Co-circulation of Usutu virus and West Nile virus in a reed bed ecosystem
© Rudolf et al. 2015
Received: 27 August 2015
Accepted: 3 October 2015
Published: 12 October 2015
Mosquito-borne flaviviruses are a major public health threat in many countries worldwide. In Central Europe, West Nile virus (WNV) and Usutu virus (USUV), both belonging to the Japanese encephalitis virus group (Flaviviridae) have emerged in the last decennium. Surveillance of mosquito vectors for arboviruses is a sensitive tool to evaluate virus circulation and consequently to estimate the public health risk.
Mosquitoes (Culicidae) were collected at South-Moravian (Czech Republic) fishponds between 2010 and 2014. A total of 61,770 female Culex modestus Ficalbi mosquitoes, pooled to 1,243 samples, were examined for flaviviruses by RT-PCR.
One pool proved positive for USUV RNA. Phylogenetic analysis demonstrated that this Czech USUV strain is closely related to Austrian and other Central European strains of the virus. In addition, nine strains of WNV lineage 2 were detected in Cx. modestus collected in the same reed bed ecosystem.
This is the first detection of USUV in Cx. modestus. The results indicate that USUV and WNV may co-circulate in a sylvatic cycle in the same habitat, characterised by the presence of water birds and Cx. modestus mosquitoes, serving as hosts and vectors, respectively, for both viruses.
Usutu virus (USUV) is a mosquito-borne virus (Japanese encephalitis group, genus Flavivirus; family Flaviviridae) that was originally isolated in Africa. In or before 1996, the virus was introduced to Europe . It circulates in nature between birds (as amplifying hosts) and bird-feeding mosquitoes, principally Culex spp., as vectors. USUV is taxonomically and ecologically very similar to West Nile virus (WNV) [2, 3]. Contrary to WNV, USUV has rarely caused human disease – only in immunocompromised persons . However, USUV antibodies were recently reported in three patients with neuroinvasive disease in Croatia .
In the Czech Republic, two strains of USUV were isolated from dead blackbirds (Turdus merula) in Brno, 2011 and 2012 . In addition, specific neutralizing antibodies against USUV were found in common coots (Fulica atra) in Moravia [7, 8].
Neutralizing antibodies against WNV were rarely found in the local human population, but five cases of West Nile fever in humans were reported after heavy floods in 1997 . More frequently WNV antibodies occur in apparently healthy wild birds in this region . Three identical strains of WNV (proposed genomic lineage 3: Rabensburg) were isolated from Culex pipiens and Aedes rossicus mosquitoes in 1997, 1999 and 2006 . In a previous study, we reported four strains of lineage 2 WNV from Culex modestus mosquitoes collected in reed beds at South-Moravian fishponds (Czech Republic) during August 2013 .
Within the scope of the joint European EDENext project we extended our virological surveillance of local Cx. modestus mosquitoes for pathogenic flaviviruses, including USUV.
Mosquito processing, RNA extraction, PCR and sequencing
Caught insects were transported to the laboratory in cooled flasks, and stored at −65 °C until examination. Mosquitoes were determined on a chilled table under a stereomicroscope according to an entomological key , and monospecific pools consisting of up to 50 female Cx. modestus (other species were not tested in this study) were homogenized in 1.5 ml of cooled phosphate-buffered saline pH 7.4 supplemented with 0.4 % bovine serum albumin (Sigma) and antibiotics (PBS-BSA), and centrifuged.
RNA was extracted from 140 μl mosquito homogenates using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Generic oligonucleotide primers targeting the NS5 region of flaviviruses were used for screening purposes . Samples positive by the above pan-flavivirus PCR were subjected to USUV [15, 16] as well as WNV  -specific RT-PCR assays for amplification of overlapping genome regions. Amplification products were directly sequenced (Microsynth, Balgach, Switzerland); the sequences were aligned and compiled, and identified by BLAST search against GenBank database.
Phylogenetic and molecular evolutionary analyses of concatenated sequences were conducted using neighbor-joining, maximum likelihood, minimum evolution, UPGMA and maximum parsimony algorithms (MEGA version 6, with 1000 replicates for bootstrap testing).
The original mosquito homogenates of PCR-positive samples were inoculated intracerebrally (i.c., 20 μl) into specific pathogen free suckling ICR mice (SM). Brains of dead animals were homogenized in PBS-BSA, centrifuged, and passaged (i.c.) in a new batch of SM. Bacterial sterility of the suspensions was checked in meat-peptone and thioglycollate broths incubated at 37 °C . All experiments with laboratory mice were conducted in accordance with the Czech Animal Protection Act no. 246/1992, and the protocols were approved by the Institutional and Central Care and Use Committees at the Academy of Sciences of the Czech Republic in Prague and by the Veterinary Service in Brno. The facility is accredited by the Czech National Committee on Care and Use of Laboratory Animals (6630/2008-10001).
Results and discussion
Numbers of female Culex modestus mosquitoes examined for flaviviruses in individual years and at three study sites
Also in South Moravia (Czech Republic), USUV strains had been previously isolated, namely from blackbirds, which were found dead in 2011 and 2012, respectively . The 2011 isolate was sequenced in two regions: partial E + NS1 (GenBank: JX236666) and partial NS5-5′UTR (GenBank: JX236667). In the E + NS1 region there is a 1371 nt overlap (nts 1610–2980 referring to the USUV sequence AY453411) between the 2011 blackbird-derived USUV sequence  and the 2013 Cx. modestus-derived sequences (this paper), and in the E protein coding region of this overlap the two genomes differ in (only) three nucleotides at positions 1872 (T vs. C), 2322 (C vs. T), and 2419 (C vs. T), resulting in a 99.78 % identity between the two Czech USUV strains in this genomic region; none of the substitutions lead to putative amino acid changes.
Furthermore, WNV genomic lineage 2 (WNV-2) was detected in nine pools of Cx. modestus mosquitoes collected in August 2013: #13-104 (collected at Nový fishpond); #13-329 (coll. at Nesyt fishpond); #13-479 (coll. at Mlýnský fishpond); #13-502 (coll. at Mlýnský fishpond) (these four records were reported in a previous study: ); #13-670 (coll. at Mlýnský fishpond); #13-743 (coll. at Nesyt fishpond); #13-853 (coll. at Mlýnský fishpond); #13-859 (coll. at Nesyt fishpond); #13-862 (coll. at Nesyt fishpond); the overall minimum prevalence rate of WNV in Cx. modestus was therefore 0.146 per 1,000 mosquitoes, about ten times higher than that for USUV. All WNV RNA positive original mosquito homogenates were then inoculated into SM. While the homogenates #13-329, #13-670, #13-743, and #13-853 did not kill any mice, the five others did: #13-104 killed 6 of 11 inoculated SM within 7–8 days post inoculation (DPI), average survival time (AST) of SM was 7.7 days; #13-479 killed 8 of 9 inoculated SM (6–7 DPI; AST 6.1 d); #13-502 killed specifically 7 of 10 SM (6–8 DPI; AST 6.4 d); #13-859 killed 5 of 11 SM (6–7 DPI; AST 6.4 d); and #13-862 killed all 11 inoculated SM (6–7 DPI; AST 6.7 d).
To the best of the authors’ knowledge, this is the first detection of USUV in Cx. modestus. It indicates that USUV may co-circulate with WNV in certain habitats – this phenomenon was demonstrated previously in northern Italy, where the principal mosquito vector of USUV (and WNV as well) is Cx. pipiens [3, 17–21]. A comprehensive review on the co-circulation of the two arboviruses in Europe has recently been written . Contrary to northern Italy, where USUV occurs in Culex mosquitoes much more frequently than WNV, reverse proportion was found in South Moravia in this study.
Interestingly, both viruses (USUV, WNV) were detected in South Moravia in 2013, but not in the years 2010, 2011, 2012 and 2014. This result could be affected by the number of Cx. modestus mosquitoes examined in individual years, which was much higher in 2013 than in the other years (Table 1). Moreover, mosquitoes were not collected in August 2014 (only in July).
Our previous finding that the common coot (Fulica atra) relatively often reveals specific antibodies to USUV [7, 8] might indicate a specific role of this avian species in the circulation of USUV in wetlands.
This is the first detection of USUV in Cx. modestus. The results indicate that USUV and WNV may co-circulate in a sylvatic cycle in the same habitat, characterised by the presence of water birds and Cx. modestus mosquitoes, serving as hosts and vectors, respectively, for both viruses. The present finding suggests that USUV (similar to WNV) may circulate in two types of ecosystems: (i) sylvatic cycle between Cx. pipiens/ Cx. modestus and water birds - such as coots, based on a previous serosurvey study ; (ii) urban cycle involving Cx. pipiens and blackbirds or occasionally some other synanthropic avian species.
This study was funded by the EU grant FP7-261504 EDENext. The publication is catalogued by the EDENext Steering Committee as EDENext428. The contents of this paper are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission.
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