Usutu Virus Sequences in Culex pipiens (Diptera: Culicidae), Spain

To the Editor: Usutu virus (USUV) is an arbovirus and a member of the mosquito-borne cluster within the Flavivirus genus. USUV belongs to the Japanese encephalitis virus antigenic group, which is closely related to pathogens such as West Nile virus (WNV) (1). 
 
USUV has been isolated from a human in the Central African Republic and from several mosquito species from tropical and subtropical Africa (2). In late summer 2001, USUV emerged in central Europe and caused deaths in several species of resident birds in Austria (3). However, monitoring of USUV in dead birds from 2003 through 2005 showed that the absolute numbers of USUV–associated bird deaths declined, although USUV detection persisted in bird tissues (4). This decrease in USUV-associated bird deaths was attributed to herd immunity in the bird population (5). In the summer of 2005, USUV was detected in a blackbird in Hungary. The complete genomic sequence of the Hungarian USUV strain shared 99.9% identity with the strain circulating in Austria since 2001 (6). On the other hand, neutralizing antibodies against USUV have been detected in sera of resident and migrant birds in the United Kingdom without causing an obvious reduction in the bird population (7). 
 
From May through October 2006, monitoring of flaviviruses in mosquitoes was performed in the northeast region of Spain (Catalonia). This monitoring was implemented in the 3 main wetlands of the region: Aiguamolls de la Emporda (Girona Province) near France, where WNV was detected in dead horses in 2000 (8); Delta del Llobregat (Barcelona Province); and Delta de l’Ebre (Tarragona Province). Mosquitoes were collected by mosquito control services in these areas. Female mosquitoes were classified and grouped in pools according to date, species, and localization. During this period, 436 pools belonging to 9 mosquito species were collected. The most abundant species was Culex pipiens (n = 168). 
 
Viral RNA was recovered from mosquito pools by homogenization and viral RNA extraction with QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), and then generic reverse transcription (RT)-nested PCR was used to identify flaviviruses (9). This procedure was used to amplify a specific fragment of the NS5 gene within the flavivirus genome. The 143-bp amplification product was detected by electrophoresis and purified by using QIAquick PCR Purification Kit (QIAGEN). Sequencing reactions were performed with ABI Prism BigDye Terminator Cycle Sequencing v.3.1 Ready Reaction (Applied Biosystems, Foster City, CA, USA), and analyzed by using an ABI PRISM model 3730 automated sequencer (Applied Biosystems). Assembly of the consensus sequences and translation into amino acid sequences was performed with Larsergene DNASTAR group of programs (DNASTAR Inc., Madison, WI, USA). Comparisons with published sequences of known flaviviruses were performed by searches with FASTA program in EMBL database (available from www.ebi.ac.uk/embl/) to identify the detected agent and to study the level of homology. 
 
One pool of Cx. pipiens captured in the middle of August 2006 from Delta del Llobregat, in a typical Mediterranean climate that contained 3 female mosquitoes, was positive for flaviviruses. That positive pool was obtained from the center of the village of Viladecans, where different common migratory and sedentary birds such as Passer domesticus, Hirundo rustica, or Delichon urbica feed and nest. The Spanish USUV sequence showed 97.97% homology to USUV strain SAAR-1776 from South Africa; it showed 94.94% similarity with USUV strain Vienna 2001 from Austria and USUV strain Budgapest from Hungary, with 2-nt and 5-nt differences, respectively (Figure). All of these were synonymous mutations and thus did not result in amino acids replacements. The homology data showed that the Spanish strain belongs to USUV species and is more related to the African USUV isolates than to central European isolates. 
 
 
 
Figure 
 
Comparison at nucleotide level of sequenced fragment among related Usutu virus (USUV). Dot indicates coincident nucleotide. The partial nucleotide sequence of detected Spanish USUV has been deposited in the GenBank database under accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM909649","term_id":"187453067","term_text":"AM909649"}} ... 
 
 
 
To date, no bird deaths observed in Barcelona Province have been associated with viral encephalitis. However, this region is where the USUV-specific RT-PCR–positive samples were obtained from Cx. pipiens mosquitoes. One possible explanation for these findings is that Spanish USUV could be naturally avirulent for birds because the African strains of USUV appear to be in Africa. Alternatively, USUV and other related viruses such as WNV may have been circulating in Spain for many years, as a result of regular reintroduction by birds migrating from Africa. Under such circumstances, natural genetic resistance, herd immunity, and cross-protective immunity caused by related viruses likely provided at least some protection against symptomatic infections. The discovery of USUV-specific RNA, most related to the African strains of USUV, in Cx. pipiens in Spain extends previous evidence (7,10) that USUV and related flaviviruses such as WNV are being introduced into western Europe from Africa, presumably by migratory birds.

enteric fever in the United Kingdom has been seriously jeopardized. In cases of treatment failures, commonly used alternative antimicrobial agents have included third-generation cephalosporins such as ceftriaxone. The macrolide antimicrobial azithromycin is also being increasingly used, particularly for patients with hypersensitivity to penicillins (5). With this in mind, 50 S. Typhi and 40 S. Paratyphi A strains isolated from January 2005 through December 2006, which exhibited resistance to ciprofl oxacin at 0.125 mg/L, were tested for resistance to azithromycin by Etest. Results indicated that none of the isolates of S. Typhi exhibited MICs >8 mg/L, which corresponded to the MIC to azithromycin of a drug-sensitive control strain of S. Typhi (range 4-8 mg/L, MIC 90 6 mg/L). For S. Paratyphi A, none of the isolates exhibited MICs >12 mg/L, corresponding to that of a drugsensitive control strain of this serovar (range 6-12 mg/L, MIC 90 10 mg/L). Although there are no defi nitive data on resistance levels for azithromycin in relation to treatment of typhoid and paratyphoid, these fi ndings suggest that resistance to this antimicrobial agent in terms of treatment effi cacy has not yet been jeopardized.
These results indicate that the availability of effective antimicrobial agents for the treatment of typhoid and paratyphoid infection is becoming increasingly limited for patients in the United Kingdom. Nevertheless, despite the dramatic upsurge in the oc-currence of strains with decreased susceptibility, ciprofl oxacin still remains the drug of choice for many physicians. It is reassuring that in cases of treatment failure, third-generation cephalosporins such as ceftriaxone and macrolide antimicrobial agents such as azithromycin appear to be viable alternatives.

Usutu Virus Sequences in Culex pipiens (Diptera: Culicidae), Spain
To the Editor: Usutu virus (USUV) is an arbovirus and a member of the mosquito-borne cluster within the Flavivirus genus. USUV belongs to the Japanese encephalitis virus antigenic group, which is closely related to pathogens such as West Nile virus (WNV) (1).
USUV has been isolated from a human in the Central African Republic and from several mosquito species from tropical and subtropical Africa (2). In late summer 2001, USUV emerged in central Europe and caused deaths in several species of resident birds in Austria (3). However, monitoring of USUV in dead birds from 2003 through 2005 showed that the absolute numbers of USUV-associated bird deaths declined, although USUV detection persisted in bird tissues (4). This decrease in USUV-associated bird deaths was attributed to herd immunity in the bird population (5 2001  170  24  23  23  35  0  232  28  27  27  23  2  2002  150  18  17  17  35  1  149  10  9  10  39  3  2003  218  20  20  21  43  1  177  17  18  17  65  12  2004  215  23  23  24  47  2  221  5  5  5  70  14  2005  222  29  29  29  62  2  217  7  7  7  60  12  2006  240  23  24  24  68  2  278  2  3  2  in the United Kingdom without causing an obvious reduction in the bird population (7). From May through October 2006, monitoring of fl aviviruses in mosquitoes was performed in the northeast region of Spain (Catalonia). This monitoring was implemented in the 3 main wetlands of the region: Aigüamolls de la Empordà (Girona Province) near France, where WNV was detected in dead horses in 2000 (8); Delta del Llobregat (Barcelona Province); and Delta de l'Ebre (Tarragona Province). Mosquitoes were collected by mosquito control services in these areas. Female mosquitoes were classifi ed and grouped in pools according to date, species, and localization. During this period, 436 pools belonging to 9 mosquito species were collected. The most abundant species was Culex pipiens (n = 168).
Viral RNA was recovered from mosquito pools by homogenization and viral RNA extraction with QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), and then generic reverse transcription (RT)-nested PCR was used to identify fl aviviruses (9). This procedure was used to amplify a specifi c fragment of the NS5 gene within the fl avivirus genome. The 143-bp amplifi cation product was detected by electrophoresis and purifi ed by using QIAquick PCR Purifi cation Kit (QIA-GEN  (Figure). All of these were synonymous mutations and thus did not result in amino acids replacements. The homology data showed that the Spanish strain belongs to USUV species and is more related to the African USUV isolates than to central European isolates.
To date, no bird deaths observed in Barcelona Province have been associated with viral encephalitis. However, this region is where the USUVspecifi c RT-PCR-positive samples were obtained from Cx. pipiens mosquitoes. One possible explanation for these fi ndings is that Spanish USUV could be naturally avirulent for birds because the African strains of USUV appear to be in Africa. Alternatively, USUV and other related viruses such as WNV may have been circulating in Spain for many years, as a result of regular reintroduction by birds migrating from Africa. Under such circumstances, natural genetic resistance, herd immunity, and cross-protective immunity caused by related viruses likely provided at least some protection against symptomatic infections. The discovery of USUV-specifi c RNA, most related to the African strains of USUV, in Cx. pipiens in Spain extends previous evidence (7,10) that USUV and related fl aviviruses such as WNV are being introduced into western Europe from Africa, presumably by migratory birds.  (2), the small sample size, short duration of surveillance, and lack of a comparison group preclude fi rm assessment of an association between P[4]G2 predominance and vaccination.
Because Rotarix was introduced in Brazil in March 2006, most children >12 months old (66 [51%] of 129) in the study were ineligible for vaccination. Genotype P [4]G2 was the only strain identifi ed even in older children, which suggests either a change in disease ecology from vaccination or the random circulation of P [4]G2 strains in the community. Ongoing hospitalbased surveillance during 2006 in 3 regional countries that had not introduced rotavirus vaccine (El Salvador, Guatemala, and Honduras) showed that P [4]G2 was the predominant circulating strain (prevalence 68%-81%). Thus, as previously documented (3,4), the predominance of P[4]G2 strains after Rotarix introduction in Brazil could represent a natural shift unrelated to vaccination.
Evaluation of vaccine effectiveness against specifi c strains will allow full assessment of the public health impact of vaccination. Although the data are sparse in the study from Gurgel et al., a comparison of the odds of vaccination among rotavirus-positive (cases) versus rotavirus-negative (controls) children shows 80% vac-cine effectiveness against P [4]G2 strains among infants <1 year of age, in accordance with recently published data from a controlled trial (5). To further elucidate vaccine impact, we are providing support for vaccine effectiveness studies in Nicaragua and El Salvador and conducting strain monitoring before and after licensure throughout Latin America.

Letters
Letters commenting on recent articles as well as letters reporting cases, outbreaks, or original research are welcome. Letters commenting on articles should contain no more than 300 words and 5 references; they are more likely to be published if submitted within 4 weeks of the original article's publication. Letters reporting cases, outbreaks, or original research should contain no more than 800 words and 10 references. They may have one Figure or Table and should not be divided into sections. All letters should contain material not previously published and include a word count.