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Evidence of West Nile virus lineage 2 circulation in Northern Italy
Veterinary Microbiology 158 (2012) 267–273
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Evidence of West Nile virus lineage 2 circulation in Northern Italy G. Savini a,*, G. Capelli b, F. Monaco a, A. Polci a, F. Russo c, A. Di Gennaro a, V. Marini a, L. Teodori a, F. Montarsi b, C. Pinoni a, M. Pisciella a, C. Terregino b, S. Marangon b, I. Capua b, R. Lelli a a
Istituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy c Regione Veneto, Venice, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 December 2011 Received in revised form 2 February 2012 Accepted 9 February 2012
A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Culex pipiens Collared dove Streptopelia decaocto NS3 NS5 Phylogenetic analysis West Nile virus lineage 2
1. Introduction West Nile virus (WNV) is a RNA virus included in the Japanese Encephalitis serogroup within the Flaviviridae family. In its natural cycle birds normally act as amplifying hosts whereas mosquitoes, mainly of the genera Culex, Aedes and Ochlerotatus, play the vector role. In this cycle humans, horses, and other mammals are regarded as incidental or dead-end hosts. Based on phylogenetic analyses eight distinct lineages have been recently proposed for the strains of WNV (Mackenzie and Williams,
* Corresponding author at: Deptartment of Virology, National Reference Center for West Nile Disease, OIE Reference Laboratory for Bluetongue, Istituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332440; fax: +39 0861 332251. E-mail address: [email protected] (G. Savini). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2012.02.018
2009; Vazquez et al., 2010). Of these, isolates grouped in lineages 1 and 2 are nowadays by far the most widespread. Lineage 1 strains have been reported in North America, North Africa, Europe, and Australia, whereas strains of lineage 2 have only recently shown to be capable of spreading outside of their historical geographic range which has been for long time confined between Southern Africa and Madagascar (Lanciotti et al., 1999; Burt et al., 2002). In 2004, a lineage 2 strain has been firstly recorded in Hungary (Bakonyi et al., 2006) where it became endemic (Bakonyi et al., 2006; Erdelyi et al., 2007) before extending in Austria in 2008 and 2009 (Wodak et al., 2011). It is now two years that a strain of lineage 2 has been circulating in Greece (Papa et al., 2011a,b; Valiakos et al., 2011; Chaskopoulou et al., 2011) and, just few months ago, a WNV RNA belonging to lineage 2 was detected in a human patient in the Center of Italy (Bagnarelli et al., 2011).
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Strains of both lineages have been shown to be able to cause severe disease in birds, horses and humans (Lanciotti et al., 1999; Bakonyi et al., 2006; Venter and Swanepoel, 2010). Neuroinvasiveness and virulence of a WND strain have been recently related to the genotype and/or the immunological status of the infected population (Burt et al., 2002; Venter and Swanepoel, 2010; Papa et al., 2011a). The nervous form of WNV when occurring is characterized by meningoencephalitis, encephalitis, ataxia, weakness, recumbency, and muscle fasciculation. This article reports the first detection of a lineage 2 strain in local birds and indigenous mosquitoes in Northern Italy. 2. Materials and methods The activities described in this report were part of the extensive National surveillance plan for monitoring Flavivirus which is in place since 2002. The program involved horses, insects as well as wild and domestic birds as described by Calistri et al. (2010). In 2009, the entomological and serological surveillance was locally expanded by the Veneto region. 2.1. Entomological survey Sixty-one CDC-CO2 traps were placed in rural and periurban sites of all the 11 provinces of the Veneto (49 traps) and Friuli Venezia Giulia (12 traps) regions. Traps operated from May through October and were activated every 15 days for one night, from sunset to the next morning. Collected mosquitoes were immediately refrigerated, taken to the laboratory, counted, identified using standard taxonomic keys (Romi et al., 1997; Severini et al., 2009) and pooled in 50 specimens maximum, according to species, site and date and then stored at 80 8C. 2.2. Bird surveillance Within the framework of WN virus surveillance activities in wild birds, an episode of high mortality in doves was investigated. At the end of September 2011, inhabitants of rural areas near Treviso, a municipality in the Veneto region, noticed an unusual mortality of wild collared doves (Streptopelia decaocto) and birds of other species (i.e., blackbirds). Unfortunately, no other data were available to the local veterinary authorities on these episodes of mortality. Only one dead bird was collected by a private citizen and carried to the Institute of Padua for diagnostic investigations. A complete necropsy was performed and samples of brain, liver, kidney, spleen, lung and intestine were taken, homogenized and suspended in phosphate-buffered saline with antibiotics for viral detection. 2.3. Virus isolation From the homogenized dove tissues and the RNA positive mosquito samples, virus isolation attempts were carried out on different cell lines as described by Savini et al. (2011). In brief, homogenized tissue and mosquito
samples suspended in phosphate-buffered saline with antibiotics were centrifuged (1000 g, 10 min). After centrifugation, the supernatants were inoculated onto confluent chicken embryo fibroblast, chicken embryo liver and Vero cell cultures. Cell cultures were incubated at 37 8C for 7 days and were checked daily for the occurrence of cytopathic effects (CPE). Three blind passages were made in absence of cytopathic effect. At the end of the third passage and in case of CPE, the presence of WNV in the supernatants was confirmed or excluded either by electron microscopy or by Immunofluorescence using a monoclonal antibody (produced by ICT, Italy) against WNV. Samples of brain, liver, kidney, spleen, lung and intestine of the dove were also processed in 9–10 day old SPF embryonated chicken eggs (OIE, 2008). 2.4. Molecular analysis 2.4.1. Real time RT-PCR and nested RT-PCR Viral RNA was extracted from a pool of kidney, heart and brain and was tested by real time RT-PCR (RRT-PCR) for Avian Influenza (AI) (Spackman et al., 2002), Newcastle Disease (ND) (Wise et al., 2004), Usutu and West Nile (WN) viruses (Tang et al., 2006). Lung and intestine were also tested for ND and AI by RRT-PCR. Mosquito samples were also screened for the presence of WNV by using one-step SYBR Green-Based RRT-PCR (Ravagnan et al., 2011). This method which uses specific primers (MAMD and cFD2; Scaramozzino et al., 2001) designed on the conserved region of the non-structural NS5 gene of the WNV, is capable of detecting several Flaviviruses. Positive results were then confirmed either by using the commercial Kit TaqvetTM West Nile WNV (LSI, Lissieu, France) which is able to detect WNV RNA of the lineage 1 and 2 strains and the RT-PCR as described by Bakonyi et al. (2006) targeting NS5 gene. To ascertain which lineage the WNV RNA found in the samples belonged to, lineage specific RT-PCRs were used. For determining the presence of lineage 1 strains, the Lanciotti et al. (2000) method modified as described by Monaco et al. (2009) was used, whereas for discriminating lineage 2 strains, the method as recently described by Chaskopoulou et al. (2011), which targets the NS3 gene of the WNV genome, was used. Briefly, total RNA was reverse transcribed by the kit ‘‘TaqMan1 Reverse Transcription Reagents’’ (Applied Biosystem, USA) using random hexamers. The reverse transcription was carried out in 20 ml of mix containing 0.5 ml of Multiscribe RNA enzyme (50 U/ ml), 2 ml of 10 RT buffer, 4.4 ml of the 25 mM MgCl2 buffer, 4 ml of dNTPs 2.5 mM, 1 ml of random hexamer (50 mM) primer, 0.4 ml of Rnase inhibitor (20 U/ml) and 5 ml of RNA. The reaction was incubated 10 min at 25 8C then at 48 8C for 30 min followed by final incubation at 95 8C for 5 min to inactivate the residual activity of the reverse transcriptase. Viral RNA was amplified by a nested RT-PCR. In brief, the first amplification generated a 778 bp amplicon by using the external primer pair WN-NS3up1 (50 -GCTGGCTTCGAACCTGAAATGTTG-30 ) and WN-NS3do1 (50 -CAATGATGGTGGGTTTCACGCT-30 ). The second amplification product used the internal primer pair WNNS3up2 (50 -GCAAGATACTTCCCCAAATCATCAAGG-30 ) and
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WN-NS3do2 (50 -TGTCTGGGATCTCTGTTTGCATGTC-30 ) to amplify a 423 bp region of the NS3 coding region of the WNV genome. Both reactions were carried out in 50 ml of final volume containing 5 ml of buffer 10, 3 ml of MgCl2 (25 mM), 1 ml of dNTPs (10 mM), 0.3 ml of TaqGold and 1 ml of each primer (50 mM). The reaction was incubated at 95 8C for 10 min followed by 30 cycles of denaturation for 30 s at 94 8C, annealing at 60 8C for 30 s, extension at 72 8C for 1 min and final extension at 72 8C for 7 min. Amplified products were visualized on 1% agarose gel stained with Syber Safe (Invitrogen, USA) and the bands were observed under UV light.
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mosquito species (81%) and was present in all the 61 sites monitored. Species composition and relative abundance were similar in the two regions, confirming the area as environmental homogenous and highly suitable for C. pipiens survival. Only female mosquito samples identified at the species level (n = 78,791) were screened by the one step RRT-PCR for the presence of Flavivirus. Males (n = 766; 0.0001%) or mosquitoes not identified at the species level (n = 5400; 6.3%) were not tested. In total, 2732 pools were tested and 5 of them (0.18%) were found positive for WNV RNA from late July until mid September (Table 2). At necropsy, the dove appeared undernourished but no significant gross lesions were observed. Avian Influenza, Newcastle Disease and Usutu viruses were not detected by either molecular methods or virus isolation on target organs. The pool of kidney, heart and brain resulted positive for WN virus by both RRT-PCR and RT-PCR, conversely the attempt to isolate the virus was unsuccessful. All positive reactions were confirmed by the lineage 1 and 2 commercial RT-PCR. When the specific RT-PCRs were used, RNA of lineage 1 WNV was found in 3 C. pipiens pools while RNA of lineage 2 strain was found either in 2 pools of C. pipiens collected in the province of Udine and in the bird tissues. The NS3 gene of the lineage 2 strains was then partially sequenced and the sequence aligned. The nucleotide sequences confirmed the presence of lineage 2 strains. Results of the alignment of the partial NS3 gene sequence are shown in Fig. 2. No differences were observed between the Italian strains. The sequences also showed 100% similarity with those of the Hungarian strain but differed by 2 nucleotides in positions 80 and 142 from the 2010 Greek isolate. When the nucleotide sequences were translated to putative amino acid sequences, they differed by one amino acid only at position 249 of the NS3 protein. At this site in the Italian and Hungarian strains histidine
2.4.2. Amplification and sequencing PCR products were purified with the Qiaquick PCR Purification kit (Qiagen, Germany) and used for direct sequencing in both directions using the internal primers previously described. The sequencing reaction was set up using the Big Dye Terminator kit (Applied Biosystems, USA), the excess of dyes was removed using Cleanseq (Beckman Coulter, USA) and the nucleotide sequences were determined using the DNA sequencer ABI PRISM 3100 (Applied Biosystems, USA). Raw sequence data were assembled using Contig Express (Vector NTI suite 9.1, Invitrogen, USA) and translated into amino acid sequences using Vector NTI suite 9.1 (Invitrogen, USA). Consensus sequences from the virus detected in Italian mosquitoes and bird tissues were aligned with the WNV lineage 2 isolated in 2004 in Hungary (DQ116961), in 2011 in Greece (HQ537483) and to the reference strain isolated in 1937 in Uganda (M12294) with ClustalW (Thompson et al., 1994) (Table 1). 3. Results Overall, 85,398 mosquitoes were collected belonging to 14 species. Culex pipiens was the most abundant
Table 1 Strains of West Nile viruses included in the NS3 partial sequencing. Name
Strain
Location
Year
lineage
Source
GenBank
CpITA11/1 CpITA11/2 SdeITA/11 HUM/ITA11 HUN04 GRE10 UGA37 ITA09
Italy 2011/21612 Italy 2011/21617 Italy 2011/23743 Italy 2011 Goshawk-Hungary/04 Nea Santa Greece 2010 B956 Italy/2009/J-225677
Italy Italy Italy Italy Hungary Greece Uganda Italy
2011 2011 2011 2011 2004 2010 1937 2009
2 2 2 2 2 2 2 1
Culex pipiens Culex pipiens Streptopelia decaocto Homo sapiens Accipiter gentilis Culex pipiens Homo sapiens Garrulus glandarius
– – – JN858070 DQ116961 HQ537483 AY532665 JF719068
Table 2 Details of the West Nile virus infected Culex pipiens collected during the 2011 entomological survey. Site code
Province
Region
WN area code
Collection date
WNV lineage
MIR (%)
198 217 210 178 211
Venezia Pordenone Udine Treviso Udine
VEN FVG FVG FVG FVG
AS AS ACV Outside ACV
July 27th July 27th August 19th September 14th September 14th
1 1 2 1 2
1.695 1.852 0.813 1.852 1.333
VEN, Veneto region; FVG, Friuli Venezia Giulia region ACV, area with WN viral circulation; AS, WN surveillance area; outside, area outside the AS area. MIR, minimum infection rate (positive pools/mosquitoes tested 100) for the site and date of collection.
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Fig. 1. Areas where lineage 2 strains of West Nile virus have been detected in Northern Italy.
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replaces proline. The blast of the NS5 nucleotide sequence also showed a similarity of 99% with the West Nile Hungarian 2004 isolate. 4. Discussion This report provided further evidence that a lineage 2 strain is circulating in Italy. It was found in two pools of C. pipiens and in the tissues of a resident collared dove found dead in North East of Italy. This is the first time that a lineage 2 strain has been detected in mosquitoes and birds in Italy. A human case caused by a lineage 2 strain infection was recently described in Ancona (Bagnarelli et al., 2011). It was a mild case characterized by a period of high fever with no response to antibiotics. The strain which was detected in the urine sample, when sequenced, showed 99% identity to the complete genome of isolate goshawkHungary/04 and to the more recent Nea Santa-Greece2010 (Bakonyi et al., 2006; Papa et al., 2011b; Bagnarelli et al., 2011). According to the NS3 partial sequences, the strains detected in mosquitoes and bird tissues in this study, were identical and also showed 100% similarity with the same fragment sequences of the strain detected in Italy and of the strains which circulated in Hungary in 2004. Therefore, it appears that the same strain of lineage 2 WNV was likely responsible for the dove and mosquito infections even if the areas where the bird and the mosquitoes were collected were not contiguous (Fig. 1). In contrast to the previous Italian lineage 2 reported case, this lineage 2 strain detection occurred in an higher risk area compared to, where lineage 1 WNV has circulated and/or is circulating. It is currently difficult to track down the exact route of entrance of the virus. The phylogenetic findings and the sample collection dates imply a southward expansion of the virus from the Central European countries. The areas where the infected mosquitoes were collected are adjacent to Slovenia and/or are part of the flying paths of the long and short migration routes. Migratory birds from Africa have often been implicated in the emergence of the WNV outbreaks in Europe (Hubalek and Halouzka, 1999; Rappole et al., 2000; Murgue et al., 2001; Malkinson et al., 1998; Rappole and Hubalek, 2003). In this case, considering the high genetic similarity with the Hungarian isolate, it is more likely that birds migrating either along the south-eastern migration route from Europe and western Asia to Africa, or along short migration routes from Central to Southern Europe, have introduced the virus. A similar infection pathway was also proposed for the spread of Usutu virus in Italy (Savini et al., 2011). Since in a month period the same strain virus was detected in local birds and indigenous mosquitoes in two different areas situated 50 km apart, it looks as if the Italian lineage 2 strain established itself with the tendency to spread westward. Many prerequisites are required to maintain a WNV strain in an environment. Vector competency and presence of susceptible vertebrate host capable of transmitting the infection to vectors, are indeed key parameters for the maintenance and spread of WNV in a given environment. The areas where the lineage 2 strains have been detected, have already proven to have these prerequisites as lineage 1 WNV and also Usutu virus, which
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share the same life cycle, have circulated and/or are circulating. Although further studies carried out in nonepidemic periods are needed to test this hypothesis, it is then more than likely that this lineage 2 strain will establish and further spread in the neighboring areas. As a consequence, in the next season it is highly probable that both lineages will contemporaneously circulate in the same areas. Whether this will result in an enhancement or reduction of their virulence is hard to say. Similarly, it is difficult to predict the occurrence of possible genetic recombination phenomena (Pickett and Lefkowitz, 2009). Because cross protection between both lineages has been observed in horses (Minke et al., 2011), the circulation of both lineages could eventually result in a rapid endemization of the area with no severe clinical consequences. According to the phylogenetic analysis, the Italian as the Hungarian lineage 2 strains differed from that isolated in Greece as they have histidine rather than proline at location 249 of the NS3 protein. This site has been involved
Fig. 2. Phylogenetic analysis of the Italian sequences. Sequence dataset was analyzed using BioEdit version 7.0.9 and nucleotide alignment was performed with Clustal-W (Thompson et al., 1994). Phylogenetic analysis was conducted by using distance (NJ) method in PHYLIP (the PHYlogeny Inference Package) program version 3.67. The reliability of the phylogenetic trees was confirmed by performing bootstrap analysis with 1000 replicate values. The tree obtained was rooted with ITA09 strain. The asterisk indicates significant bootstrapping values (>60%). ITA09: West Nile virus (WNV) strain isolated in Italy in 2009; UGA37: B956 lineage 2 prototype strain; GRE10: Greek WNV strain isolated in 2010; HUN04: Hungarian isolate detected in 2004; HumITA11: Italian WNV strain detected in a human patient in 2011; SdeITA11: Italian WNV strain detected in a wild collared dove in 2011; Cp1ITA11: WNV strain detected in a pool of C. pipiens caught in Bagnara Arsa (UD); Cp2ITA11: WNV strain detected in a pool of C. pipiens caught in Palazzolo della Stella (UD).
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in genetic modulation of WNV pathogenicity, using American crows (Corvus brachyrhynchos) as avian model of WNV disease (Brault et al., 2007). The influence of histidine in position 249 of the NS3 on the virulence of a strain is still unknown. In line with those which circulated in Hungary and Austria which were able to cause encephalitis and death in a sparrow hawks (Accipiter nisus), goshawks (Accipiter gentilis) (Bakonyi et al., 2006; Erdelyi et al., 2007) and gyrfalcon (Falco rusticolus), the Italian lineage 2 strain found in the tissues of a collared dove was likely the cause of the bird death. In addition to the dove, the lineage 2 strain was also detected in pools of C. pipiens. Considering that C. pipiens has been demonstrated to serve both as an enzootic and a bridge vector for humans (Hamer et al., 2008), it is highly probable that lineage 2 WNV will infect humans again. However is not possible to predict what will be the impact of this infection even if the clinical expression of the lineage 2 human case reported in Italy was relatively mild. 5. Conclusions This study demonstrated that a WNV strain belonging to lineage 2 is circulating in North-East of Italy. As it was found either in local collared dove and in indigenous mosquito (C. pipiens), it seems to have developed strategies of adaptation and become established. Its NS3 partial sequence is identical to the other Italian isolate detected in a human patient as well as to Hungarian isolate with which it seems to share similar features including the pathogenicity for birds. Comprehensive investigations on the occurrence, ecology, and epidemiology of the different WNV strains circulating in Italy should be of highest priority. Because of its zoonotic potential, veterinarians, physicians, and public health officials need to work more closely together to control, prevent, and better estimate the epidemiological impact and possible threat to animal and public health of WNV infections. Greater communication and collaboration between these different professional figures at both, central and local level would, ultimately, improve prevention and control strategies with great benefit for human and veterinary public health. References Bagnarelli, P., Marinelli, K., Trotta, D., Monachetti, A., Tavio, M., Del Gobbo, R., Capobianchi, M.R., Menzo, S., Nicoletti, L., Magurano, F., Varaldo, P.E., 2011. Human case of autochthonous West Nile virus lineage 2 infection in Italy, September 2011. Euro Surveill. 16 (43) pii=20002. Available from: http://www.eurosurveillance.org/ViewArticle.aspx? ArticleId=20002. Bakonyi, T., Ivanics, E., Erdely, K., Ursu, K., Ferenczi, E., Weissenbock, H., Nowotny, N., 2006. Lineage 1 and 2 strains of encephalitic West Nile virus Central Europe. Emerg. Infect. Dis. 12, 618–623. Brault, A.C., Huang, C.Y., Langevin, S.A., Kinney, R.M., Bowen, R.A., Ramey, W.N., Panella, N.A., Holmes, E.C., Powers, A.M., Miller, B.R., 2007. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat. Genet. 39, 1162–1166. Burt, F.J., Grobbelaar, A.A., Leman, P.A., Anthony, F.S., Gibson, G.V., Swanepoel, R., 2002. Phylogenetic relationships of southern African West Nile virus isolates. Emerg. Infect. Dis. 8, 820–826. Calistri, P., Giovannini, A., Savini, G., Monaco, F., Bonfanti, L., Ceolin, C., Terregino, C., Tamba, M., Cordioli, P., Lelli, R., 2010. West Nile virus transmission in 2008 in North-Eastern Italy. Zoonoses Public Health 57, 211–219.
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Evidence of West Nile virus lineage 2 circulation in Northern Italy G. Savini a,*, G. Capelli b, F. Monaco a, A. Polci a, F. Russo c, A. Di Gennaro a, V. Marini a, L. Teodori a, F. Montarsi b, C. Pinoni a, M. Pisciella a, C. Terregino b, S. Marangon b, I. Capua b, R. Lelli a a
Istituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy c Regione Veneto, Venice, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 December 2011 Received in revised form 2 February 2012 Accepted 9 February 2012
A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Culex pipiens Collared dove Streptopelia decaocto NS3 NS5 Phylogenetic analysis West Nile virus lineage 2
1. Introduction West Nile virus (WNV) is a RNA virus included in the Japanese Encephalitis serogroup within the Flaviviridae family. In its natural cycle birds normally act as amplifying hosts whereas mosquitoes, mainly of the genera Culex, Aedes and Ochlerotatus, play the vector role. In this cycle humans, horses, and other mammals are regarded as incidental or dead-end hosts. Based on phylogenetic analyses eight distinct lineages have been recently proposed for the strains of WNV (Mackenzie and Williams,
* Corresponding author at: Deptartment of Virology, National Reference Center for West Nile Disease, OIE Reference Laboratory for Bluetongue, Istituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332440; fax: +39 0861 332251. E-mail address: [email protected] (G. Savini). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2012.02.018
2009; Vazquez et al., 2010). Of these, isolates grouped in lineages 1 and 2 are nowadays by far the most widespread. Lineage 1 strains have been reported in North America, North Africa, Europe, and Australia, whereas strains of lineage 2 have only recently shown to be capable of spreading outside of their historical geographic range which has been for long time confined between Southern Africa and Madagascar (Lanciotti et al., 1999; Burt et al., 2002). In 2004, a lineage 2 strain has been firstly recorded in Hungary (Bakonyi et al., 2006) where it became endemic (Bakonyi et al., 2006; Erdelyi et al., 2007) before extending in Austria in 2008 and 2009 (Wodak et al., 2011). It is now two years that a strain of lineage 2 has been circulating in Greece (Papa et al., 2011a,b; Valiakos et al., 2011; Chaskopoulou et al., 2011) and, just few months ago, a WNV RNA belonging to lineage 2 was detected in a human patient in the Center of Italy (Bagnarelli et al., 2011).
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Strains of both lineages have been shown to be able to cause severe disease in birds, horses and humans (Lanciotti et al., 1999; Bakonyi et al., 2006; Venter and Swanepoel, 2010). Neuroinvasiveness and virulence of a WND strain have been recently related to the genotype and/or the immunological status of the infected population (Burt et al., 2002; Venter and Swanepoel, 2010; Papa et al., 2011a). The nervous form of WNV when occurring is characterized by meningoencephalitis, encephalitis, ataxia, weakness, recumbency, and muscle fasciculation. This article reports the first detection of a lineage 2 strain in local birds and indigenous mosquitoes in Northern Italy. 2. Materials and methods The activities described in this report were part of the extensive National surveillance plan for monitoring Flavivirus which is in place since 2002. The program involved horses, insects as well as wild and domestic birds as described by Calistri et al. (2010). In 2009, the entomological and serological surveillance was locally expanded by the Veneto region. 2.1. Entomological survey Sixty-one CDC-CO2 traps were placed in rural and periurban sites of all the 11 provinces of the Veneto (49 traps) and Friuli Venezia Giulia (12 traps) regions. Traps operated from May through October and were activated every 15 days for one night, from sunset to the next morning. Collected mosquitoes were immediately refrigerated, taken to the laboratory, counted, identified using standard taxonomic keys (Romi et al., 1997; Severini et al., 2009) and pooled in 50 specimens maximum, according to species, site and date and then stored at 80 8C. 2.2. Bird surveillance Within the framework of WN virus surveillance activities in wild birds, an episode of high mortality in doves was investigated. At the end of September 2011, inhabitants of rural areas near Treviso, a municipality in the Veneto region, noticed an unusual mortality of wild collared doves (Streptopelia decaocto) and birds of other species (i.e., blackbirds). Unfortunately, no other data were available to the local veterinary authorities on these episodes of mortality. Only one dead bird was collected by a private citizen and carried to the Institute of Padua for diagnostic investigations. A complete necropsy was performed and samples of brain, liver, kidney, spleen, lung and intestine were taken, homogenized and suspended in phosphate-buffered saline with antibiotics for viral detection. 2.3. Virus isolation From the homogenized dove tissues and the RNA positive mosquito samples, virus isolation attempts were carried out on different cell lines as described by Savini et al. (2011). In brief, homogenized tissue and mosquito
samples suspended in phosphate-buffered saline with antibiotics were centrifuged (1000 g, 10 min). After centrifugation, the supernatants were inoculated onto confluent chicken embryo fibroblast, chicken embryo liver and Vero cell cultures. Cell cultures were incubated at 37 8C for 7 days and were checked daily for the occurrence of cytopathic effects (CPE). Three blind passages were made in absence of cytopathic effect. At the end of the third passage and in case of CPE, the presence of WNV in the supernatants was confirmed or excluded either by electron microscopy or by Immunofluorescence using a monoclonal antibody (produced by ICT, Italy) against WNV. Samples of brain, liver, kidney, spleen, lung and intestine of the dove were also processed in 9–10 day old SPF embryonated chicken eggs (OIE, 2008). 2.4. Molecular analysis 2.4.1. Real time RT-PCR and nested RT-PCR Viral RNA was extracted from a pool of kidney, heart and brain and was tested by real time RT-PCR (RRT-PCR) for Avian Influenza (AI) (Spackman et al., 2002), Newcastle Disease (ND) (Wise et al., 2004), Usutu and West Nile (WN) viruses (Tang et al., 2006). Lung and intestine were also tested for ND and AI by RRT-PCR. Mosquito samples were also screened for the presence of WNV by using one-step SYBR Green-Based RRT-PCR (Ravagnan et al., 2011). This method which uses specific primers (MAMD and cFD2; Scaramozzino et al., 2001) designed on the conserved region of the non-structural NS5 gene of the WNV, is capable of detecting several Flaviviruses. Positive results were then confirmed either by using the commercial Kit TaqvetTM West Nile WNV (LSI, Lissieu, France) which is able to detect WNV RNA of the lineage 1 and 2 strains and the RT-PCR as described by Bakonyi et al. (2006) targeting NS5 gene. To ascertain which lineage the WNV RNA found in the samples belonged to, lineage specific RT-PCRs were used. For determining the presence of lineage 1 strains, the Lanciotti et al. (2000) method modified as described by Monaco et al. (2009) was used, whereas for discriminating lineage 2 strains, the method as recently described by Chaskopoulou et al. (2011), which targets the NS3 gene of the WNV genome, was used. Briefly, total RNA was reverse transcribed by the kit ‘‘TaqMan1 Reverse Transcription Reagents’’ (Applied Biosystem, USA) using random hexamers. The reverse transcription was carried out in 20 ml of mix containing 0.5 ml of Multiscribe RNA enzyme (50 U/ ml), 2 ml of 10 RT buffer, 4.4 ml of the 25 mM MgCl2 buffer, 4 ml of dNTPs 2.5 mM, 1 ml of random hexamer (50 mM) primer, 0.4 ml of Rnase inhibitor (20 U/ml) and 5 ml of RNA. The reaction was incubated 10 min at 25 8C then at 48 8C for 30 min followed by final incubation at 95 8C for 5 min to inactivate the residual activity of the reverse transcriptase. Viral RNA was amplified by a nested RT-PCR. In brief, the first amplification generated a 778 bp amplicon by using the external primer pair WN-NS3up1 (50 -GCTGGCTTCGAACCTGAAATGTTG-30 ) and WN-NS3do1 (50 -CAATGATGGTGGGTTTCACGCT-30 ). The second amplification product used the internal primer pair WNNS3up2 (50 -GCAAGATACTTCCCCAAATCATCAAGG-30 ) and
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WN-NS3do2 (50 -TGTCTGGGATCTCTGTTTGCATGTC-30 ) to amplify a 423 bp region of the NS3 coding region of the WNV genome. Both reactions were carried out in 50 ml of final volume containing 5 ml of buffer 10, 3 ml of MgCl2 (25 mM), 1 ml of dNTPs (10 mM), 0.3 ml of TaqGold and 1 ml of each primer (50 mM). The reaction was incubated at 95 8C for 10 min followed by 30 cycles of denaturation for 30 s at 94 8C, annealing at 60 8C for 30 s, extension at 72 8C for 1 min and final extension at 72 8C for 7 min. Amplified products were visualized on 1% agarose gel stained with Syber Safe (Invitrogen, USA) and the bands were observed under UV light.
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mosquito species (81%) and was present in all the 61 sites monitored. Species composition and relative abundance were similar in the two regions, confirming the area as environmental homogenous and highly suitable for C. pipiens survival. Only female mosquito samples identified at the species level (n = 78,791) were screened by the one step RRT-PCR for the presence of Flavivirus. Males (n = 766; 0.0001%) or mosquitoes not identified at the species level (n = 5400; 6.3%) were not tested. In total, 2732 pools were tested and 5 of them (0.18%) were found positive for WNV RNA from late July until mid September (Table 2). At necropsy, the dove appeared undernourished but no significant gross lesions were observed. Avian Influenza, Newcastle Disease and Usutu viruses were not detected by either molecular methods or virus isolation on target organs. The pool of kidney, heart and brain resulted positive for WN virus by both RRT-PCR and RT-PCR, conversely the attempt to isolate the virus was unsuccessful. All positive reactions were confirmed by the lineage 1 and 2 commercial RT-PCR. When the specific RT-PCRs were used, RNA of lineage 1 WNV was found in 3 C. pipiens pools while RNA of lineage 2 strain was found either in 2 pools of C. pipiens collected in the province of Udine and in the bird tissues. The NS3 gene of the lineage 2 strains was then partially sequenced and the sequence aligned. The nucleotide sequences confirmed the presence of lineage 2 strains. Results of the alignment of the partial NS3 gene sequence are shown in Fig. 2. No differences were observed between the Italian strains. The sequences also showed 100% similarity with those of the Hungarian strain but differed by 2 nucleotides in positions 80 and 142 from the 2010 Greek isolate. When the nucleotide sequences were translated to putative amino acid sequences, they differed by one amino acid only at position 249 of the NS3 protein. At this site in the Italian and Hungarian strains histidine
2.4.2. Amplification and sequencing PCR products were purified with the Qiaquick PCR Purification kit (Qiagen, Germany) and used for direct sequencing in both directions using the internal primers previously described. The sequencing reaction was set up using the Big Dye Terminator kit (Applied Biosystems, USA), the excess of dyes was removed using Cleanseq (Beckman Coulter, USA) and the nucleotide sequences were determined using the DNA sequencer ABI PRISM 3100 (Applied Biosystems, USA). Raw sequence data were assembled using Contig Express (Vector NTI suite 9.1, Invitrogen, USA) and translated into amino acid sequences using Vector NTI suite 9.1 (Invitrogen, USA). Consensus sequences from the virus detected in Italian mosquitoes and bird tissues were aligned with the WNV lineage 2 isolated in 2004 in Hungary (DQ116961), in 2011 in Greece (HQ537483) and to the reference strain isolated in 1937 in Uganda (M12294) with ClustalW (Thompson et al., 1994) (Table 1). 3. Results Overall, 85,398 mosquitoes were collected belonging to 14 species. Culex pipiens was the most abundant
Table 1 Strains of West Nile viruses included in the NS3 partial sequencing. Name
Strain
Location
Year
lineage
Source
GenBank
CpITA11/1 CpITA11/2 SdeITA/11 HUM/ITA11 HUN04 GRE10 UGA37 ITA09
Italy 2011/21612 Italy 2011/21617 Italy 2011/23743 Italy 2011 Goshawk-Hungary/04 Nea Santa Greece 2010 B956 Italy/2009/J-225677
Italy Italy Italy Italy Hungary Greece Uganda Italy
2011 2011 2011 2011 2004 2010 1937 2009
2 2 2 2 2 2 2 1
Culex pipiens Culex pipiens Streptopelia decaocto Homo sapiens Accipiter gentilis Culex pipiens Homo sapiens Garrulus glandarius
– – – JN858070 DQ116961 HQ537483 AY532665 JF719068
Table 2 Details of the West Nile virus infected Culex pipiens collected during the 2011 entomological survey. Site code
Province
Region
WN area code
Collection date
WNV lineage
MIR (%)
198 217 210 178 211
Venezia Pordenone Udine Treviso Udine
VEN FVG FVG FVG FVG
AS AS ACV Outside ACV
July 27th July 27th August 19th September 14th September 14th
1 1 2 1 2
1.695 1.852 0.813 1.852 1.333
VEN, Veneto region; FVG, Friuli Venezia Giulia region ACV, area with WN viral circulation; AS, WN surveillance area; outside, area outside the AS area. MIR, minimum infection rate (positive pools/mosquitoes tested 100) for the site and date of collection.
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Fig. 1. Areas where lineage 2 strains of West Nile virus have been detected in Northern Italy.
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replaces proline. The blast of the NS5 nucleotide sequence also showed a similarity of 99% with the West Nile Hungarian 2004 isolate. 4. Discussion This report provided further evidence that a lineage 2 strain is circulating in Italy. It was found in two pools of C. pipiens and in the tissues of a resident collared dove found dead in North East of Italy. This is the first time that a lineage 2 strain has been detected in mosquitoes and birds in Italy. A human case caused by a lineage 2 strain infection was recently described in Ancona (Bagnarelli et al., 2011). It was a mild case characterized by a period of high fever with no response to antibiotics. The strain which was detected in the urine sample, when sequenced, showed 99% identity to the complete genome of isolate goshawkHungary/04 and to the more recent Nea Santa-Greece2010 (Bakonyi et al., 2006; Papa et al., 2011b; Bagnarelli et al., 2011). According to the NS3 partial sequences, the strains detected in mosquitoes and bird tissues in this study, were identical and also showed 100% similarity with the same fragment sequences of the strain detected in Italy and of the strains which circulated in Hungary in 2004. Therefore, it appears that the same strain of lineage 2 WNV was likely responsible for the dove and mosquito infections even if the areas where the bird and the mosquitoes were collected were not contiguous (Fig. 1). In contrast to the previous Italian lineage 2 reported case, this lineage 2 strain detection occurred in an higher risk area compared to, where lineage 1 WNV has circulated and/or is circulating. It is currently difficult to track down the exact route of entrance of the virus. The phylogenetic findings and the sample collection dates imply a southward expansion of the virus from the Central European countries. The areas where the infected mosquitoes were collected are adjacent to Slovenia and/or are part of the flying paths of the long and short migration routes. Migratory birds from Africa have often been implicated in the emergence of the WNV outbreaks in Europe (Hubalek and Halouzka, 1999; Rappole et al., 2000; Murgue et al., 2001; Malkinson et al., 1998; Rappole and Hubalek, 2003). In this case, considering the high genetic similarity with the Hungarian isolate, it is more likely that birds migrating either along the south-eastern migration route from Europe and western Asia to Africa, or along short migration routes from Central to Southern Europe, have introduced the virus. A similar infection pathway was also proposed for the spread of Usutu virus in Italy (Savini et al., 2011). Since in a month period the same strain virus was detected in local birds and indigenous mosquitoes in two different areas situated 50 km apart, it looks as if the Italian lineage 2 strain established itself with the tendency to spread westward. Many prerequisites are required to maintain a WNV strain in an environment. Vector competency and presence of susceptible vertebrate host capable of transmitting the infection to vectors, are indeed key parameters for the maintenance and spread of WNV in a given environment. The areas where the lineage 2 strains have been detected, have already proven to have these prerequisites as lineage 1 WNV and also Usutu virus, which
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share the same life cycle, have circulated and/or are circulating. Although further studies carried out in nonepidemic periods are needed to test this hypothesis, it is then more than likely that this lineage 2 strain will establish and further spread in the neighboring areas. As a consequence, in the next season it is highly probable that both lineages will contemporaneously circulate in the same areas. Whether this will result in an enhancement or reduction of their virulence is hard to say. Similarly, it is difficult to predict the occurrence of possible genetic recombination phenomena (Pickett and Lefkowitz, 2009). Because cross protection between both lineages has been observed in horses (Minke et al., 2011), the circulation of both lineages could eventually result in a rapid endemization of the area with no severe clinical consequences. According to the phylogenetic analysis, the Italian as the Hungarian lineage 2 strains differed from that isolated in Greece as they have histidine rather than proline at location 249 of the NS3 protein. This site has been involved
Fig. 2. Phylogenetic analysis of the Italian sequences. Sequence dataset was analyzed using BioEdit version 7.0.9 and nucleotide alignment was performed with Clustal-W (Thompson et al., 1994). Phylogenetic analysis was conducted by using distance (NJ) method in PHYLIP (the PHYlogeny Inference Package) program version 3.67. The reliability of the phylogenetic trees was confirmed by performing bootstrap analysis with 1000 replicate values. The tree obtained was rooted with ITA09 strain. The asterisk indicates significant bootstrapping values (>60%). ITA09: West Nile virus (WNV) strain isolated in Italy in 2009; UGA37: B956 lineage 2 prototype strain; GRE10: Greek WNV strain isolated in 2010; HUN04: Hungarian isolate detected in 2004; HumITA11: Italian WNV strain detected in a human patient in 2011; SdeITA11: Italian WNV strain detected in a wild collared dove in 2011; Cp1ITA11: WNV strain detected in a pool of C. pipiens caught in Bagnara Arsa (UD); Cp2ITA11: WNV strain detected in a pool of C. pipiens caught in Palazzolo della Stella (UD).
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in genetic modulation of WNV pathogenicity, using American crows (Corvus brachyrhynchos) as avian model of WNV disease (Brault et al., 2007). The influence of histidine in position 249 of the NS3 on the virulence of a strain is still unknown. In line with those which circulated in Hungary and Austria which were able to cause encephalitis and death in a sparrow hawks (Accipiter nisus), goshawks (Accipiter gentilis) (Bakonyi et al., 2006; Erdelyi et al., 2007) and gyrfalcon (Falco rusticolus), the Italian lineage 2 strain found in the tissues of a collared dove was likely the cause of the bird death. In addition to the dove, the lineage 2 strain was also detected in pools of C. pipiens. Considering that C. pipiens has been demonstrated to serve both as an enzootic and a bridge vector for humans (Hamer et al., 2008), it is highly probable that lineage 2 WNV will infect humans again. However is not possible to predict what will be the impact of this infection even if the clinical expression of the lineage 2 human case reported in Italy was relatively mild. 5. Conclusions This study demonstrated that a WNV strain belonging to lineage 2 is circulating in North-East of Italy. As it was found either in local collared dove and in indigenous mosquito (C. pipiens), it seems to have developed strategies of adaptation and become established. Its NS3 partial sequence is identical to the other Italian isolate detected in a human patient as well as to Hungarian isolate with which it seems to share similar features including the pathogenicity for birds. Comprehensive investigations on the occurrence, ecology, and epidemiology of the different WNV strains circulating in Italy should be of highest priority. Because of its zoonotic potential, veterinarians, physicians, and public health officials need to work more closely together to control, prevent, and better estimate the epidemiological impact and possible threat to animal and public health of WNV infections. Greater communication and collaboration between these different professional figures at both, central and local level would, ultimately, improve prevention and control strategies with great benefit for human and veterinary public health. References Bagnarelli, P., Marinelli, K., Trotta, D., Monachetti, A., Tavio, M., Del Gobbo, R., Capobianchi, M.R., Menzo, S., Nicoletti, L., Magurano, F., Varaldo, P.E., 2011. Human case of autochthonous West Nile virus lineage 2 infection in Italy, September 2011. Euro Surveill. 16 (43) pii=20002. Available from: http://www.eurosurveillance.org/ViewArticle.aspx? ArticleId=20002. Bakonyi, T., Ivanics, E., Erdely, K., Ursu, K., Ferenczi, E., Weissenbock, H., Nowotny, N., 2006. Lineage 1 and 2 strains of encephalitic West Nile virus Central Europe. Emerg. Infect. Dis. 12, 618–623. Brault, A.C., Huang, C.Y., Langevin, S.A., Kinney, R.M., Bowen, R.A., Ramey, W.N., Panella, N.A., Holmes, E.C., Powers, A.M., Miller, B.R., 2007. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat. Genet. 39, 1162–1166. Burt, F.J., Grobbelaar, A.A., Leman, P.A., Anthony, F.S., Gibson, G.V., Swanepoel, R., 2002. Phylogenetic relationships of southern African West Nile virus isolates. Emerg. Infect. Dis. 8, 820–826. Calistri, P., Giovannini, A., Savini, G., Monaco, F., Bonfanti, L., Ceolin, C., Terregino, C., Tamba, M., Cordioli, P., Lelli, R., 2010. West Nile virus transmission in 2008 in North-Eastern Italy. Zoonoses Public Health 57, 211–219.
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