:195–206 Ha SK, Choi C, Chae C (2004) Development of an optimized protocol for the detection of classical swine fever virus in formalini-fixed, paraffin-embedded tissues by seminested reverse transcriptionpolymerase chain reaction and comparison with in situ hybridization. Res Vet Sci 77:163–169 Hubalek Z, Halouzka J (1999) West Nile fever-a re-emerging mosquitoborne viral disease in Europe. Emerg Infect Dis 5:643–650
441 Kapoor H, Signs K, Somsel P, Downes FP, Clark PA, Massey JP (2004) Persistence of West Nile Virus (WNV) IgM antibodies in cerebrospinal fluid from patients with CNS disease. J Clin Virol 31:3289–3291 Long MT, Jeter W, Hernandez J, Sellon DC, Gosche D, Gillis K, Bille E, Gibbs EP (2006) Diagnostic performance of the equine IgM capture ELISA for serodiagnosis of West Nile virus infection. J Vet Intern Med 20:608–613 Magurano F, Remoli ME, Baggieri M, Fortuna C, Marchi A, Fiorentini C, Bucci P, Benedetti E, Ciufolini MG, Rizzo C, Piga S, Salcuni P, Rezza G, Nicoletti L (2012) Circulation of West Nile virus lineage 1 and 2 during an outbreak in Italy. Clin Microbiol Infect 18: E545–E547 Monaco F, Savini G, Calistri P, Polci A, Pinoni C, Bruno R, Lelli R (2011) 2009 West Nile disease epidemic in Italy: First evidence of overwintering in Western Europe? Res Vet Sci 91:321–326 Pai A, Kleinman S, Malhotra K, Lee-Haynes L, Pietrelli L, Saldanha J (2008) Performance characteristics of the Food and Drug Administration-Licensed Roche Cobas TaqScreen West Nile virus assay. Transfusion 48:2184–2189 Pesko KN, Ebel GD (2012) West Nile virus population genetics and evolution. Infect Genet Evol 12:181–190 Petersen LR, Brault AC, Nasci RS (2013) West Nile virus: Review of the literature. JAMA 310:308–315 Rizzo C, Salcuni P, Nicoletti L, Ciufolini MG, Russo F, Masala R, Frongia O, Finarelli AC, Gramegna M, Gallo L, Pompa MG, Rezza G, Salmaso S, Declich S (2012). Epidemiological surveillance of West Nile neuroinvasive diseases in Italy, 2008 to 2011. Euro Surveill 17(20) Rossini G, Cavrini F, Pierro A, Macini P, Finarelli A, Po C, Peroni G, Di Caro A, Capobianchi M, Nicoletti L, Landini M, Sambri V (2008) First human case of West Nile virus neuroinvasive infection in Italy, September 2008—Case report. EuroSurveill 9:13(41) Sambri V, Capobianchi MR, Cavrini F, Charrel R, Donoso-Mantke O, Escadafal C, Franco L, Gaibani P, Gould EA, Niedrig M, Papa A, Pierro A, Rossini G, Sanchini A, Tenorio A, Varani S, Vázquez A, Vocale C, Zeller H (2013) Diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies. Viruses 25: 2329–2348 Scaramozzino N, Crance JM, Jouan A, DeBriel DA, Stoll F, Garin D (2001) Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcriptionPCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. J Clin Microbiol 39:1922–1927 Schuffenecker I, Peyrefitte CN, el Harrak M, Murri S, Leblond A, Zeller HG (2005) West Nile virus in Morocco, 2003. Emerg Infect Dis 11: 306–309 Smithburn KC, Hughes TP, Burke AW, Paul JH (1940) A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med 20:471–492 www.centronazionalesangue.it/sites/default/files/prot._n._1172.cns_. 2011_ulteriori_indicazioni_wnv.pdf. Accessed on May 2014 www.epicentro.iss.it/problemi/westnile/bollettino/WN_News_2013_13. pdf. Accessed on May 2014 www.saluter.it. Accessed on May 2014 Ziermann R, Sanchez-Guerrero SA (2008) PROCLEIX West Nile virus assay based on transcription-mediated amplification. Expert Rev Mol Diagn 8:239–245
MINI REVIEW
Review of West Nile virus epidemiology in Italy and report of a case of West Nile virus encephalitis Serena Delbue & Pasquale Ferrante & Sara Mariotto & Gianluigi Zanusso & Antonino Pavone & Mauro Chinaglia & Roberto L’Erario & Salvatore Monaco & Sergio Ferrari
Received: 4 July 2014 / Accepted: 25 July 2014 / Published online: 20 August 2014 # Journal of NeuroVirology, Inc. 2014
Abstract West Nile virus (WNV) is a flavivirus that causes neurological disorders in less than 1 % of infected subjects. Human cases of WNV-associated fever and/or neurological disorders have been reported in Italy since 2008. The first outbreak occurred in the northeastern region of Italy surrounding the Po River and was caused by the Po River lineage 1 strain, and since then, WNV infections have been reported in several regions of central Italy. Although the virus is highly genetically conserved, stochastic mutations in its genome may lead to the emergence of new strains, as was observed in Italy in 2011 with the identification of two new lineage 1 strains, the WNV Piave and WNV Livenza strains. To help further define WNV epidemiology in Italy, we describe a case of an Italian man living in the Po River area who developed fatal encephalitis in 2009 due to infection with the WNV Piave strain. This finding supports the notion that the Piave strain has been circulating in this area of Italy for 2 years longer than was previously believed.
S. Delbue : P. Ferrante (*) Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Pascal 36, 20133 Milan, Italy e-mail: [email protected] P. Ferrante Foundation Ettore Sansavini, Health Science Foundation, Lugo, Italy S. Mariotto : G. Zanusso : S. Monaco : S. Ferrari Department of Neurological and Movement Sciences, University of Verona, P.le L.A. Scuro 10, 37134 Verona, Italy A. Pavone Neurology Unit, Garibaldi Hospital, Via Palermo 636, 95122 Catania, Italy M. Chinaglia : R. L’Erario Department of Neurosciences, Neurology Unit, Hospital of Rovigo, Viale Tre Martiri 89, 45100 Rovigo, Italy
Keywords West Nile virus . Lineage . Viral encephalitis
Introduction West Nile virus (WNV) is an arthropod-borne virus that is transmitted to humans by mosquitos primarily of the genus Culex in a cycle involving birds as amplifying hosts. The WNV virion is an icosahedral particle that is surrounded by an envelope and contains a single-stranded positive-sense RNA genome ranging in size from 10,842 to 11,057 nucleotides that encodes seven non-structural and three structural proteins (Deubel et al. 2001). Based on nucleotide sequence data, WNV strains are phylogenetically classified into five genetic lineages, but only lineages 1 and 2, which have a nucleotide sequence identity of approximately 75 %, have been associated with major epidemics. Lineages 1 and 2 are primarily distributed throughout North Africa, Central/ Southern Europe and North America and Sub-Saharan Africa, and Central/Eastern Europe and Greece, respectively. This virus, which belongs to the Flaviviridae family within the Flavivirus complex, was first isolated from the blood of a febrile patient in 1937 in the West Nile district of Uganda (Smithburn et al. 1940). Since, WNV has spread worldwide and has been responsible for both sporadic cases and outbreaks of symptomatic disease. Initially, WNV-associated fever was considered to be a minor asymptomatic disease but became a public health concern at the end of the 1990s when several outbreaks of infection occurred, causing severe and sometimes fatal neurological disorders (ND) (Pesko and Ebel 2012; Petersen et al. 2013). Pathogenesis of WNV infection WNV is maintained in a bird-mosquito-bird transmission cycle and occasionally infects humans. Although WNV has
438
been detected in many different mosquito species, only a few of the genus Culex (i.e., Culex pipiens, Culex quinquefasciatus, and Culex tarsalis) can spread the virus to humans. The predominant and preferred reservoirs of the virus are birds, which develop viremia that is high enough to infect the mosquitos that feed on them. Bites from infected mosquitos account for nearly all infections in non-avian vertebrate hosts, including humans and equines, which are considered to be “dead-end” hosts because they develop low-level viremia that is insufficient for transmission to mosquitos. The incubation period for WNV infection is usually 3– 15 days after a bite from an infected mosquito. However, symptoms of infection, such as fever, headache, generalized weakness, myalgia, anorexia, nausea, and morbilliform or maculopapular rash, occur in approximately 25 % of infected people. The disease may last a few days, generally with complete recovery (Petersen et al. 2013). Approximately 1 % of subjects who are infected via mosquito bites develop neuroinvasive diseases with clinical manifestations consisting of meningitis, encephalitis, and acute flaccid paralysis. In these cases, the illness may last weeks to months and may cause death, especially among elderly individuals (over 70 years old) or immunocompromised patients. The fatality rate among patients with neuroinvasive diseases is approximately 10 %. Treatment of WNV infection is supportive, and thus far, no human vaccine is available. The most efficient preventive measure is the use of mosquito repellents and communitybased mosquito control programs.
Epidemiology of WNV in Italy The presence of WNV in Europe was first revealed in 1958 when specific antibodies were detected in two Albanians (Bárdo et al. 1959). The first cases of WNV fever in Italy were described approximately 10 years later through personal communications (Hubalek and Halouzka 1999); however, the first laboratory-confirmed human case of neuroinvasive infection was reported in 2008 and occurred in a rural area near the Po River (northeastern Italy), which is an area that is heavily infested with both Culex and Aedes albopictus vectors and where an outbreak of WNV among horses was concurrently reported (Rossini et al. 2008). By the end of 2008, WNV infection had caused fever, aseptic encephalitis, and meningitis in seven patients living in the Emilia-Romagna and Veneto regions. Five cases of asymptomatic WNV infection were also reported in the same year (Barzon et al. 2009). In the summer of 2009, 18 cases of neuroinvasive disease due to WNV were again diagnosed in Emilia-Romagna and Veneto in addition to the Lombardia region. During these two outbreaks, genomic sequences of WNV were obtained from magpie isolates that
J. Neurovirol. (2014) 20:437–441
were found to belong to the lineage 1 clade 1a within the European Mediterranean/North African cluster (Schuffenecker et al. 2005; Magurano et al. 2012). Notably, the isolates, which are termed the Po River strains, showed high levels of similarity, and the virus was able to maintain its pathogenic characteristics over the winter season. These observations allowed for the formulation of the hypothesis that between 2008 and 2009, WNV established an endemic cycle in Italy through local birds and vectors (Monaco et al. 2011). During the following 2 years, several human cases of neuroinvasive WNV disease and fever were reported in the areas surrounding the Po River in addition the Friuli-Venezia Giulia region (northeastern Italy) and Sardinia Island (five cases of neuroinvasive WNV disease in 2011 and two cases in 2012) (Magurano et al. 2012). In 2011, two new WNV lineage 1 strains, the Piave and the Livenza strains, spread throughout the Veneto region, whereas the Po River strain simultaneously disappeared (Barzon et al. 2012a, 2013a). Interestingly, the first human cases of autochthonous WNV lineage 2 infection that were related to Hungarian-Greek strains were also reported in Italy in September 2011 in the Marche and Sardinia regions (Central Italy). The largest human outbreak of WNV infection in Italy was reported in 2012 with a total of 42 human cases of neuroinvasive disease and fever, mainly occurring in northeastern Italy (Veneto and Friuli Venezia Giulia regions), which were most likely caused by the hot climate that facilitated the rapid spread of the vectors. These infections were mainly sustained by lineage 1 Livenza (Barzon et al. 2013b). Finally, as of November 2013, the Italian Health Institute (ISS) reported 40 cases of WNV infection due to both lineages 1 and 2, occurring mainly in the Veneto and Emilia-Romagna regions, which were the areas that were affected by the first outbreaks in 2008–2009 (http://www.epicentro.iss.it/ problemi/westnile/bollettino/WN_News_2013_13.pdf). In the period between 2008 and 2011, a case fatality rate of 16 % was reported compared with that of 8 % in Israel and 9 % in the USA; additionally, an association between age and severe disease was observed (Rizzo et al. 2012).
Diagnostic and surveillance issues The diagnosis of WNV infection is primarily based on clinical evidence and laboratory data that are obtained by commercially available serological and/or molecular tests. The detection of a specific IgM immune response in either the cerebrospinal fluid (CSF) or serum remains to be the most widely used and reliable approach for indicating the early stages of an infection. This test, which is known as the IgM antibody capture enzyme immunoassay (MAC-EIA), allows for the identification of the presence of IgM against the virus for up to 47 days post-infection in serum and up to 199 days post-
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infection in CSF (Kapoor et al. 2004). The calculated sensitivity of the test is 91.7 %, and the specificity is 99.2 % (Long et al. 2006). Subsequently, the diagnosis of infection should be confirmed by indirect IgG EIA or by an immunofluorescence assay that is performed on samples that are collected at different stages of the disease and during convalescence. IgG seroconversion should be indicated by at least a fourfold increase in serum antibodies. Because of broad antigenic crossreactivity among flaviviruses and to improve the specificity of these tests, diagnoses should be confirmed using the plaque reduction neutralization test (PRNT). However, the PRNT test requires viable virus isolates and a high biosafety level laboratory, which are not always available for routine use. Additionally, WNV genomes may also be detected in the blood, CSF, or urine of the patients with suspected WNV infections using reverse transcription PCR (RT-PCR), RTnested PCR, or real-time RT-PCR. The sensitivity of the test depends on the specific techniques that are employed and on the target sequence and varies from the equivalent of 0.1 plaque-forming units (PFU) to 0.01 PFU. In cases of WNV neuroinvasive disease, the viral load in clinical specimens should be higher than in asymptomatic infected subjects from day 2 through day 18 post-infection (Sambri et al. 2013). In light of the wide circulation of WNV in several regions of Italy, a national plan for human surveillance has been defined for affected areas where laboratory-confirmed infections in horses and/or humans had been reported in previous years or during the surveillance period (between June 15 and November 15, which was the period with the highest vector activity). Moreover, if human cases of WNV-associated neuroinvasive disease are detected during the same year, immediate nucleic acid amplification test screening of all blood and hematopoietic stem cell donations is implemented. This screening is performed using commercial nucleic acid amplification tests (NATs) that reach high levels of sensitivity (40.3 genome copies/mL) and that are fully automated on high-throughput instruments and allow for the testing of hundreds of samples per day (Rossini et al. 2008). Thus far, two different commercially licensed tests are available, including one that is based on real-time RT-PCR and a second that is based on transcription-mediated amplification technology (TMA) (Pai et al. 2008; Ziermann and SanchezGuerrero 2008). The main limitation of these tests is the genetic variability of the Italian WNV strains, particularly after the recent appearance of lineage 2. At the national level, all blood, tissue, and solid organ donors who have travelled to an affected area are temporarily deferred for 28 days http://www.centronazionalesangue.it/sites/default/files/prot._ n._1172.cns_.2011_ulteriori_indicazioni_wnv.pdf. Additionally, several Italian regions, such as EmiliaRomagna, that are mainly affected by WNV circulation have developed their own laboratory surveillance schemes and protocols (www.saluter.it).
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Case report To obtain further insight into WNV epidemiology in Italy, we describe here a case of fatal and neuroinvasive encephalitis that was caused by the WNV lineage 1a/Piave strain, occurring in 2009 in the Veneto region of Italy. Although this is a single case, it importantly demonstrates the introduction of this particular strain into Italy 2 years earlier than was previously believed. In August 2009, an 82-year-old man living in the Veneto region of northeastern Italy was admitted to the hospital because of altered mental status, disequilibrium, and frequent falls. The patient had hypertension and stable ischemic heart disease and a negative history of travelling outside of his area of residency. A computed tomography (CT) scan of the brain showed a slight bilateral hypodensities in the basal ganglia and white matter that were interpreted as ischemic lesions. An electroencephalogram (EEG) showed diffuse bilateral slowing of cortical background activity. Upon neurological examination, the patient was somnolent with right-sided hemiparesis. Three weeks after admission, the patient developed septic fever. A lumbar puncture yielded clear, colorless CSF showing 2 cells/ mL and protein levels of 74 mg/dL. Antimicrobial drug therapy consisting of quinolones and a combination of piperacillin and tazobactam was administered. A repeat CT scan of the brain showed the presence of a new hypodense lesion in the right caudate nucleus. On the twenty-fifth day, the patient became comatose. Magnetic resonance imaging (MRI) of the brain revealed bilateral hyperintense signal abnormalities in the temporo-parietal regions and corona radiata; basal ganglia, cerebellum, and pons in the T2-weighted; and fluid attenuation recovery (FLAIR) images in addition to hyperintense signals in the white matter surrounding the occipital horns of the lateral ventricles in the diffusion-weighted images (DWI) (Fig. 1). Encephalitis was suspected, but in-house CSF PCR failed to show the presence of the HSV1, HSV2, VZV, enterovirus, or WNV genomes. Serum but not CSF IgM and IgG antibodies against WNV were detected using a commercial enzymelinked immunosorbent assay (ELISA) kit, and a positive plaque reduction neutralization test on the serum substantiated the serological diagnosis. Unfortunately, the results were obtained after death and 1 month after admission to the hospital. A general autopsy showed increased adrenal gland size with the presence of a diffuse large B cell primary bilateral adrenal lymphoma. A neuropathological examination revealed focal lesions of different sizes at the level of the internal capsule, putamen, insula, centrum semiovale, thalamus, and medulla oblongata. A microscopic examination demonstrated that the leptomeninges were infiltrated with lympho-monocytes. A variable degree of lymphocyte and monocyte infiltration was
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Fig. 1 Brain MRI of the patient obtained 25 days after admission shows bilateral hyperintense signals in basal ganglia in T2-weighted sequences (a); in DWI-weighted axial (b), and coronal FLAIR images (c, d); hyperintense signal abnormalities are present in the white matter surrounding the occipital horns. Neuropathological findings in regions indicated by dashed squares in c (parietal cortex, e hematoxylin and eosin stain, 25× original magnification) and d (cerebellum, f, 25×) showing cellular inflammation and necrotic foci. Perivascular mononuclear infiltrates (g, 50×), gliosis (h, 100×), and neuronophagia (i, 100×) were also observed in basal ganglia (50×)
present in all brain areas. Inflammation and liquefactive necrosis prevailed in the cortical gray matter, thalamus, basal ganglia, substantia nigra, cerebral peduncles, inferior olivary
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nuclei, and cerebellar cortex. Glial nodules, perivascular cuffing by mononuclear cells, neuron loss, and neuronophagia were also observed in the involved brain areas (Fig. 1). Total RNA was isolated from tissues that were collected from both the basal ganglia and the cerebellar cortex of the brain using a previously described protocol (Ha et al. 2004). RT-semi-nested PCR against a portion of the NS5 gene sequence of WNV was performed (Scaramozzino et al. 2001). Because nested PCR is prone to contamination, strict precautionary measures were taken to avoid cross-contamination from prior PCRs. The nucleic acids were extracted, amplification mixtures were prepared and nested PCRs were run in separate rooms, and multiple negative controls (which contained water instead of the DNA template) were included with each assay batch. All RNA samples that were extracted from brain tissues revealed the presence of WNV. A sequencing analysis of the amplified region identified a 96 % rate of homology with the previously described strain Italy/2011/Piave (Barzon et al. 2012b) (Fig. 2). In the present case, the combination of fever and mental status changes in addition to MRI signal abnormalities in the basal ganglia, thalamus, and pons in an elderly man suggested viral encephalitis after ruling out ischemic brain disease and other inflammatory and infectious disorders of the CNS. The possibility of WNV neuroinvasive disease was considered due to the season, age of the patient, and the occurrence of two outbreaks of WNV infection affecting humans, horses, and birds in the Po River area in 2008 and 2009 (Barzon et al. 2013b). WNVencephalitis was diagnosed based on positive ELISA and PRNT assays. In contrast, RT-PCR on CSF did not detect the viral genome, which was later amplified from the brain tissues that were obtained during autopsy. The subsequent sequencing analysis of the amplified fragment allowed for the strain to be identified as Italy/2011/Piave. Interestingly, the infection that was caused by this strain was generally not recognized in this area of Italy until 2 years later, but the case described here demonstrates that it was already circulating and causing deaths along the Po River in northeastern Italy in 2009. We hypothesize that the epidemiology of WNV in Italy is more complex than what has been reported thus far
Fig. 2 Schematic representation of the WNV NS5 gene fragment nucleotide sequence. The WNV infecting the brain was identified as strain Italy/2011/ Piave (JQ928175.1) with deletions of three bases at nt 9,227; 9,233; and 9,234 and with a point mutation at nt 9,244
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and that perhaps additional strains could be identified (Barzon et al. 2013a). Because WNV is generally present at low levels in the body fluids of patients with ND, it is difficult to perform biomolecular analyses on the infectious viral strain and to study the phylogeny of WNV. Therefore, the case we report here is of particular interest because it was possible to isolate and sequence the viral strain and obtain rare genetic information by studying the brain tissue of the patient. Funding statement This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sector. Conflict of interest The authors declare that they have no conflict of interest
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