Molecular epidemiology and host genetics of norovirus and rotavirus infections in Portuguese elderly living in aged care homes.

Norovirus (NoV) and rotavirus group A (RVA) are major agents of acute gastroenteritis worldwide. This study aimed to investigate their epidemiological profile in Portuguese elderly living in long-term care facilities and to assess the host genetic factors mediating infection susceptibility. From November 2013 to June 2015, 636 faecal specimens from 169 elderly, mainly asymptomatic, living in nursing homes in Greater Lisbon and Faro district, Portugal, were collected. NoV and RVA were detected by real-time polymerase chain reaction and NoV genotyped by phylogenetic analysis. NoV detection rate was 7.1% (12 of 169). Three GI.3 and one GII.6 strains were genotyped. RVA detection rate was 3.6% (6 of 169), exclusively in asymptomatic individuals. Host genetic factors associated with infection susceptibility were described on 250 samples by saliva-based enzyme-linked immunosorbent assays. The Lewis-negative phenotype was 8.8% (22 of 250) and the rate of nonsecretors was 16.8% (42 of 250). Association to NoV and RVA infection was performed in the subgroup of individuals (n = 147) who delivered both faecal and saliva samples. The majority of NoV- and RVA-positive individuals (90.9% and 83.3%, respectively) were secretor-positive, with Lewis B phenotype. In a subset of individuals, FUT2 and FUT3 genes were genotyped to assess mutations and validate the secretor and Lewis phenotypes. All sequenced nonsecretors were homozygous for FUT2 nonsense mutation G428A. In this study, low detection rates of NoV and RVA infections were found during two winter seasons. However, even in the absence of any outbreak, the importance of finding these infections in a nonepidemic situation in long-term care facilities may have important implications for infection control.

Molecular epidemiology of rotavirus in four provinces of Angola before vaccine introduction.

Angola is a sub-Saharan country in southern Africa highly affected by diarrhoeal disease with limited epidemiological data regarding etiologic agents. This study was performed during 2012-2013, prior to rotavirus vaccine introduction, with the objective to detect and characterize the rotavirus strains circulating in four provinces of the country: Huambo, Luanda, Zaire, and Cabinda. A high rotavirus detection rate (35%, 117/334) was observed. G1 was the most common G-genotype (83.6%), whereas P[8] (50.9%) followed by P[6] (38.8%) were the most common P-types. G1P[8] was identified as the predominant combination (50%), followed by the unusual G1P[6] (29.3%). Strains such G2P[4], G8P[6], G9P[6], and G12P[6] were also found in lower frequencies (5.2-1.7%). The P[6] strains did not cluster in the phylogenetic trees according to their geographic origin or even the corresponding G-genotype, suggesting a limited number of recent introductions and extensive reassortment events. Our results represent the first report on rotavirus genotype profiles in Angola, showing a wide circulation of the unusual genotype G1P[6], and underline the importance of RV surveillance after the vaccine introduction. J. Med. Virol. 88:1511-1520, 2016. © 2016 Wiley Periodicals, Inc.

High rate of detection of G8P[6] rotavirus in children with acute gastroenteritis in São Tomé and Príncipe.

The burden of rotavirus infections greatly affects the low-income African countries. In the absence of epidemiological data on pediatric diarrhea in São Tomé and Príncipe (STP), a study was conducted from August to December 2011. Rotavirus antigen was detected in 36.7 % of the collected fecal samples (87/237). G8P[6] was identified as the predominant genotype (71.1 % detection rate), while G1P[8] represented only 8.4 %. Phylogenetic analysis of VP7 G8 strains showed clustering within lineage G8d, while VP4 P[6] strains clustered within lineage 1a. Our results represent the first report on rotavirus from STP and show one of the highest detection rates of G8 rotaviruses worldwide.

Characterization of an insect-specific flavivirus (OCFVPT) co-isolated from Ochlerotatus caspius collected in southern Portugal along with a putative new Negev-like virus.

We describe the isolation and characterization of an insect-specific flavivirus (ISF) from Ochlerotatus caspius (Pallas, 1771) mosquitoes collected in southern Portugal. The RNA genome of this virus, tentatively designated OCFVPT, for O. caspius flavivirus from Portugal, encodes a polyprotein showing all the features expected for a flavivirus. As frequently observed for ISF, the viral genomes seems to encode a putative Fairly Interesting Flavivirus ORF (FIFO)-like product, the synthesis of which would occur as a result of a -1 translation frameshift event. OCFVPT was isolated in the C6/36 Stegomyia albopicta (= Aedes albopictus) cell line where it replicates rapidly, but failed to replicate in Vero cells in common with other ISFs. Unlike some of the latter, however, the OCFVPT genome does not seem to be integrated in the mosquito cells we tested. Phylogenetic analyses based on partial ISF NS5 nucleotide sequences placed OCFVPT among recently published viral strains documented from mosquitoes collected in the Iberian Peninsula, while analyses of ORF/E/NS3/or NS5 amino acid sequences cluster OCFVPT with HANKV (Hanko virus), an ISF recently isolated from O. caspius mosquitoes collected in Finland. Taking into account the genetic relatedness with this virus, OCFVPT is not expected to be overtly cytopathic to C6/36 cells. The cytopathic effects associated with its presence in culture supernatants are postulated to be the result of the replication of a co-isolated putative new Negev-like virus.

Hepatitis C virus subtypes circulating among intravenous drug users in Lisbon, Portugal.

Hepatitis C virus (HCV) infects 2-3% of the world population and intravenous drug consumption is the leading cause of transmission in industrialized countries. The unavailability of data on the molecular epidemiology of HCV infection in Portugal prompted the study of HCV subtypes circulating among intravenous drug users residing in the Lisbon metropolitan area and sampled about 10 years apart (1998-2000 and 2008-2009). Partial coding sequences for E1 and/or NS5B were obtained from 124 individuals with HCV viremia, both mono-infected and co-infected with HIV. Phylogenetic analysis showed that, for both time periods, the most prevalent subtypes were 1a and 3a, found, altogether, in 64.9% and 71.6% of the individuals, respectively for the first and the second sampling periods. However, genotype 4 viruses (subtypes 4a and 4d), introduced later, as inferred by comparison of intra-subtype genetic distances, were also relatively frequent even one decade ago (24.6%). This HCV subtype profile for Portuguese intravenous drug users is in agreement with those described for other southern European countries when in association with drug consumption. With the exception of subtype 1b, phylogenetic trees did not show clustering of the Portuguese sequences, but rather phylogenetic mixing of HCV sequences from different geographic origins, as described previously in other Western countries and suggestive of a large international transmission network. Consistent with the low recombination rates reported for HCV, only one sample revealed discordant subtypes for the two regions analyzed (4d in E1 and 4a in NS5B), representing a potential new recombinant that deserves further analysis.

A new genotype 2 subcluster identified among GBV-C strains circulating in the Lisbon metropolitan area of Portugal.

The rate of infection by the GBV-C virus was investigated in a group of 214 individuals at high risk of infection with parenterally transmitted viruses, and all living in the Lisbon metropolitan area (Portugal). RNA was extracted from plasma samples, and a fragment of the 5'-UTR was amplified by RT-PCR, disclosing a high prevalence of infection (40.7%). Most probably due to similar modes of viral transmission, the majority of GBV-C (+) individuals were found to be coinfected with HIV and/or HCV. A genomic region covering part of the E1/E2 glycoprotein coding sequence was amplified from approximately half of the GBV-C positive samples (44/87). Phylogenetic analysis of nucleotide sequences showed segregation of Portuguese GBV-C strains with genotype 1 (G1, n = 10) and genotype 2 (G2, n = 24) references. Genotype 1 was significantly associated with the African descent of those infected. Curiously, some of the strains assigned to genotype 2 were shown to form a separate cluster (designated G2*) in both neighbor-joining and Bayesian phylogenetic trees, which was confirmed by multivariate principal coordinate analysis. However, analysis of the distribution of intra- and intergenotype genetic distances support the hypothesis that rather than corresponding to a new viral genotype, G2* is a geographical subcluster within the genotype 2 radiation. J. Med. Virol. 82:452-459, 2010. (c) 2010 Wiley-Liss, Inc.

Multiple Phlebovirus (Bunyaviridae) genetic groups detected in Rhipicephalus, Hyalomma and Dermacentor ticks from southern Portugal.

In the last decade, various research groups have reported a large diversity of new tick-borne phleboviruses, mostly prompted by the discovery of important human pathogens such as the Heartland and severe fever with thrombocytopenia syndrome viruses. Since these analyses have rarely been conducted using ticks collected from Southern Europe, this study was carried out so as to bring new insights into the diversity of phleboviruses circulating in Southern Portugal. Tick specimens were collected from the vegetation (questing ticks) or directly from animals (feeding ticks), and the majority analysed in pools using a detection strategy targeting the large (L) viral genomic segment. A high number of pools revealed the presence of phebovirus sequences, regardless of gender (male/female), origin (questing/feeding) or even species of the tick analysed. These sequences apparently formed three different groups in phylogenetic trees, and encoded L proteins characterized by group-specific amino acid residues. Furthermore, under the conditions used, these viruses failed to replicate in both Vero and DH82 cells. The impact these viruses may have on human/animal health will be addressed in the future.

Genetic characterization of human immunodeficiency virus type 1 from Beira, Mozambique.

In this report, we examined the genetic diversity of HIV-1 strains circulating in the city of Beira, the second largest metropolitan area in Mozambique. A total of 131 blood samples, collected between August and October 2003 from antiretroviral-naïve individuals, were characterized with a combined approach consisting of heteroduplex mobility assay (HMA) subtyping for gag (n=74) and/or env (n=117) genes, and DNA sequence analysis of proviral env (C2V3C3, n=52), LTR (n=30) and/or pol (n=43) genomic regions. Aside from the identification, by bootscanning analysis, of a viral strain with a C/A1 mosaic C2V3C3 structure, classified as subtype A by env HMA, phylogenetic inference studies of the sequence data demonstrated the circulation of genetically diverse subtype C viruses, predominantly of the R5 type. Inspection of the LTR sequences revealed a pattern of structural and regulatory elements typical of subtype C, with 63.3% of the viruses showing three NF-kappaB binding sites. Analysis of the predicted protease sequences enabled us to detect a single primary mutation (I84V, n=1) associated with resistance to protease inhibitors (PI), while secondary mutations were highly prevalent, some of them in combinations which may confer PI resistance. Although an unexpectedly high rate (11.6%) of reverse transcriptase key mutations (V75A, K103N, Y181C, M184I, or P236L) was detected in the sequences analyzed, our data suggest the non-epidemic circulation of resistant viruses, and the absence of multi-class drug resistant viral strains.

Natural polymorphisms of HIV type 2 pol sequences from drug-naive individuals.

Until today, the susceptibility of human immunodeficiency virus type 2 (HIV-2) to protease and nucleosidic reverse-transcriptase inhibitors (PI and NRTI, respectively) has not been clearly documented. In this report we studied HIV-2 proviral sequences (n = 30) from drug-naive patients. Our results revealed that several amino acid positions in the protease and reverse transcriptase coding sequence harbored residues that have been associated with drug resistance in HIV-1-infected patients. In particular, the M46I substitution in the protease was detected in 90% of the sequences analyzed, which, together with the other substitutions identified, may indicate a reduced susceptibility of HIV-2-infected drug-naive patients to PI. Furthermore, interpretation of genotypic data with four available algorithms, developed for interpretation of HIV-1 sequence data, suggested nonoverlapping profiles of drug resistance.

Clinical and laboratory profile of Zika virus infection in dengue suspected patients: A case series.

BACKGROUND: The Zika virus (ZIKV) is an emerging arthropod-borne virus related to the dengue virus (DENV), and shows a similar clinical profile as other arboviral diseases, such as dengue and chikungunya virus (CHIKV). Historically, ZIKV has been associated with sporadic cases of human infection, but is now responsible for outbreaks worldwide. In Brazil, cases have been reported since 2015, with some cases causing severe disease. OBJECTIVE: To identify clinical symptoms of Zika in patients in Dengue suspected patients. STUDY DESIGN: Description of a series of cases, wherein we analyzed 100 clinical samples collected from patients who exhibited acute febrile disease for ≤5days, from January to February 2016. RESULTS: In this study, we report 13 cases of ZIKV infection in adults presenting dengue-like symptoms in a DENV endemic area. All patients presented with fever, with myalgia being the second most frequently observed symptom. Two patients had rashes, but none of them had conjunctivitis. Other less frequent manifestations included headache, arthralgia, diarrhea, and nausea. CONCLUSION: The co-circulation of ZIKV and DENV is a serious public health concern, since it represents both a clinical and diagnostic challenge in endemic areas, as well as in the field of travel medicine.

West Nile virus in Southern Portugal, 2004.

West Nile virus (WNV) genomic RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in six out of 57 mosquito pools collected in Southern Portugal, during the summer of 2004, yielding an infection rate (IR) of 2.8/1,000 mosquitoes. Phylogenetic analysis of a 217-nucleotide fragment of the NS5 coding region, amplified from Culex pipiens s.l. and Culex univittatus unfed females, demonstrated a close relationship with WNV strains circulating in the Mediterranean basin (Italy, 1998; France, 2000; Morocco, 2003). The data in this short report demonstrate the presence of WNV in mosquitoes in Southern Portugal and the need of permanent surveillance for viral activity within the country.

Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture.

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.

Molecular epidemiology of HIV type 1 infection in Portugal: high prevalence of non-B subtypes.

In this study, we have investigated the diversity of current HIV-1 strains circulating in the metropolitan area of Lisbon, Portugal. A total of 217 HIV-1-positive blood samples, collected between October 1998 and December 2000, was genetically characterized in the gp120 C2V3C3 region (n = 205) or part of the gp41 N-terminal segment (n = 12) by heteroduplex mobility assay (HMA) and/or DNA sequencing. The HMA subtyping efficiency (number of samples unambiguously subtyped by HMA divided by the total number of samples subtyped) was 65.9% (143 of 217), with indeterminate migration patterns of subtype A and G strains contributing significantly to this value. On the overall, subtype B was the most prevalent (50.2%), followed by subtypes G (21.7%), A (17.5%), and F (5.5%), whereas subtypes C, D, H, and J accounted altogether for 5.1% of the infections. Non-B subtypes were responsible for 77.4 and 33.1% of the infections among African immigrants and Portuguese subjects, respectively. Angolan individuals (n = 25) were the only ones infected with all the HIV-1 subtypes documented, probably reflecting a high degree of viral genetic diversification in their country of origin. Phylogenetic analysis showed a predominance of IbNG-like viruses among subtype A sequences and two new major subclusters within subtype G (G(P) and G(P)'). The majority of the Portuguese G sequences described formed a well-defined subcluster (G(P)), supported by bootstrap values >90%, phylogenetically distant from clade G sequences in databases. gag (p24/p7) sequence analysis of these variants confirmed the maintenance of the subtype G subclusters. The multiple subclustering observed for the major clades A, B, D, and G, as well as the variety of subtypes found, indicate a high diversity of HIV-1 variants circulating in Portugal and suggest a need for continuous epidemiologic surveillance.

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal.

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.

Prevalence and partial characterization of genotypes of the human TT virus infecting Portuguese individuals.

The prevalence and genotype distribution of human TT viruses (TTV) were analyzed in 312 Portuguese individuals. Detection of TTV DNA was carried out by polymerase chain reaction (PCR) through the combined use of N22 and UTR-specific primers and revealed a prevalence of infection of 74%. Detection of TTV DNA was not statistically associated to the use of intravenous drugs, infection with HBV, HCV, HIV-1, HIV-1 viral load or CD4 cell count (in HIV-1 infected individuals). Our data suggest that, in the population studied, the prevalence of TTV infection does not seem to be related to intravenous viral transmission. Phylogenetic analysis of 49 plasmid clones harboring N22-specific sequences revealed the circulation of genotypes: 1 (27%, subtype G1a and G1b), 2 (51%, subtype G2b and G2c) and 4 (22%), as well as multiple genotype infections (G1b-G2b and G1a-G4). To our knowledge, this is the first report of TTV detection and partial characterization of TTV genetic variants in Portuguese individuals. Our results show that TTV infection is widespread in Portugal as in other parts of the world.

Novel multiregion hybridization assay for the identification of the most prevalent genetic forms of the human immunodeficiency virus type 1 circulating in Portugal.

The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs, technical features, and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. Multiregion hybridization assays (MHA) represent an alternative for a consistent genetic analysis of large numbers of viral strains. Classically, MHA rely on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and on their characterization with clade-specific TaqMan probes (also known as hydrolysis probes). In this context, the aim of our study was the development of a technical variant of an MHA (vMHA(B/G/02)) for genotyping the most prevalent genetic forms of HIV-1 circulating in Portugal. Different sets of primers were designed for universal and clade-specific amplifications of several sections of the viral genome: gag, pol(Pr), pol(RT), vpu, env(gp120), and env(gp41). vMHA(B/G/02) was implemented using a real-time PCR-based approach, with detection dependent on the use of SYBR Green I. As an alternative, a technically less demanding strategy based on conventional PCR and agarose gel analysis of the reaction products was also developed. This method performed with overall good sensitivity and specificity (>91%) when a convenience sample of 45 plasma-derived HIV-1 strains was analyzed. Apart from the detection of subtype B, G, CRF02_AG, and CRF14_BG viruses, several unique B/G recombinant were also detected. Curiously, recombinant viruses including CRF02_AG sequences were not detected in the group of samples analyzed.

GB virus C (GBV-C) evolutionary patterns revealed by analyses of reference genomes, E2 and NS5B sequences amplified from viral strains circulating in the Lisbon area (Portugal).

GBV-C is a non-pathogenic virus that is largely dispersed in different human populations. The phylogenetic analysis of the 5'-untranslated region (5'UTR) of the GBV-C genome has led to the segregation of viral strains into six genotypes, but incongruent results are frequently obtained depending on the genome region analyzed. In this report, different phylogenetic approaches and multivariate statistics were combined to disclose evolutionary patterns that contribute to shape GBV-C evolution. The data here presented indicate: (i) that the phylogenetic noise was mostly determined by the size of the analyzed sequence, rather than by its position on the viral genome; (ii) that most genomic segments in the coding sequence seemed to evolve under a similar evolution model, which was different from that which best fits the 5'UTR, with overall large heterogeneity of rate change across the sequence; (iii) that due to saturation of transversions occurring in the 5'UTR at genetic distances <0.10, care should be taken in drawing conclusions about the tree topologies involving the deeper branches, especially when using distance-based methods; (iv) that a non-uniform distribution of InSi and dS occurs over the viral ORF highlighting regions of the viral genome with remarkably low levels of silent substitutions, and implying that the observed differences may contribute to the detected phylogenetic incongruences; and finally (v) that genetic recombination clearly impacts the GBV-C evolution extensively, this being shown for both reference genomes and NS5B GBV-C sequences amplified from Portuguese residents.

Genetic characterization of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal.

We describe the full genetic characterization of an insect-specific flavivirus (ISF) from Culex theileri (Theobald) mosquitoes collected in Portugal. This represents the first isolation and full characterization of an ISF from Portuguese mosquitoes. The virus, designated CTFV, for Culex theileri flavivirus, was isolated in the C6/36 Stegomyia albopicta (=Aedes albopictus) cell line, and failed to replicate in vertebrate (Vero) cells in common with other ISFs. The CTFV genome encodes a single polyprotein with 3357 residues showing all the features expected for those of flaviviruses. Phylogenetic analyses based on all ISF sequences available to date, place CTFV among Culex-associated flaviviruses, grouping with recently published NS5 partial sequences documented from mosquitoes collected in the Iberian Peninsula, and with Quang Binh virus (isolated in Vietnam) as a close relative. No CTFV sequences were found integrated in their host's genome using a range of specific PCR primers designed to the prM/E, NS3, and NS5 region.

Species Composition and WNV Screening of Mosquitoes from Lagoons in a Wetland Area of the Algarve, Portugal.

The aim of this study was to evaluate mosquito abundance, species diversity, larval and adult population dynamics in seven lagoons integrated in the wetland coastal system of the Algarve, Portugal, in the summer of 2007, as well as the screening of these for West Nile virus (WNV). WNV has been isolated from mosquitoes in this region, in the summer of 2004, next to the putative area of infection of two linked human WN cases. Adult mosquitoes were collected with CDC traps baited with CO(2), and potential breeding sites were surveyed for immature stages. Morphological identification of 1,432 adult mosquitoes and 85 larvae revealed the presence of 10 species: Anopheles atroparvus, Anopheles algeriensis, Coquillettidia richiardii, Culex modestus, Culex pipiens, Culex theileri, Culex univittatus, Culiseta longiareolata, Aedes caspius, and Aedes detritus. Adult mosquito peak densities were recorded in July, contrasting with null larval breeding in the same month in the surveyed biotopes. Most abundant species were C. pipiens (52%), C. theileri (29%), and A. caspius (11%). Lagoon Salgados and Quinta das Salinas, exhibited the highest similarity of culicid fauna, despite being most distant from each other, Female mosquitoes (1,249 specimens) screened by RT-PCR, did not reveal WNV products. However, previous detection of WNV activity in this area, susceptible to re-introductions, demands for continued vigilance.

Mosquito surveys and West Nile virus screening in two different areas of southern Portugal, 2004-2007.

Longitudinal mosquito surveys were carried out in southern Portugal from 2004 to 2007, in a wetland area (Comporta, District of Setúbal) and around the perimeter of a dam irrigation plant that created the largest artificial lake in Europe, 250 km(2) (Alqueva, Districts of Evora and Beja). Our aim was to study the diversity, abundance, and seasonal dynamics of mosquitoes, comparing these two different areas, to screen mosquitoes for West Nile Virus (WNV), an arboviral agent already detected in Portugal, because these areas are populated with abundant avian fauna. Monthly collections of adult mosquitoes were carried out by Centers for Disease Control light-traps with CO(2) and by indoor resting collections. Mosquitoes were identified and screened for arboviruses by reverse transcriptase (RT)-polymerase chain reaction directed toward amplification of a 217-bp fragment of the NS5 gene. Mosquito peak densities were observed in July-August in Comporta and May-June, with a plateau in July-October, in Alqueva. However, densities were far higher in Comporta area (220,821 specimens) than in Alqueva area (9442 specimens), with a clear difference in species distribution, as in Comporta the predominant species was Culex theileri (85%), followed by Aedes caspius (6%), Anopheles atroparvus (4%), and Culex pipiens sensu latu (s.l.) (3%), whereas in Alqueva the predominant species was Cx. pipiens s.l. (56%), followed by An. atroparvus (18%), Cx. theileri (14%), and Culiseta longiareolata (9%). Female mosquitoes (8842 in 175 pools) of the species Ae. caspius, An. atroparvus, Culex mimeticus, Cx. pipiens Sensu latu (s.l.), Cx. theileri, and Culex univittatus were screened and found to be negative for WNV genomic RNA. Although there was no detection of WNV sequences in mosquitoes, vigilance should continue as the circulation of virus has been previously detected more than once in Portugal, in humans, animals, and mosquitoes, and in other surrounding Mediterranean countries.