Influenza Virus Genotypes Circulating In Central Greece During 2012-2014 And Vaccine Strain Match

摘要

During the period 2012-2014 in central Greece, influenza A(H1N1), A(H3N2) and B virus genotypes were detected and isolated from individuals with influenza illness. Influenza A(H1N1) and A (H3N2) viruses were the dominant virus type in circulation as it was detected in 9% and 12% of pharyngeal swabs examined by real-time RT-PCR during both seasons, respectively, while type B viruses were detected in only 3% of the samples examined. Influenza activity in central Greece, as was determined by number of reported influenza cases and influenza positive samples detected, was markedly increased in the 2013-14, as compared to the 2012-13 season. Influenza A(H1N1)pdm09 viruses were detected during the 2013-14 season along with A (H3N2) and type B viruses. All type A(H1N1), A (H3N2) and type B influenza isolates analyzed by cell culture reacted to a high titer (> 640) against antisera to vaccine-like viruses of the same period, indicating satisfactory influenza vaccine protection against circulating seasonal and pandemic influenza viral strains. Introduction Influenza type A and B viruses possess a segmented RNA genome which promotes the periodic emergence of influenza viral variants responsible for influenza epidemics and significant disease burden in the community. (1, 2). The surface antigens of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) are under selective pressure from the host immune system, constituting the basic mechanism of antigenic drift evident in influenza viruses (3, 4 )Influenza surveillance programs aim to reduce the impact of influenza by efficient vaccination programs. Global influenza activity is associated with increased morbidity and mortality indices due mainly to complications that result from infection, especially among individuals belonging to the high-risk groups (2, 5). Yearly vaccination with the trivalent influenza vaccine is recommended at the beginning of the influenza season in order to reduce the impact of the disease in the community (7).The present study reports laboratory data relating to influenza virus molecular epidemiology in central Greece and constitutes the first systematic survey of influenza viruses in central Greece. Materials and Methods Clinical specimenDuring the period 2012-14 a total of 865 pharyngeal swabs were collected from non-vaccinated individuals of all ages based on the gravity of symptoms associated with influenza-like illness (ILI), 3-4 days following the onset of symptoms, which included high fever (>38.50C), arthrralgia, myalgia, cough, headache, general malaise. Sampling took place during the influenza season in Greece from December to April, each year by health professionals at the University Hospital and by physicians of the sentinel network, Larissa, Thessaly, Greece. Sample storage and transport was accomplished in virus transport medium (24,9 gr bacto-tryptose, 5 gr gelatin, 0,5 gr streptomycin, 500,000 U penicilin and 50 mg fungizone) per liter. Storage was at +40C for up to 4 days. Long-term storage of the clinical samples was at -70oC.Real-time RT-PCR (rt-RT-PCR)Total RNA was extracted from 200ml of processed clinical sample with TRIzol (Gibco BRL, Life Technologies, NY, USA), according to manufacturer’s recommendations, in the presence of 20mg glycogen, molecular biology grade (Boehringer, Germany). Reactions in real-time RT-PCR were performed in a 7500 Applied Biosystem thermocycler, at a total reaction volume of 25μl (including 6.5μl RNA). Five different master mixes were prepared for each sample (influenza A, influenza B, H1, H3 and internal control). Forty reaction cycles were performed as follows: 450C for 10 sec, 950C for 10 min and 720C for 1min. Primers and probes used in the rt-RT-PCR reactions are shown in Table 1.Virus isolation and identificationFollowing screening by real-time RT-PCR, all positive samples were inoculated into sensitive Mardin-Darby Canine Kidney (MDCK) cell culture and incubated at 37oC for 7-10 days, as previously