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 5 205) or part of the gp41 N-terminal segment (n 5 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 5 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 9). 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. 313

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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.

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.

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. INJECTING DRUG USERS (IDUs) form one of the major risk groups for HIV-1 infection in Portugal. In the period of 1997-2001, 62% of the reported AIDS cases were related to this transmission class 1 but, following the general trends in most Western European countries, the incidence of HIV-1 infection among Portuguese IDUs has started to decrease.Phylogenetic analyses of HIV-1 isolates distributed worldwide showed, overall, 23 distinct HIV-1 subtypes (A-D, F-H, J, and K) and circulating recombinant forms (CRF01-14) within group M (HIV Sequence Database, http://hiv-web.lanl.gov/). These HIV-1 subtypes are geographically distributed nonrandomly, with subtype B being predominant in Europe and North America. We have reported multiple env HIV-1 subtypes circulating in Portugal, with a high prevalence of non-B subtypes, both in African and Portuguese individuals. 2 The aim of the present study was to investigate the genetic diversity of incident HIV-1 strains among IDUs living in Lisbon. From May 1999 through December 2000, after obtaining informed consent, blood samples were collected from 81 HIV-1 seropositive individuals attending three different drug addiction recovery centers and enrolled in a methadone therapy program. Seventy additional seropositive IDUs had been part of a previous broader surveillance study.2 Among these 151 IDUs, the mean age was 31 years, ranging from 18 to 49 years; the great majority (88.1%) were of Portuguese nationality, and 29.1% were women. Proviral DNA from uncultured peripheral blood mononuclear cells was extracted and we proceeded to the analysis of the env C2V3C3 (nucleotides 6982-7246, based on HXB2) or the gp41 (nucleotides 7878-8282 of HXB2) regions by het-

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.

Highlights► A new insect flavivirus (CTFV) was isolated from Culex theileri mosquitoes. ► CTFV does not replicate in Vero cells. ► Phylogenetic analyses place CTFV among Culex-associated flaviviruses. ► CTFV seems to be dispersed in the Iberian Peninsula. ► CTFV sequences were not found in theirs hosts’ genome.

Phylogenetic analysis showed that, for both time periods, the most prevalent subtypes 11 were 1a and 3a, found, altogether, in 64.9 and 71.6% of the individuals, respectively for 12 the first and the second sampling periods. However, genotype 4 viruses (subtypes 4a 13 and 4d), introduced later, as inferred by comparison of intra-subtype genetic distances, 14 were also relatively frequent even one decade ago (24.6%). This HCV subtype profile 15 for Portuguese intravenous drug users is in agreement with those described for other 16 southern European countries when in association with drug consumption. With the 17 exception of subtype 1b, phylogenetic trees did not show clustering of the Portuguese 18 sequences, but rather phylogenetic mixing of HCV sequences from different geographic 19 origins, as described previously in other Western countries and suggestive of a large 20 international transmission network. Consistent with the low recombination rates 21 reported for HCV, only one sample revealed discordant subtypes for the two regions 22 analyzed (4d in E1 and 4a in NS5B), representing a potential new recombinant that 23 deserves further analysis. 24 25