Anna Pierro

West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention

West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis antigenic group, has caused sporadic outbreaks in humans, horses and birds throughout many of the warmer regions of Europe for at least 20 years. Occasional cases of West Nile encephalitis have also been associated with infected blood transfusions and organ donations. Currently, WNV appears to be expanding its geographical range in Europe and causing increasing numbers of epidemics/outbreaks associated with human morbidity and mortality. This brief review reports on the current epidemic situation regarding WNV in Europe, highlighting the clinical, diagnostic and preventive measures available for controlling this apparently emerging human pathogen.

Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease.

Chikungunya virus in North-Eastern Italy: a seroprevalence survey.

After an outbreak of Chikungunya infection in Emilia-Romagna Region (North-eastern Italy), a survey was performed to estimate the seroprevalence of antibody to Chikungunya virus and the proportion of asymptomatic infections, to identify factors associated with infection, and evaluate the performance of the surveillance system. The method used was a survey on a random sample of residents of the village with the largest number of reported cases. The prevalence was 10.2% (33 of 325), being higher in older people and males, and lower when window screens and insect repellents were used. Only 18% of infected persons were fully asymptomatic, 85% of the 27 symptomatic confirmed cases satisfied the surveillance case definition, and 63% of the persons meeting the criteria for suspect case were identified by the active surveillance system. This study provides basic parameters for modeling the transmission potential of outbreaks and planning control measures for Chikungunya infection in temperate settings.

Mosquito, bird and human surveillance of West Nile and Usutu viruses in Emilia-Romagna Region (Italy) in 2010.

Background In 2008, after the first West Nile virus (WNV) detection in the Emilia-Romagna region, a surveillance system, including mosquito- and bird-based surveillance, was established to evaluate the virus presence. Surveillance was improved in following years by extending the monitoring to larger areas and increasing the numbers of mosquitoes and birds tested. Methodology/Principal Findings A network of mosquito traps, evenly distributed and regularly activated, was set up within the surveyed area. A total of 438,558 mosquitoes, grouped in 3,111 pools and 1,276 birds (1,130 actively sampled and 146 from passive surveillance), were tested by biomolecular analysis. The survey detected WNV in 3 Culex pipiens pools while Usutu virus (USUV) was found in 89 Cx. pipiens pools and in 2 Aedes albopictus pools. Two birds were WNV-positive and 12 were USUV-positive. Furthermore, 30 human cases of acute meningoencephalitis, possibly caused by WNV or USUV, were evaluated for both viruses and 1,053 blood bags were tested for WNV, without any positive result. Conclusions/Significance Despite not finding symptomatic human WNV infections during 2010, the persistence of the virus, probably due to overwintering, was confirmed through viral circulation in mosquitoes and birds, as well as for USUV. In 2010, circulation of the two viruses was lower and more delayed than in 2009, but this decrease was not explained by the relative abundance of Cx. pipiens mosquito, which was greater in 2010. The USUV detection in mosquito species confirms the role of Cx. pipiens as the main vector and the possible involvement of Ae. albopictus in the virus cycle. The effects of meteorological conditions on the presence of USUV-positive mosquito pools were considered finding an association with drought conditions and a wide temperature range. The output produced by the surveillance system demonstrated its usefulness and reliability in terms of planning public health policies.

Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses

Background Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. Methodology/Principal Findings An rRT-PCR assay targeting the 5′ untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1–4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. Conclusions/Significance This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

Detection of Usutu-Virus-Specific IgG in Blood Donors from Northern Italy

We developed a novel enzyme-linked immunosorbent assay to detect the specific IgG response to Usutu virus (USUV) in humans, by evaluating 359 blood donors who were living in northeastern Italy. Our results demonstrate the presence of an anti-USUV response in 4 subjects with no history of other flavivirus infection.

A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid

BACKGROUND Usutu virus (USUV), a flavivirus that belongs to the Japanese encephalitis virus (JEV) family, has recently emerged as a human pathogen, necessitating new diagnostic tools. OBJECTIVE The development and assessment of a real-time RT-PCR assay to detect USUV in human samples. STUDY DESIGN Based on USUV genomic sequences from GenBank, USUV-specific primers and probes that target the NS5 gene were designed. The sensitivity was evaluated in a 10-fold dilution series of plasmid that contained the amplicon and in a dilution series of a quantified human USUV isolate. The specificity was determined by testing various concentrations of related ArBo viruses, including flaviviruses and phleboviruses. Human RNAse P was also amplified in the assay. One hundred four human specimens from patients who suffered from viral meningoencephalitis were evaluated. RESULTS The real-time RT-PCR assay had a sensitivity of 50 genomic copies per reaction (corresponding to 2200 copies/ml) and 1 PFU/ml of USUV isolate. USUV isolates from Austria were identified with identical efficiency, and no ArBo viruses, other than USUV, were detected. USUV was also identified in 3 cerebrospinal fluid samples. All human samples were positive for RNAse P. CONCLUSIONS This PCR assay is recommended for all cases in which a rapid and clinically accurate diagnosis of human USUV infection is required.

False-Positive Transcription-Mediated Amplification Assay Detection of West Nile Virus in Blood from a Patient with Viremia Caused by an Usutu Virus Infection

ABSTRACT Detection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus.

Phylogenetic analysis of West Nile virus isolates, Italy, 2008-2009.

To determine the lineage of West Nile virus that caused outbreaks in Italy in 2008 and 2009, several West Nile virus strains were isolated from human specimens and sequenced. On the basis of phylogenetic analyses, the strains isolated constitute a distinct group within the western Mediterranean cluster.

Absence of Neuroinvasive Disease in a Liver Transplant Recipient Who Acquired West Nile Virus (WNV) Infection from the Organ Donor and Who Received WNV Antibodies Prophylactically

We describe the first case of West Nile virus (WNV) infection in Europe with transmission from donor to recipient following liver transplantation. The infection was detected in the recipient 3 days after transplantation, during the asymptomatic phase. We also report an innovative prophylactic strategy based on infusion of WNV hyperimmune plasma and gamma globulins that could be effective in preventing the appearance of a neuroinvasive disease.