Dottore di ricerca

ciclo: XXXIV

supervisore: GEN(Med) E.I. Florigio LISTA
co-supervisore: Prof.ssa Isabella SCREPANTI

Titolo della tesi: Detection and Genotyping of Yellow Fever Virus and Prevalence of West Nile and Usutu virus antibodies in human sera, Lazio, 2018, and Molise, 2020

Detection and Genotyping of Yellow Fever Virus (YFV) Yellow fever is a severe hemorrhagic illness with a high mortality rate. The causative agent, a mosquito-borne RNA virus, is endemic in tropical regions of Africa and South America. Despite the use of a safe and effective vaccine for over a half-century, an alarming resurgence of yellow fever outbreaks has been recorded in endemic areas in the last decades. Active surveillance, immunization programs and early detection of yellow fever outbreaks are essential tools to fight and minimize the spread of infections. This study aimed to develop a high sensitivity-specificity reverse-transcription PCR for the detection of all the yellow fever virus strains. Eighty-two YFV complete genomes were downloaded from GenBank. Sequences alignment was performed and several pairs of primer were designed and tested on a conserved region of the yellow fever virus genome. The vaccinial YFV 17D 204 strain was used as template in PCR set-up. To evaluate the sensitivity and specificity of the novel method, a panel of samples, provided by the EVD-LabNet ( during the External Quality Assurance (EQA) on molecular detection of YFV, were analyzed. Moreover, to further validate the specificity of the novel method, the genomes of other Flavivirus (TBEV, USUV, ZIKA, DENV1, DENV2, WNVL1, WNVL2, JEV) were analyzed. The new Real-Time PCR has shown a very high sensitivity and specificity. The employed primers were also evaluated in a classical endpoint PCR. The method did not show a significant loss of performance. Sequences alignment and phylogenetic analysis revealed that the amplified region carries nucleotide variants some of which are YF lineage-restricted. To test the feasibility of YFV lineage assignment by the analysis of this region, two representative samples of the EQA panel were amplified and sequenced. Amplicon analysis allowed to correctly assign the yellow fever virus lineage of the analyzed samples. This novel method might represent a further improvement in YFV molecular analysis. The method showed a high sensitivity (LOD 20 copies/reaction), and no cross-reactivity has been observed with other Flaviviruses. Moreover, the opportunity to choose between a Real-Time PCR or a classic endpoint PCR could better meet the needs of laboratories. Finally, sequencing analysis of the resulting amplicon allows identifying the yellow fever virus lineage. Prevalence of West Nile and Usutu virus antibodies in human sera West Nile virus (WNV) is an emerging and re-emerging zoonotic flavivirus first identified in and endemic to Africa. Since its isolation in Uganda in 1937, the West Nile virus (WNV) has been responsible for thousands cases of morbidity and mortality in birds, horses, and humans. In humans, the clinical presentation ranges from asymptomatic (approximately 80% of infections) to a self-limiting febrile illness (20% of infected individuals). About 1% of infected individuals develop neurological symptoms, which may fall in encephalitis/paralysis and death. In Italy, human cases of West Nile virus (WNV) infection have been recorded since 2008, and seasonal outbreaks have occurred almost annually. Usutu virus (USUV) is an emerging Flavivirus isolated in 1959 (Usutu River, Swaziland). Although WNV is much more widespread and plays a much larger role in human health, the two viruses are characterized by similar envelope antigens and clinical manifestations, and present overlapping features in terms of geographic range of transmission, host, and vector species. This study aimed to evaluate the seroprevalence of WNV and USUV, in the Italian regions Lazio and Molise. Five hundred human serum samples were collected between May 30 and October 31, 2018, from healthy blood donors (BDs) attending the University Hospital Polyclinic of Tor Vergata of Rome, Lazio region. Five hundred twenty-six human serum samples were collected between February 14 and September 29, 2020, from healthy blood donors attending the Molise Regional Health public company. Aliquots of five sera (50 µl each) were pooled and nucleic acids were extracted and reverse transcription (RT) reactions were performed. None of them were positive for flavivirus RNA. All the pooled sera from Lazio and Molise were screened by an in-house enzyme-linked immunosorbent assay (ELISA) for detecting WNV and USUTUV antibodies. The reactive sera were then tested using the Plaque Reduction Neutralization Test (PRNT) for WNV and USUV. WNV and USUV seroprevalence by PRNT was null in Molise, whereas WNV and USUTU seroprevalence in Lazio was 0.40% and null, respectively.

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