Prevalence of Usutu and West Nile virus antibodies in human sera, Modena, Italy, 2012.

A collection of 3069 human sera collected in the area of the municipality of Modena, Emilia Romagna, Italy, was retrospectively investigated for specific antibodies against Usutu (USUV) and West Nile viruses (WNV). All the samples resulting positive using a preliminary screening test were analyzed with the plaque reduction neutralization test. Overall, 24 sera were confirmed as positive for USUV (0.78%) and 13 for WNV (0.42%). The results suggest that in 2012, USUV was circulating more than WNV in North-eastern Italy.

Molecular evidence of Plasmodium vivax infection in Duffy negative symptomatic individuals from Dschang, West Cameroon.

BACKGROUND: Plasmodium vivax infection is known to be rare in West/Central Africa, the most accepted explanation being the lack of expression of erythroid Duffy antigen in the local human populations. Duffy negativity prevents the parasite to exploit the entry mechanism on the red blood cell surface. However, there are a growing number of reported vivax infections in Duffy-negative individuals. Data on P. vivax circulation in Cameroon are limited. The aim of the study was to evaluate the P. vivax presence, and its association with the Duffy genotype in West Cameroon. RESULTS: Overall, 484 blood samples were collected consecutively from febrile outpatients attending the Dschang's Hospital (West Cameroon) during a 3-months period. Plasmodium vivax infection was detected by PCR in 5.6% (n = 27/484) of the cases, representing 38.6% (n = 27/70) of all Plasmodium infections detected. All P. vivax infected individuals showed a Duffy-negative genotype, and the frequency of Duffy-positive individuals in the whole tested population was 1.7%. CONCLUSIONS: The results of this study confirm the circulation of P. vivax in Cameroon, as well as that the lack of expression of Duffy-antigen does not confer full protection against vivax malaria acquisition.

Co-circulation of Dengue and Chikungunya Viruses, Al Hudaydah, Yemen, 2012.

We investigated 400 cases of dengue-like illness in persons hospitalized during an outbreak in Al Hudaydah, Yemen, in 2012. Overall, 116 dengue and 49 chikungunya cases were diagnosed. Dengue virus type 2 was the predominant serotype. The co-circulation of these viruses indicates that mosquitoborne infections represent a public health threat in Yemen.

Durability of neutralizing antibodies against yellow fever virus after vaccination in healthy adults.

In accordance with the World Health Organization, one dose of yellow fever vaccine may guarantee protection lifelong in healthy adults. However, relatively little information is still available from ad hoc studies. We evaluated the persistence of neutralizing antibodies, which are considered to be an immune correlate of protection, in a large number of military personnel vaccinated up to 47 years before. Overall, 322 individuals were studied. The median time from vaccination to blood collection for neutralizing antibody evaluation was 9 years, ranging from <1 to 47 years. Of the 322 participants, 319 had neutralizing antibodies (99.1 %). The highest median PRNT50 value was observed in those vaccinated ≤1 year before (median PRNT50 = 320). In conclusion, our study confirms on a larger scale that, in healthy adults, neutralizing antibodies may persist as long as 47 years after a single yellow fever vaccines dose.

West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals.

BACKGROUND: West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals. RESULTS: We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed -1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. CONCLUSIONS: N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

Evidence of a humoral response to a novel protein WARF4 embedded in the West Nile virus NS4B gene encoded by an alternative open reading frame.

West Nile virus (WNV) is a flavivirus that is maintained in a bird-mosquito transmission cycle. Humans, horses and other non-avian vertebrates are usually incidental hosts. However, WNV is a neurotropic virus, which requires an efficient humoral response for the control of a neuroinvasive infection. The WNV genome encodes three structural (capsid, premembrane/membrane and envelope) and seven non-structural proteins. Bioinformatic analysis performed on the WNV genomes detected a conserved alternative open reading frame restricted to the lineage I virus. To quickly verify the existence of this putative protein, entitled West Nile Alternative Reading Frame 4 (WARF4), we produced a prokaryotic recombinant source of WARF4 and verified its immunogenicity in vivo by analyzing 43 horse serum samples, of which 15 were positive for antibodies to WNV premembrane and envelope (prM-E) proteins. Specific antibodies to WARF4 were significantly detected in 5 out of the 15 serum samples testing positive for antibodies to prM-E WNV proteins. Our findings provide evidence of a significant antibody response to the WARF4 protein in the serum of the horse testing positive for antibodies to prM-E proteins, thus indicating that this antigen might be a potential tool for further characterization of the immune response of WNV infections in humans as well.

A label-free impedimetric aptasensor for the detection of Bacillus anthracis spore simulant.

Herein, we report an impedimetric DNA-based aptamer sensor for a single-step detection of B. anthracis spore simulant (B. cereus spore). Specifically, we designed a miniaturized label-free aptasensor for B. cereus spores based on a gold screen-printed electrode functionalized with B. cereus spores-binding aptamer (BAS-6R). Several parameters were optimized to fabricate the aptasensor such as the concentration of DNA aptamer solution (0.5 µM), the time (48 h), the temperature (4 °C), and the pH (7.5) for aptamer immobilization on the working electrode surface. Once the aptasensor was developed, it was tested against B. cereus spores 14579 evaluating the effect of incubation time and MgCl2 concentration. Under the optimized conditions (incubation time equal to 3 h and absence of MgCl2), B. cereus spores 14579 were detected with a linear range between 104 CFU/ml and 5 × 106 CFU/ml and a detection limit of 3 × 103 CFU/ml. Furthermore, the study of selectivity toward B. cereus 11778, B. subtilis, Legionella pneumophila, and Salmonella Typhimurium has demonstrated the capability of this sensor to detect B. cereus spores, proving the suitability of the DNA-based sensing element combined with a portable instrument for a label-free measurement on site of B. anthracis spore simulant.

Rapid molecular detection and genotyping of West Nile Virus lineages 1 and 2 by real time PCR and melting curve analysis.

Following its spread in the USA, West Nile Virus (WNV) has reemerged in the Mediterranean basin with a renewed pathogenicity. The introduction of WNV lineage 2 in Europe and its co-circulation with lineage 1 has resulted in a continuously changing epidemiological scenario, highlighting the importance of differential detection of the two lineages. The paper describes a new real-time PCR method for the detection and genotyping of the two main lineages of WNV. The method requires a single pair of primers and probes and is based on the analysis of highly conserved consensus sequences detected in the 5' terminus of the viral genome.

Phylogeny of Dengue and Chikungunya viruses in Al Hudayda governorate, Yemen.

Yemen, which is located in the southwestern end of the Arabian Peninsula, is one of countries most affected by recurrent epidemics caused by emerging vector-borne viruses. Dengue virus (DENV) outbreaks have been reported with increasing frequency in several governorates since the year 2000, and the Chikungunya virus (CHIKV) has been also responsible of large outbreaks and it is now a major public health problem in Yemen. We report the results of the phylogenetic analysis of DENV-2 and CHIKV isolates (NS1 and E1 genes, respectively) detected in an outbreak occurred in Al-Hudayda in 2012. Estimates of the introduction date of CHIKV and DENV-2, and the phylogeographic analysis of DENV-2 are also presented. Phylogenetic analysis showed that the Yemen isolates of DENV belonged to the lineage 2 Cosmopolitan subtype, whereas CHIKV isolates from Yemen belonged to the ECSA genotype. All the CHIKV isolates from Yemen were statistically supported and dated back to the year 2010 (95% HPD: 2009-2011); these sequences showed an alanine in the aminoacid position 226 of the E1 protein. Phylogeographic analysis of DENV-2 virus showed that cluster 1, which included Yemen isolates, dated back to 2003 Burkina Faso strains (95% HPD 1999-2007). The Yemen, cluster dated back to 2011 (95% HPD 2009-2012). Our study sheds light on the global spatiotemporal dynamics of DENV-2 and CHIKV in Yemen. This study reinforces both the need to monitor the spread of CHIKV and DENV, and to apply significant measures for vector control.