Zastupljenost i karakterizacija influenca A virusa izolovanih iz respiratornih uzoraka pacijenata sa teritorije Južnobačkog okruga

Abstract

U radu je ispitana zastupljenost influenca A virusa, njihova antigenska i genetička svojstva i osetljivost na antivirotik oseltamivir. Ispitivanje je sprovedeno u toku četiri uzastopne sezone, od 2010/2011 do 2013/2014 i obuhvatilo je 887 briseva nosa i grla pacijenata sa simptomima gripa, sa teritorije Južnobačkog okruga. Svi uzorci su testirani na prisustvo influenca A(H1N1)pdm09, A(H3N2), A(H1N1), A(H5) i A(H7) i influenca B virusa, real-time RT PCR testom. Pozitivni uzorci iz sezona 2012/2013 i 2013/2014, podvrgnuti su izolaciji na MDCK ćelijskim kulturama, a zatim je izvršeno ispitivanje sposobnosti dobijenih izolata da aglutiniraju eritrocite kokoške, čoveka i zamorca u reakciji virusne hemaglutinacije. Antigenska svojstva izolata sa hemaglutinacionim titrom ≥40, ispitana su reakcijom inhibicije hemaglutinacije. Genetičkoj karakterizaciji, sekvenciranjem hemaglutinin i neuraminidaza gena, podvrgnuti su reprezentativni izolati iz sezona 2012/2013 i 2013/2014. Za ispitivanje osetljivosti odabranih izolata virusa na oseltamivir upotrebljen je hemiluminiscentni test inhibicije aktivnosti neuraminidaze. Ukupno 46,3% (411/887) uzoraka bilo je influenca pozitivno, od čega je 73% (300/411) bilo influenca A pozitivno, a 27% (111/411)influenca B pozitivno (p<0,0001). Influenca A(H1N1)pdm09 podtip je detektovan u 48% (144/300), a A(H3N2) podtip u 52% (156/300) influenca Apozitivnih uzoraka. Najveći procenat influencaA pozitivnih zabeležen je u uzrastnoj grupi 5-14 godina (48,2%, 77/160) i kod pacijenata sa lakšim kliničkim manifestacijama gripa (43,7%, 153/350). Influenca A(H1N1)pdm09 podtip preovladavao je u uzrastnoj grupi 15-29 godina (66%, 31/47, p=0,0400) i 30-64 godina (55,9%,71/127, p=0,0215), kao i kod pacijenata sa teškom akutnom respiratornom bolešću (63,5%, 80/126, p<0,0001), fatalnih slučajeva (100%,9/9, p=0,0039) i pacijenata sa hroničnim bolestima i stanjima (68,8%, 84/122, p<0,0001). Influenca A(H3N2) podtip dominirao je kod dece uzrasta do 4 godine (72,2%,13/18, p=0,0381) i 5-14 godina (75,3%, 58/77, p<0,0001), kod pacijenata sa lakšim oblikom bolesti (69,3%,106/153, p<0,0001) i bez hroničnih bolesti ili stanja (66,3%, 118/178, p<0,0001). Najznačajniji predikcioni faktori komplikacija influence bili su: prisustvo hroničnih bolesti ili stanja i uzrast ≥15 godina. Prisustvo hroničnih bolesti ili stanja nosilo je 34 puta, a uzrast ≥15 godina 10 puta veći rizik od nastanka teških oblika bolesti. Izolacija influenca virusa na MDCK ćelijskim kulturama, bila je uspešna u 34,3% (70/204) slučajeva, pri čemu je u grupi uzoraka sa real-time RT-PCR Ct vrednostima <30 ona iznosila 80,5% (62/77), kod uzoraka sa Ct vrednostima 30-34 svega 8,7% (8/92), a izolacija iz uzoraka sa Ct vrednostima >34 nije bila moguća. U reakciji hemaglutinacije, najbolji rezultati su postignuti sa eritrocitima zamorca, koje je u titru ≥40 aglutiniralo 56% (14/25) A(H1N1)pdm09 virusa i 62,5% (15/24) A(H3N2) virusa. Sa humanim eritrocitima dobar titar dalo je 16% (4/25) influenca A(H1N1)pdm09 i 8,3% (2/24) A(H3N2) virusa, a sa kokošijim eritrocitima 8% (2/25) A(H1N1)pdm09 virusa i nijedan virus A(H3N2) podtipa. Rezultati antigenske karakterizacije pokazali su da je svih 23 influenca virusa A(H1N1)pdm09 podtipa, iz sezona 2012/2013 i 2013/2014, antigenski bilo slično referentnom, vakcinalnom virusu A/California/7/2009. Nasuprot tome, samo 1 od 7 ispitanih A(H3N2) virusa iz sezone 2012/2013, antigenski je bio sličan vakcinalnom virusu A/Victoria/361/2011, a samo 2 od 20 iz sezone 2013/2014 antigenski je bilo slično vakcinalnom A/Texas /50/2012 virusu. Filogenetska analiza hemaglutinin gena influenca A(H1N1)pdm09 virusa iz sezone 2012/2013, pokazala je da su u našoj sredini, bili prisutni virusi iz dve različite genogrupe, 6C i 7, dok su naredne sezone svi analizirani virusi pripadali genogrupi 6B. Virusi iz naše sredine bili su filogenetski srodni A(H1N1)pdm09 virusima iz drugih evropskih zemalja. Svi ispitani A(H3N2) virusi iz sezone 2012/2013 i2013/2014, pripadali su genetičkoj grupi 3C.3.Filogenetski su bili srodni sa virusima iz drugih gografskih regiona Evrope. Svih 20 izolata influenca A(H1N1)pdm09 podtipa i 23 A(H3N2) podtipa pokazali su normalnu inhibiciju aktivnosti neuraminidaze pod dejstvom oseltamivira.ekvenciranje neuraminidaza gena jednog A(H3N2) virusa, koji je imao 8 puta redukovanu inhibiciju aktivnosti neuraminidaze oseltamivirom, ukazalo jena prisustvo retke mutacije Q391H, povezane sa rezistencijom na inhibitore neuraminidaze. Rezultati ovog rada ukazali su na značaj influenca A virusa kao etioloških uzročnika akutnih respiratornih obolenja u našoj sredini, naročito za osobe sa hroničnim bolestima koje su pod povećanim rizikom od razvoja teških oblika gripa. U ovom istraživanju stečena su i saznanja koja imaju praktičnu primenu u postupku antigenske karakterizacije influenca A virusa, koja je jedna od ključnih faza u procesu pripreme vakcine protiv gripa. Značajna antigenska razlika A(H3N2) virusa koji su cirkulisali u sezonama 2012/2013 i 2013/2014 u odnosu na viruse koji su bili u sastavu vakcina u datim sezonama, ukazala je na neophodnost unapređenja proizvodnje vakcine protiv gripa. Dobijeni su i prvipodaci orezistenciji na antivirotik oseltamivir, kao i o filogenetskim odnosima i genetičkim grupama virusa koji su cirkulisali u našoj sredini.

In this study we investigated the representation, antigenic and genetic properties, and sensitivity to antiviral drug oseltamivir of influenza A viruses. The study was conducted during 4 consecutiveseasons 2010/2011 - 2013/2014, and included 887 nasal and throat swabs taken from patients with influenza-like symptoms from South Backa district. All samples were tested for influenza A(H1N1)pdm09, A(H3N2), A(H1N1), A(H5), A(H7) and influenza B viruses, by real-time RT-PCR. Isolation on MDCK cell culture was performed with positive samples from seasons 2012/2013 and 2013/2014, and virus isolates were tested for ability to agglutinate guinea pig, chicken and human red blood cells in reaction of virus hemagglutination. Antigenic properties of isolates with hemagglutination titre ≥40, were investigated using reaction of hemagglutination inhibition. Genetic characterization was performed by sequencing of neuraminidase and hemagglutination genes of representative isolates from seasons 2012/2013 and 2013/2014. Testing for sensitivity to oseltamivir was done with chemiluminescent neuraminidase inhibition assay. Total of 46,3% (411/887) of samples were influenza positive, out of which 73% (300/411) were influenza A positive and 27% (111/4111, p<0,0001) were influenza B positive. Influenza A(H1N1)pdm09 subtype was detected in 48% (144/300), and A(H3N2) subtype in 52% (156/300) of influenza A positive samples. The highest proportion of influenza A positive samples wasfound in age group 5-14 years (48,2%, 77/160) and among patients with uncomplicated influenza (43,7%, 153/350). Influenza A(H1N1)pdm09 subtype predominated in age group 15-29 years (66%, 31/47, p=0,0400) and 30-64 years (55,9%,71/127, p=0,0215), in patients with severe acute respiratory illness (63,5%, 80/126, p<0,0001), in fatal cases (100%, 9/9, p=0,0039), and among patients with underlying chronic diseases and conditions (68,8%,84/122, p<0,0001). Influenza A(H3N2) subtype predominated in age group ≤4 years (72,2%, 13/18, p=0,0381) and 5-14 years (75,3%,58/77, p<0,0001), in patients with mild form of influenza (69,3%,106/153, p<0,0001), and in group of patients without chronic diseases and conditions (66,3%,60/478, p<0,0001). The most significant risk factors for severe influenza were: the presence of underlying diseases and conditions and age ≥15 years. Patients with chronic illnesses and conditions had 34 times higher and patients ≥15 years of age 10 times higher risk from severe influenza. Isolation rate of influenza A viruses in MDCK cell cultures was 34,3% (70/204). For samples with real time RT-PCR Ct values <30 isolation rate was 80,5% (62/77), for samples with Ct values 30-34 it was 8,7% (8/92), while isolation of viruses from samples with Ct values >34 was not successful. In the reaction of virus hemagglutination, the best results were achieved with guinea pig red blood cells which agglutinated in titre ≥40, 56% (14/25) of influenza A(H1N1)pdm09 viruses and 62,5% (15/24) of A(H3N2) viruses. With human erythrocytes, good titre gave 16% (4/25) of influenza A(H1N1)pdm09 and 8,3% (2/24)of A(H3N2) viruses and with chicken erythrocytes 8% (2/25) A(H1N1)pdm09 viruses and none of the A(H3N2) viruses. Results of the antigenic characterization of 23 influenza A(H1N1)pdm09 viruses, showed that they were antigenically similarto referent, vaccine virus A/California/7/2009. On the contrary, only 1 out of 7 influenza A(H3N2) viruses from season 2012/2013,was antigenically similar to A/Victoria/361/2011 vaccine virus, and only 2 out of 20 from season 2013/2014 were antigenically similar to A/Texas/50/2012 vaccine virus. Filogenetic analysis of hemagglutinin genes indicated co-circulation of 2 distinct genetic groups, 6C and 7, of A(H1N1)pdm09 viruses during the season 2012/2013, while during the season 2013/2014 all tested viruses were from genetic group 6B. Influenza A(H1N1)pdm09 viruses from our region, were closely related to viruses from other European countries. All influenza A(H3N2) viruses from season 2012/2013 and 2013/2014 belonged to genetic clade 3C.3 and were closely related to viruses from different European countries. Total of 20 A(H1N1)pdm09 isolates and 23 A(H3N2) isolates were tested for sensitivity to oseltamivir, and all of them showed normal inhibition of neuraminidase activity with oseltamivir. Sequencing of neuraminidase gene of one A(H3N2) virus with 8-fold reduced inhibition by oseltamivir, revealed rare mutation Q391H associated with antiviral resistance. Results of this study indicate the significance of influenza A viruses as etiological factors of acute respiratory diseases in our area, especially for persons with chronic medical conditions who are at higher risk for severe influenza. Data gathered during the process of virus isolation and investigation of hemagglutination abilities of isolated viruses, have practical application in antigenic testing of influenza A viruses which is one of the key points of process of anti-flu vaccine production. Significant antigenic difference between influenza A(H3N2) viruses from seasons 2012/2013 and 2013/2014 and vaccine viruses, emphasis the importance of vaccine production improvement. During this study, the first data about antiviral resistance, filogenetic relationships and genetic groups of influenza viruses from our region, were obtained.