Fátima Amaro

Detection of mosquito-only flaviviruses in Europe.

The genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy, Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.

Sympatric occurrence of Culex pipiens (Diptera, Culicidae) biotypes pipiens, molestus and their hybrids in Portugal, Western Europe: feeding patterns and habitat determinants

Culex (Culex) pipiens (Diptera: Culicidae) has two recognized biotypes, pipiens and molestus, which differ in physiology and behaviour; this difference may influence vectorial capacity for West Nile virus (WNV). Our goal was first to determine the presence of Cx. pipiens populations in 31 locations in Portugal and to subsequently analyse their host‐feeding preferences and habitat determinants. Molecular identification of Cx. pipiens forms and their hybrids was performed in 97 females; bloodmeal sources were identified in 59 engorged specimens. Overall, 61.9% of specimens were identified as Cx. pipiens f. pipiens, 20.6% as Cx. pipiens f. molestus, and 17.5% as hybrid forms. Culex pipiens f. pipiens fed preferentially on birds, and Cx. pipiens f. molestus on humans. Hybrid forms fed mostly on birds, but human bloodmeals were common. With reference to habitat, Cx. pipiens f. pipiens and hybrid forms were positively correlated with peri‐urban habitats. Our results confirm the sympatric presence of different Cx. pipiens biotypes in 14 of the 31 locations studied. Peri‐urban areas were a common habitat of all biotypes and may represent zones of hybridization. The feeding preferences and sympatric distribution of the Cx. pipiens biotypes observed in Portugal favour the epizootic circulation of WNV and the occurrence of disease outbreaks of WNV.

Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects.

Several flaviviruses are important pathogens for humans and animals (Dengue viruses, Japanese encephalitis virus, Yellow-fever virus, Tick-borne encephalitis virus, West Nile virus). In recent years, numerous novel and related flaviviruses without known pathogenic capacity have been isolated worldwide in the natural mosquito population. However, phylogenetic studies have shown that genomic sequences of these viruses diverge from other flaviviruses. Moreover, these viruses seem to be exclusive of insects (they do not seem to grow on vertebrate cell lines), and were already defined as mosquito-only flaviviruses or insect-specific flaviviruses. At least eleven of these viruses were isolated worldwide, and sequences ascribable to other eleven putative viruses were detected in several mosquito species. A large part of the cycle of these viruses is not well known, and their persistence in the environment is poorly understood. These viruses are detected in a wide variety of distinct mosquito species and also in sandflies and chironomids worldwide; a single virus, or the genetic material ascribable to a virus, was detected in several mosquito species in different countries, often in different continents. Furthermore, some of these viruses are carried by invasive mosquitoes, and do not seem to have a depressive action on their fitness. The global distribution and the continuous detection of new viruses in this group point out the likely underestimation of their number, and raise interesting issues about their possible interactions with the pathogenic flaviviruses, and their influence on the bionomics of arthropod hosts. Some enigmatic features, as their integration in the mosquito genome, the recognition of their genetic material in DNA forms in field-collected mosquitoes, or the detection of the same virus in both mosquitoes and sandflies, indicate that the cycle of these viruses has unknown characteristics that could be of use to reach a deeper understanding of the cycle of related pathogenic flaviviruses.

Borrelia hispanica in Ornithodoros erraticus, Portugal

Tick-borne relapsing fever (TBRF) is a spirochetal infection caused by the genus Borrelia. The disease is distributed in the Old and New World with many different species reported. In Europe, TBRF is caused by B. hispanica transmitted to man by Ornithodoros erraticus, a soft tick usually found in old premises to shelter pig herds. In Portugal, the first human case of TBRF was reported in 1942 but since the beginning of the 1960s, the disease has rarely been described and seems to either have disappeared or have been undiagnosed. Therefore, in 2009 a survey was undertaken to evaluate the presence of the tick in this type of premises and to evaluate its role as a reservoir of Borrelia. The work was carried out where the ticks were previously reported in the Alentejo and Algarve regions. Of 63 pigpens surveyed, O. erraticus was collected from 19% (n = 12) of these pigpens using CO(2) traps. To evaluate potential Borrelia hosts, both pigs (n = 25) and small rodents (n = 10) inhabiting these pigpens were surveyed for Borrelia presence, by whole blood PCR and/or tissue culture, respectively. All results for pigs and rodents were negative for the presence of B. hispanica. PCR assays targeting the 16S rRNA gene and intergenic spacer region of Borrelia were used. Sequence analysis of the positive samples confirmed the presence of B. hispanica in 2.2% (n = 5) of ticks from a pigpen in Alentejo. These results confirm natural, but albeit low, persistence of this agent in Portugal.

Molecular Characterization of a New Isolate of Borrelia lusitaniae Derived from Apodemus sylvaticus in Portugal

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

Mosquito Surveillance for Prevention and Control of Emerging Mosquito-Borne Diseases in Portugal — 2008-2014

Mosquito surveillance in Europe is essential for early detection of invasive species with public health importance and prevention and control of emerging pathogens. In Portugal, a vector surveillance national program—REVIVE (REde de VIgilância de VEctores)—has been operating since 2008 under the custody of Portuguese Ministry of Health. The REVIVE is responsible for the nationwide surveillance of hematophagous arthropods. Surveillance for West Nile virus (WNV) and other flaviviruses in adult mosquitoes is continuously performed. Adult mosquitoes—collected mainly with Centre for Disease Control light traps baited with CO2—and larvae were systematically collected from a wide range of habitats in 20 subregions (NUTS III). Around 500,000 mosquitoes were trapped in more than 3,000 trap nights and 3,500 positive larvae surveys, in which 24 species were recorded. The viral activity detected in mosquito populations in these years has been limited to insect specific flaviviruses (ISFs) non-pathogenic to humans. Rather than emergency response, REVIVE allows timely detection of changes in abundance and species diversity providing valuable knowledge to health authorities, which may take control measures of vector populations reducing its impact on public health. This work aims to present the REVIVE operation and to expose data regarding mosquito species composition and detected ISFs.

Detection of antibodies against Anaplasma phagocytophilum in Algerian mice (Mus spretus), Portugal.

The recent detection of Anaplasma phagocytophilum in Portugal stimulated further research on the agents enzootic cycle, which usually involves rodents. Thus a total 322 rodents belonging to five species, including 30 Apodemus sylvaticus (wood mouse), 65 Mus musculus (house mouse), 194 M. spretus (algerian mouse), 5 Rattus norvegicus (brown rat) and 28 R. rattus (black rat), were studied by indirect immunofluorescent assay (IFA) and/or polymerase chain reaction (PCR) for A. phagocytophilum exposure in four sampling areas of mainland and two areas of Madeira Island, Portugal. Overall, 3.6% (7/194) of M. spretus presented with IFA-positive results. Seropositive mice were detected in all three mainland sampling areas where this species was captured, with prevalence of 5.2% (5/96) and 5.0% (1/20) for the Ixodes-areas of Arrábida and Mafra, and 1.3% (1/78) for Mértola, a difference that was not statistically significant (p > 0.05). The majority of IFA-positive mice were detected in spring when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021), although the significance of this association was not evident after Bonferroni correction. Nevertheless, neither the seropositive M. spretus, nor additional samples of 10% seronegative rodents from mainland, and 16% of rodents collected in Madeira Island showed evidence of A. phagocytophilum active infections when spleen and/or lung samples were tested by PCR. Either the M. spretus results represents residual antibodies from past A. phagocytophilum infections, present infections with limited bacteremia, or cross-reactions with closely related agents deserves more investigation.

Ultrastructural and immunofluorescence studies of Zika infection

Zika virus is a flavivirus transmitted by mosquitoes from the Aedes genus, sexually, or by vertical transmission. It was discovered in 1947 in Africa [1] and it is thought to produce, in some cases, microcephaly in newborns and the Guilan–Barre syndrome in adults [2]. The otherwise low pathogenicity of the virus for humans precluded its detailed study. Recently, outbreaks in Pacific islands, with a virus related to Asian strains, were followed by a large outbreak in Brazil, which easily spread to all Latin-American countries and Cape Verde in África [1]. Ultrastructural information on Zika’s effects on cells is very limited [3]. Also an early histopathological study signaled alterations on other organs, which have not been further explored [4]. We studied the ultrastructure and immunocytochemistry of the zika infection of Vero E6 cells and of inoculated adult and newborn mice. Infected Vero E6 cells were collected for electron microscopy 2, 3, and 4 days after infection. Newborn mice were inoculated intracerebrally with 0.10 ml of Zika MR766 Vero E6-infected cell supernatant. Two adult mice were inoculated with a single intramuscular injection. Control mice were inoculated with 0.10 ml of uninfected Vero E6 cell supernatant. Immunofluorescence antigen detection assay was accomplished using sections of paraffin-embedded histologic samples. Specimens of the same blocks were used for H+E. About 10 μl of Zika human-positive sera were incubated for 30 min at 37°C, washed twice in phosphate-buffered saline (PBS), and then incubated for 30 min after the addition of fluorescein-conjugated rabbit immunoglobulin to human IgG (Dako, Santa Clara, California). Slides were then washed, dried, and covered after mounting with 10% glycerol in PBS. Slides were examined using an Olympus fluorescence microscope BH2 (10×40). Electron microscopy was performed with standard embedding and thin section methods described elsewhere [5]. Infected Vero cells showed abundant particles within smooth membranes close to the rough endoplasmic reticulum and Golgi apparatus (Figure 1(a). Numerous particle clusters were seen adhering to the cell membrane. The infected brain disclosed numerous virus particle clusters between cells (Figure 1(b)) and a few intracellular particles within vacuoles. The brain of the inoculated newborn mice showed hemorrhagic areas and the livers were atrophic. By immunofluorescence, virus antigen was detected in neurons, but also in the endothelium of blood vessels, both in the brain and in the liver (Figure 2). No virus particles were detected up to now in the liver.

Electron- microscopy characterization of cells infected with a new phlebovirus isolated in sandflies from South Portugal

This work was partially supported by an FCT project: New arboviruses isolated in Portugal. Risk assessment and public health application (PTDC/SAU-SAP/119199/2010). We wish to thank Ligia Chainho for the technical support.