Seroprevalence of West Nile and dengue virus in the human population of the Bolivian Chaco.

To determine the seroprevalence of antibodies against dengue virus (DENV) and West Nile virus (WNV) in the human population of the Bolivian Chaco, we tested 256 inhabitants of two rural communities. The seroprevalence, confirmed by plaque reduction neutralization test, was 7.8% and 2.7% for DENV and WNV, respectively.

Seroprevalence survey of arboviruses in workers from Tuscany, Italy.

BACKGROUND: Arthropod-borne viruses (Arbovirus) play an important role among emerging and re-emerging infectious diseases and in the spreading of infections in new geographic areas. Although some arboviral infections may be asymptomatic or mild flu-like illnesses, many occur as severe forms of meningitis and meningoencephalitis. OBJECTIVES: To assess whether arboviral infections may be associated with occupational risk, in a population of agricultural and forestry workers potentially at high risk for arthropods bite and sting. METHODS: A seroprevalence survey for arboviruses belonging to the genera Flaviviruses (West Nile, Tick-borne encephalitis and Usutu viruses) and Phlebovirus (Toscana virus) was carried out in Grosseto province (Tuscany, Italy). One hundred and one serum samples of occupationally exposed workers and 100 serum samples of not exposed workers were analyzed using commercial and home-made serological assays. Serological data were obtained in 2012 and analyzed according to demographic characteristics, recollection of insect-bites, and time spent in outdoor activities. RESULTS: A total seropositivity of 10% (21/201) was observed for Toscana virus. No difference in seroprevalence for Toscana virus was observed among the exposed (10/101) versus the not exposed (11/100) workers. No seropositivity for West Nile, Usutu and Tick-borne encephalitis viruses was detected. CONCLUSIONS: Although circulation of Toscana virus is recognized in the study area, our results did not reveal a higher risk for workers exposed to arthropods bite and sting. Health surveillance programs remain useful to monitor the potential emergence of arboviruses.

Climate change, vector-borne diseases and working population.

INTRODUCTION: Risks associated with climate change are increasing worldwide and the global effects include altered weather and precipitation patterns, rising temperatures and others; human health can be affected directly and indirectly. This paper is an overview of literature regarding climate changes, their interaction with vector-borne diseases and impact on working population. MATERIALS AND METHODS: Articles regarding climate changes as drivers of vector-borne diseases and evidences of occupational cases have been picked up by public databank. Technical documents were also included in the study. RESULTS: Evidences regarding the impact of climate changes on vector-borne diseases in Europe, provided by the analysis of the literature, are presented. DISCUSSION: Climate-sensitive vector-borne diseases are likely to be emerging due to climate modifications, with impacts on public and occupational health. However, other environmental and anthropogenic drivers such as increasing travelling and trade, deforestation and reforestation, altered land use and urbanization can influence their spread. Further studies are necessary to better understand the phenomenon and implementation of adaptation strategies to protect human health should be accelerated and strengthened.

Phleboviruses detection in Phlebotomus perniciosus from a human leishmaniasis focus in South-West Madrid region, Spain.

BACKGROUND: Phlebotomus-borne (PhB-) viruses are distributed in large areas of the Old World and are widespread throughout the Mediterranean basin, where recent investigations have indicated that virus diversity is higher than initially suspected. Some of these viruses are causes of meningitis, encephalitis and febrile illnesses. In order to monitor the viral presence and the infection rate of PhB-viruses in a recently identified and well characterized human zoonotic leishmaniasis focus in southwestern Madrid, Spain, a sand fly collection was carried out. METHODS: Sand fly insects were collected in four stations using CDC light traps during 2012-2013 summer seasons. Screening for Phlebovirus presence both via isolation on Vero cells and via polymerase chain reaction (PCR), using degenerated primers targeting a portion of the L segment, was performed. The serological identity and phylogenetic relationships on the three genomic segments of the viral isolates were carried out. RESULTS: Six viral isolates belonging to different serological complexes of the genus Phlebovirus were obtained from fifty pools on a total of 963 P. perniciosus (202 females). Phylogenetic analysis and serological assays allowed the identification of two isolates of Toscana virus (TOSV) B genotype, three isolates strongly related to Italian Arbia virus (ARBV), and one isolate of a novel putative Phlebovirus related to the recently characterized Arrabida virus in South Portugal, tentatively named Arrabida-like virus. Positive male sand fly pools suggested that transovarial or venereal transmission could occur under natural conditions. CONCLUSIONS: Our findings highlighted the presence of different Phlebovirus species in the South-West area of the Madrid Autonomous Community where an outbreak of cutaneous and visceral human leishmaniasis has been recently described. The evidence of viral species never identified before in Spain, as ARBV and Arrabida-like virus, and TOSV B genotype focus stability was demonstrated. Environmental aspects such as climate change, growing urbanization, socio-economic development could have contributed to the genesis of this wide ecological niche of PhB-viruses and Leishmania spp. The potential role of vertebrates as reservoir for the phleboviruses identified and the possibility of Phleboviruses-Leishmania co-infection in the same sand fly should be assessed. Furthermore the PhB-viruses impact on human health should be implemented.

Experimental evaluation of sand fly collection and storage methods for the isolation and molecular detection of Phlebotomus-borne viruses.

BACKGROUND: Several viruses have been recently isolated from Mediterranean phlebotomine sand flies; some are known to cause human disease while some are new to science. To monitor the Phlebotomus-borne viruses spreading, field studies are in progress using different sand fly collection and storage methods. Two main sampling techniques consist of CDC light traps, an attraction method allowing collection of live insects in which the virus is presumed to be fairly preserved, and sticky traps, an interception method suitable to collect dead specimens in high numbers, with a risk for virus viability or integrity. Sand flies storage requires a "deep cold chain" or specimen preservation in ethanol. In the present study the influence of sand fly collection and storage methods on viral isolation and RNA detection performances was evaluated experimentally. METHODS: Specimens of laboratory-reared Phlebotomus perniciosus were artificially fed with blood containing Toscana virus (family Bunyaviridae, genus Phlebovirus). Various collection and storage conditions of blood-fed females were evaluated to mimic field procedures using single and pool samples. Isolation on VERO cell cultures, quantitative Real time-Retro-transcriptase (RT)-PCR and Nested-RT-PCR were performed according to techniques commonly used in surveillance studies. RESULTS: Live engorged sand flies stored immediately at -80 °C were the most suitable sample for phlebovirus identification by both virus isolation and RNA detection. The viral isolation rate remained very high (26/28) for single dead engorged females frozen after 1 day, while it was moderate (10/30) for specimens collected by sticky traps maintained up to 3 days at room temperature and then stored frozen without ethanol. Opposed to viral isolation, molecular RNA detection kept very high on dead sand flies collected by sticky traps when left at room temperature up to 6 days post blood meal and then stored frozen in presence (88/95) or absence (87/88) of ethanol. Data were confirmed using sand fly pools. CONCLUSIONS: While the collection and storage methods investigated had not much impact on the ability to detect viral RNA by molecular methods, they affected the capacity to recover viable viruses. Consequently, sand fly collection and handling procedures should be established in advance depending on the goal of the surveillance studies.

Experimental studies on comparison of the vector competence of four Italian Culex pipiens populations for West Nile virus.

BACKGROUND: West Nile virus (WNV) is a vector-borne disease responsible for causing epidemics in many areas of the world. The virus is maintained in nature by an enzootic bird-mosquito-bird cycle and occasionally transmitted to other hosts, such as equines and humans. Culex species, in particular the ubiquitous species Culex pipiens is thought to play a major vector role both in enzootic and epizootic maintenance and transmission of WNV. Introduced in Europe in recent years, since 2008 WNV has been stably circulating mainly in the Northeastern regions of Italy, although sporadic equine and/or human cases, as well as WNV infected Cx. pipiens pools, have been recorded in other Italian areas. The scope of our study was to evaluate the potential competence of some Italian populations of Cx. pipiens to transmit WNV and to assess their ability for vertical transmission of the virus. For this purpose four Italian populations, from different areas, were experimentally infected. METHODS: After the infectious blood meal, fed females were monitored for 32 days to determine the length of viral extrinsic incubation period. WNV titre of infected mosquitoes was evaluated both by quantitative Real Time PCR and viral titration by Plaque Forming Units/ml (PFU/mL) in VERO cells. Potential Infection, Dissemination, Transmission rates (IR, DR, TR) were assessed by detection of the virus in body, legs plus wings and saliva of the fed females, respectively. RESULTS: All tested populations were susceptible to the WNV infection. The viral presence in legs and wings demonstrated the ability of WNV to disseminate in the mosquitoes. Viral RNA was detected in the saliva of tested populations. No significant differences in TR values were observed among the four studied populations. The offspring of the Cx. pipiens infected females were WNV negative. CONCLUSIONS: Our study addressed an important issue in the knowledge on the complex WNV-vector relationships in Italy, indicating that all Italian Cx. pipiens populations tested exhibited vector competence for WNV. Further studies should be performed in order to better clarify the role of other factors (vector density, climatic conditions, reservoir presence etc.) in order to predict where and when WNV outbreaks could occur.

Bovine lactoferrin inhibits Toscana virus infection by binding to heparan sulphate.

Toscana virus is an emerging sandfly-borne bunyavirus in Mediterranean Europe responsible for neurological diseases in humans. It accounts for about 80% of paediatric meningitis cases during the summer. Despite the important impact of Toscana virus infection-associated disease on human health, currently approved vaccines or effective antiviral treatments are not available. In this research, we have analyzed the effect of bovine lactoferrin, a bi-globular iron-binding glycoprotein with potent antimicrobial and immunomodulatory activities, on Toscana virus infection in vitro. Our results showed that lactoferrin was capable of inhibiting Toscana virus replication in a dose-dependent manner. Results obtained when lactoferrin was added to the cells during different phases of viral infection showed that lactoferrin was able to prevent viral replication when added during the viral adsorption step or during the entire cycle of virus infection, demonstrating that its action takes place in an early phase of viral infection. In particular, our results demonstrated that the anti-Toscana virus action of lactoferrin took place on virus attachment to the cell membrane, mainly through a competition for common glycosaminoglycan receptors. These findings provide further insights on the antiviral activity of bovine lactoferrin.

Anti-tick-borne encephalitis (TBE) virus neutralizing antibodies dynamics in natural infections versus vaccination.

Tick-borne encephalitis (TBE) virus infection elicits a life-long lasting protection. However, little is known about the neutralizing antibodies titres following natural infection. In this study, subjects with past TBE disease (n = 62) were analysed for the presence and titre of anti-TBE neutralizing antibodies, and compared with a vaccinated cohort (n = 101). Neutralizing antibody titres were higher in individuals with past TBE and did not show an age-dependent decrease when compared with vaccinees.

Viral isolates of a novel putative phlebovirus in the Marche Region of Italy.

Thirty pools from 900 (540 females and 360 males) Phlebotomus perfiliewi sandflies collected during the summer of 2012 in the Fermo area (Marche Region, central Italy) were tested for the presence of Phleboviruses. A nested polymerase chain reaction was performed using degenerated primers amplifying a fragment of the polymerase gene (large segment) and a fragment of the nucleoprotein gene (small segment) of the genus Phlebovirus. One pool was positive for Toscana virus, as expected from results of studies in the area, and six pools were positive for a putative novel Phlebovirus. Virus isolation in Vero cells was performed. Minimum field infection rates/1,000 insects processed for the novel and Toscana viruses were 6.7 and 1.0, respectively. Phylogenetic analysis of the novel Phlebovirus, tentatively named Fermo virus, placed it in the Sandfly Fever Naples virus serocomplex.

Prevalence of Toscana sandfly fever virus antibodies in neurological patients and control subjects in Sicily.

Toscana sandfly fever virus (TOSV) is an arthropod-borne virus transmitted to humans by sandfly vectors. It has been associated with human cases of meningitis and meningo-encephalitis mainly occurring during the warm season. We performed a retrospective serological study to evaluate TOSV circulation in Palermo, Sicily, and to compare TOSV seroprevalence in patients with neurological symptoms and in a control group of patients without neurological symptoms. When sera from 155 patients with and without neurological symptoms were evaluated, the rate of overall TOSV IgG reactivity was 17.4%. Patients with neurological symptoms showed a higher percentage of TOSV IgG positivity than control patients (25% versus 10.8%). TOSV exposure was confirmed by virus neutralization tests which also detected a Naples virus (SFNV) infection. TOSV should be considered as an etiologic agent in the differential diagnosis of fever and meningo-encephalitis in Sicily.

[Toscana virus in the Portuguese population: serosurvey and clinical cases]. / Vírus Toscana na população Portuguesa: vigilância sero-epidemiológica e casos clínicos.

Toscana virus (Phlebovirus genus, Bunyaviridae family) is a neurotropic virus which circulates in the Mediterranean Basin. Although Portugal has been the second country where its presence was reported, the existence of this virus in our country has been referred only sporadically, and there is a lack of knowledge regarding the prevalence of antibodies in the population. Thus, the objective of this study was to analyse the prevalence of antibodies anti-Toscana virus in the human population in our country. Sero-epidemiological investigations were performed with indirect immunofluorescence assay (IFA) and enzyme linked immunosorbent assay (ELISA) tests. The study population consisted of a control population (blood donors, n=150), a population considered at risk (n=236) and a population of individuals with symptoms and laboratory diagnostic request for vector-borne viruses. The latter population was divided into two groups: those individuals with neurological symptoms (n=165) and those without neurological symptoms (n=373). We tested sera from a total of 924 individuals. The seroprevalence of IgG antibodies in the control population was 2%. In the population considered at risk, the prevalence was 3.4%. In the population with central nervous system disease, we detected a seroprevalence of 4.2%. For the same type of antibodies and in subjects without central nervous system disease, the prevalence was 1.3%. Five cases of recent infection (3%) were detected in the population with neurological signs. Those infections have been acquired in the districts of Faro, Coimbra, Aveiro and Lisbon. The associated clinical diagnoses were meningitis, meningoencephalitis and rash. The observed seroprevalences were, in general, lower than reported in other endemic countries. Only 5 of the 29 sera which gave positive results by IFA and ELISA were confirmed by plaque reduction neutralization tests with the Italian strain ISS.Phl.3. This can indicate the presence of more than one Toscana virus serotype circulating in Portugal and emphasizes the need for more research about this etiological agent in our country.

Humoral immunity in natural infection by tick-borne encephalitis virus.

Tick-borne encephalitis (TBE) virus is one of the most important flaviviruses associated with neurological disease in Europe. Cross-reactive antibodies elicited by different flaviviruses can make difficult the interpretation of ELISA and hemagglutination-inhibition (HI) tests for the diagnosis of TBE. Neutralization tests, which are more specific, are not in common use because they are difficult to perform and standardize. A plaque reduction neutralization test (PRNT), optimized previously in vaccinated children, was evaluated in sera from acute cases of TBE, collected for diagnostic purposes, and from healthy human population and wild ruminants, collected for serosurvey purposes. The PRNT results were compared with the results of ELISA and HI tests. In acute TBE disease, most sera were positive for IgM antibodies by ELISA and with high HI antibody titers; neutralizing antibodies were detected in 71.4% of patients, at a very low titer (1:10 NT(50)) in almost all cases. Seroprevalences of 8% and 6.5% for anti-TBE ELISA antibodies were found in healthy subjects and wild ruminants, respectively. Among anti-TBE positive healthy subjects, a very low 1:10 NT(50) titer was detected in 17.4% of cases, while NT(80) titers ranging from 1:10 to 1:80 were detected in 65.2% of cases. Among wild ruminants, 90.9% of ELISA and HI positive samples showed a positive, >/=1:10 NT(80) titer. In conclusion, neutralization assays can be useful for the diagnosis and serosurveys of TBE.

Occupational risk associated with Toscana virus infection in Tuscany, Italy.

BACKGROUND: Sandfly fever virus serotype Toscana is endemic in Mediterranean countries and is a widespread public health problem as it may be associated with acute neurological diseases such as aseptic meningitis. AIM: To assess whether Toscana virus infection is associated with occupational exposure. METHODS: During the summer of 2001, a total of 678 blood samples were taken from healthy subjects residing in Tuscany (349 agricultural and forestry workers and 329 control subjects living in the same areas). Information on age, type of job and lifestyle was collected in a questionnaire, and sera were analysed using enzyme-linked immunosorbent assay for Toscana virus-specific antibodies. RESULTS: Seropositivity to antibodies to Toscana virus was 30% in the control group and 23% in the agricultural and forestry workers (P<0.05). Questionnaire responses suggested that, although the agricultural and forestry workers had a potentially greater continuous exposure to sandfly fever virus (outdoor activities, rural residence and at-risk lifestyle), these risk factors did not add significant information about greater susceptibility to disease. CONCLUSION: Our findings indicate a need for better understanding of the best preventive measures to avoid the risk related to sandfly bites, especially for people who live or work in areas at risk.

Genetic variability of the M genome segment of clinical and environmental Toscana virus strains.

Twenty-seven strains of Toscana virus, collected over a period of 23 years and isolated from several localities and from different hosts (humans, arthropods and a bat), were investigated by sequencing of a portion of the M genomic segment comprising the G(N) glycoprotein coding region. Sequence data indicated that the divergence among isolates ranged from 0 to 5.7 % at the nucleotide level and from 0 to 3.4 % at the amino acid level. Phylogenetic analysis revealed four main clusters. A close correspondence between viral strains and area/year of isolation could not be demonstrated, whilst co-circulation of different viral strains in the same area and in the same time period was observed for both patients and environmental viral isolates. Alignment of the deduced amino acid sequences and evolutionary analysis indicated that most of the sites along the gene may be invariable because of purifying and/or neutral selection.

Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children.

Vaccination against tick-borne encephalitis (TBE) virus is the measure of choice for disease control in endemic areas, as no treatment is available. In Italy, the province of Belluno is one of the most active TBE virus infection foci. In this study sera were examined from vaccinated children living in areas around Belluno in order to monitor the immune response after anti-TBE vaccination. For the assessment of neutralizing antibodies, a plaque reduction neutralization test (PRNT) was optimized and the correlation between enzyme-linked immunosorbent assay (ELISA), hemaglutination inhibition (HI), and neutralizing antibodies titers was evaluated. All children had high serum levels of TBE IgG in ELISA test after the vaccination, in agreement with previous studies. HI and PRNT titers ranged between very low and high levels. A good correlation between HI and PRNT titers, and with IgG ELISA titers, was observed. PRNT is an useful assay for monitoring protective immunity after the completion of anti-TBE vaccination. This type-specific assay is an important tool for differential diagnosis in cases where the presence of cross-reactive antibodies due to other flavivirus infections or vaccinations cannot be excluded.

Unusual presentation of life-threatening Toscana virus meningoencephalitis.

This case report describes a brother and a sister with severe meningoencephalitis caused by Toscana virus (TOSv). The clinical presentation was characterized by stiff neck, deep coma, maculopapular rash, diffuse lymphadenopathy, hepatosplenomegaly, renal involvement, tendency to bleeding, and diffuse intravascular coagulation. The boy had epididymo-orchitis. Recovery with neurologic sequelae as hydrocephalus was observed. Microbiological diagnosis was obtained by serological tests and reverse transcriptase-polymerase chain reaction. Sequencing of polymerase chain reaction products from the S and M segments was carried out. TOSv may be a causative agent in severe meningoencephalitis.