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.

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.

Circulation of West Nile virus in Portugal was demonstrated by serological surveys, and the virus was isolated in 1969 from Anopheles maculipennis s.l. A survey of the whole country was carried out (2001-2004) to assess the abundance of mosquito species and to screen them for arbovirus infection. A total of 770 collections yielded 32460 mosquitoes of 15 species. The regions with the highest abundance of mosquitoes were the coastal and estuarine districts of Santarém, Setúbal and Faro. Culex pipiens s.l., An. maculipennis s.l., Cx. theileri and Ochlerotatus caspius were the most abundant and widespread, accounting for 92% of mosquitoes caught. Anopheles maculipennis s.l. and Cx. pipiens s.l. were present all over the country. Culex theileri and. Oc. caspius were more abundant in the southern and coastal areas, respectively. A total of 2355 mosquito pools were screened by RT-PCR for flaviviruses, of which 987 pools were also screened for bunyaviruses. Culex pipiens s.l. and Cx. univittatus collected in 2004 in the southern district of Faro were found to be infected with West Nile virus. The density and proximity of these mosquitoes to the human populations may constitute a public health threat in the case of involvement in arbovirus transmission cycles.

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 subtypes 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.

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.