APPROCCI MOLECOLARI E BIOINFORMATICI INNOVATIVI PER STUDI DI EPIDEMIOLOGIA MOLECOLARE DEL MORBILLO NELL'AMBITO DEL WHO EUROPEAN REGION MEASLES STRATEGIC PLAN 2010-2020

INTRODUCTION. Measles virus belongs to the morbillivirus genus of the family Paramyxoviridae. Infection with measles virus results in an extremely contagious exanthematic disease transmitted by air. It often causes severe complications and deaths, is preventable with vaccination and presents requirements for elimination. Italy is one of the 12 European countries where measles transmission is still endemic. The Global Measles and Rubella Strategic Plan 2012–2020 has set the goal of the elimination of endemic measles in the WHO European Region. To achieve this goal, high vaccination coverage must be obtained and maintained (>95%), and a sensitive and quality surveillance system must be ensured. Measles molecular surveillance is a key component to verify the endemic measles elimination, and a crucial tool both to establish any epidemiological link between cases occur in the same period and area, and to identify the importation sources. With the progress of the elimination program, the genetic diversity of circulating measles strains decreases. Continuous air travels and the ease world trade between countries facilitate imports into a given geographical area of viral variants belonging to the same genotype. In this context, traditional laboratory methods can not distinguish endemic transmission from import events of the same viral variant. In addition, as the vaccination program moves forward, an increasing proportion of measles cases occur in vaccinated individuals. New and advanced technologies must therefore allow us to broaden knowledge on measles in vaccinated people, and allow us to identify strains potentially capable of evading the immune response. AIM. The aim of the PhD project is to study and monitor in time the molecular epidemiology of measles in view of the elimination goal, through the develop and the use of innovative molecular and bioinformatic methodologies. Therefore, the research aims to combine the traditional epidemiological methods with the molecular and bioinformatic techniques of the new era. A further objective is to study measles confirmed cases in vaccinated people, in order to assess vaccination failure and to identify any escape mutant in the measles H gene. MATERIALS AND METHODS. From March 2017 to July 2019, biological specimens from patients with suspected measles were collected and analysed as part of the Measles and Rubella Integrated Surveillance System of the Lombardy Region (MoRoNET network). Viral RNA was extracted, and a Real Time RT-PCR was carried out for the measles genome identification. Retro-transcription was performed to all the measles positive samples, and a nested RT-PCR was conducted for the amplification of the N-450 region, in order to perform the genotyping. Samples of interest (N=50) have undergone two emi-nested PCR for the amplification of the H gene. Moreover, the amplification of the complete genome through specific couples of primers (which allow to obtain 10 overlapped fragments) has been conducted on the same samples. Amplicons were sequenced and a phylogenetic analysis was conducted on N-450 region, N-450/H region and on the whole genome, using the bioinformatic programs ClustalX2, BioEdit and MEGA7. Vaccinated measles cases were studied through analysis on serological data obtained from the regional referent laboratory database. In addition, the amino acid sequence of measles H protein (obtained from the conversion of the nucleotide sequences with BioEdit) was analysed in 7 vaccinated measles cases and 80 non-vaccinated measles cases, as control group. Personal, clinical and epidemiological data of measles cases analysed in this study were obtained from the Lombardy Region database of infectious diseases, MAINF. RESULTS. Overall, from March 2017 to July 2019, 885 sus