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BackgroundSince 1985, two antigenically distinct lineages of influenza B viruses (Victoria-like and Yamagata-like) have circulated globally. Trivalent seasonal influenza vaccines contain two circulating influenza A strains but a single B strain and thus provide limited immunity against circulating B strains of the lineage not included in the vaccine. In this study, we describe the characteristics of influenza B viruses that caused respiratory illness in the population in Italy over 13 consecutive seasons of virological surveillance, and the match between the predominant influenza B lineage and the vaccine B lineage, in each season.MethodsFrom 2004 to 2017, 26,886 laboratory-confirmed influenza cases were registered in Italy, of which 18.7% were type B. Among them, the lineage of 2465 strains (49%) was retrieved or characterized in this study by a real-time RT-PCR assay and/or sequencing of the hemagglutinin (HA) gene.ResultsCo-circulation of both B lineages was observed each season, although in different proportions every year. Overall, viruses of B/Victoria and B/Yamagata lineages caused 53.3 and 46.7% of influenza B infections, respectively. A higher proportion of infections with both lineages was detected in children, and there was a declining frequency of B/Victoria detections with age. A mismatch between the vaccine and the predominant influenza B lineage occurred in eight out of thirteen influenza seasons under study. Considering the seasons when B accounted for > 20% of all laboratory-confirmed influenza cases, a mismatch was observed in four out of six seasons. Phylogenetic analysis of the HA1 domain confirmed the co-circulation of both lineages and revealed a mixed circulation of distinct evolutionary viral variants, with different levels of match to the vaccine strains.ConclusionsThis study contributes to the understanding of the circulation of influenza B viruses in Italy. We found a continuous co-circulation of both B lineages in the period 2004–2017, and determined that children were particularly vulnerable to Victoria-lineage influenza B virus infections. An influenza B lineage mismatch with the trivalent vaccine occurred in about two-thirds of cases.

The objective of this study, conducted within the I-MOVE project, was to estimate pandemic VE against laboratory confirmed cases in two Italian regions using the screening method. We calculated vaccine coverage using the number of first doses of pandemic vaccine administered and demographic data. A case was defined as a patient who was swabbed from week 44 of 2009 to week 3 of 2010 and who tested positive for A/H1N1v using RT-PCR. A case was considered vaccinated against influenza A/H1N1v if she/he had received one dose of the vaccine more than 14 days before swabbing. We used Farrington's method to build the confidence intervals. We included in the analysis 755 confirmed A/H1N1v cases. The median age of cases was 24 years (range 0–89). One case (0.1%) had received one dose of vaccine more than 14 days after swabbing. The overall crude estimated VE was 92.4% (95% CI: 46.3–98.9). It suggests that the pandemic vaccine offered good protection against medically attended laboratory-confirmed A/H1N1v.

Haemagglutinin sequences of pandemic influenza A(H1N1) viruses circulating in Italy were examined, focusing on amino acid changes at position 222 because of its suggested pathogenic relevance. Among 169 patients, the D222G substitution was detected in three of 52 (5.8%) severe cases and in one of 117 (0.9%) mild cases, whereas the D222E mutation was more frequent and evenly distributed in mild (31.6%) and severe cases (38.4%). A cluster of D222E viruses among school children confirms reported human-to-human transmission of viruses mutated at amino acid position 222.

WHO European Region Influenza Network: P.Conde, I. Costa, P. Crostovão, R. Guiomar, B. Nunes, P.Pechirra, A. Rodrigues (Portugal) BACKGROUND: Countries in the World Health Organization (WHO) European Region are reporting more severe influenza activity in the 2015-2016 season compared to previous seasons. OBJECTIVES: To conduct a rapid risk assessment to provide interim information on the severity of the current influenza season METHODS: Using the WHO manual for rapid risk assessment of acute public health events and surveillance data available from Flu News Europe, an assessment of the current influenza season from 28 September 2015 (week 40/2015) up to 31 January 2016 (week 04/2016) was made compared with the 4 previous seasons. RESULTS: The current influenza season started around week 51/2015 with higher influenza activity reported in eastern Europe compared to Western Europe. There is a strong predominance of influenza A(H1N1)pdm09 compared to previous seasons, but the virus is antigenically similar to the strain included in the seasonal influenza vaccine. Compared to the 2014/2015 season, there was a rapid increase in the number of severe cases in eastern European countries with the majority of such cases occurring among adults aged

We describe a fatal case of myopericarditis presenting with cardiac tamponade in a previously healthy 11-year-old child. Pandemic H1N1 2009 influenza A virus sequences were identified in throat and myocardial tissues and pericardial fluid, suggesting damage of myocardial cells directly caused by the virus.

Background: infectious low pathogenic avian influenza viruses (LPaIVs) have been recently detected on feathers of wild ducks. Laboratory trial results suggested that the preen oil gland secretion, covering waterbirds’ feathers, may attract and concentrate virus particles from aIV-contaminated waters to birds’ bodies. We evaluated whether ducks can become infected by the ingestion of preen oil-associated viral particles, experimentally smeared on their plumage. In addition, we compared virologic and serologic results obtained from mallards whose feathers were experimentally infected, with those from wild mallards naturally carrying aIVs on feathers.Methods: we experimentally coated 7 mallards (anas plathyrynchos) using preen oil mixed with a LPaIV (h10n7 subtype), and housed them for 45 days with a control, uncoated duck. cloacal, oropharyngeal and feather swabs were collected from all birds and examined for aIV molecular detection and isolation. Blood samples were also taken to detect influenza specific antibodies. In addition, sera from 10 wild mallards, carrying on feathers infectious LPaIV h10n7, were examined.Results: virologic and serologic results indicated that through self- and allopreening all the birds experimentally coated with the preen oil/aIV mix and the control duck ingested viruses covering feathers and became infected. Virus isolation from feathers was up to 32 days post-coating treatment. one out of 8 wild mallards showing antibodies against type a influenza virus was seropositive for h10 subtype too.Conclusions: our experimental and field results show evidences suggesting that uninfected birds carrying viruses on their feathers, including immune ones, might play an active role in spreading aIV infection in nature. For this reason, routine aIV surveillance programs, aimed at detecting intestinal and/or respiratory viruses, should include the collection of samples, such as feather swabs, enabling the detection of viruses sticky to preened birds’ bodies....

Il maiale domestico (Sus scrofa domestica), condividendo con gli uccelli e i mammiferi i recettori cellulari per i virus influenzali, svolge un ruolo epidemiologico chiave nell’ecologia del virus dell’influenza di tipo A ed in particolare nell’emergenza di nuovi ceppi, come confermato dall’origine suina del virus pandemico H1N1 del 2009. Il cinghiale (Sus scrofa) è il progenitore selvatico del maiale domestico, con cui condivide sia una stretta affinità genetica sia la suscettibilità verso numerosi agenti patogeni, tra cui i virus influenzali. La sottospecie nominale (Sus scrofa scrofa) è la più comune e diffusa in Eurasia, dove può arrecare gravi danni economici e ambientali. Se da un lato l’epidemiologia delle infezioni da virus influenzali di tipo A ed il relativo impatto economico negli allevamenti suini sono noti, vi sono ancora scarse informazioni sulla dinamica di circolazione del virus nelle popolazioni di cinghiali a vita libera. Nel presente lavoro è stata condotta un’indagine sierologica per valutare l’esposizione a virus influenzali di tipo A di una popolazione di cinghiali a vita libera distribuiti in un'area protetta della Provincia di Bologna. Metodi. Durante i periodi maggio 2002-luglio 2003 e aprile 2010-aprile 2011, sono stati raccolti 741 campioni di sangue prelevati da cinghiali catturati o abbattuti selettivamente nel Parco Regionale dei Gessi Bolognesi e Calanchi dell’Abbadessa. In questa area protetta di 4.844 ettari situata nella fascia collinare preappenninica bolognese, dal maggio 2002 è applicato un modello di gestione demografica densità-dipendente basato sulla selezione ed il mantenimento di una popolazione a densità nota strutturata in base ad una piramide di popolazione sesso-età dipendente. In funzione dell’eruzione dentaria, gli animali campionati sono stati suddivisi in tre classi di età: 1a classe, 14 mesi. Per differenziare le positività sierologiche dovute ad immunità passiva materna (IPM) da quelle indotte da una risposta sierologica attiva (RSA) post-infezione, la 1a classe è stata ulteriormente suddivisa nelle sottoclassi 1a-IPM e 1a-RSA che comprendono rispettivamente i cinghiali di età

Wild aquatic birds are reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic ones. High viral loads in feces of infected birds allow fecal-oral transmission. However, this route does not fully account for the efficiency of avian influenza virus (AIV) spread since dilution of infectious feces in water progressively decreases the chances of virus/host interaction. We investigated whether preen oil gland secretion, by which all aquatic birds make their feathers waterproof, could support a natural concentration mechanism of AIVs from water to birds’ bodies, thus favouring virus spread and persistence in the aquatic environment. First, we detected consistently both viral genome and infectious AIVs on swabs taken by rubbing preened feathers of 345 wild mallards and examined by reverse transcription (RT)-PCR and virus isolation (VI) assays. Second, in two laboratory experiments using a quantitative real-time (qR) RT-PCR, we demonstrated that feather samples (n=5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces, as shown by statistical analysis. Third, we experimentally coated 7 mallards with a preen oil-AIV mix, and housed them with a control, uncoated, duck. Through self- and/or allopreening behaviour, all birds ingested the virus, as shown by virus detection in both oro-pharyngeal and cloacal samples. Virus isolation from cloacal swabs and virus-specific antibody response confirmed the occurrence of mallards’ infection. Infectious AIVs were isolated from ducks’ body surface until 32 days after the experimental coating.Our field and experimental results indicate that AIVs can be naturally concentrated and carried on the feather surface of infected ducks (i.e., those VI-positive from both cloacal and feathers swabs) and uninfected ones (i.e., those VI-positive from feathers only). In such a context, the natural preening behaviour, by which waterbirds spread preen oil all over their plumage (self-preening) or other birds’ plumage (allo-preening), could facilitate the ingesion of AIV particles stuck on birds’ feathers, thus promoting a preening-mediated infection route. Our findings also suggest that during the time period between the virus adhesion to the bird’s body and the infection (possibly due to self- and/or allopreening), the virus could move in nature with the host by an undescribed circulation mechanism. We demonstrate here a novel viral transmission route that adds to, and possibly contributes to explain the knowledge of longdistance movements and long-term infectivity of lowly and highly pathogenic AIVs in nature.

A pandemic influenza A/H1N1v strain with the neuraminidase H274Y mutation was detected in nasal secretions of a 2-year-old leukemic patient with influenza-like illness after 18 days of treatment with oseltamivir. At baseline, no drug-resistant virus was found, while 4 days after treatment initiation a mix- ture of wild-type and mutated virus was detected. After treatment interruption, the wild type influenza virus re-emerged and became prevalent in nasal secretions after a few days, suggesting the lower fitness of the mutated virus strain. The patient slowly improved concurrently with a decrease in virus load, which resulted negative 42 days after diagnosis. No other drug-resistant influenza A/H1N1v virus strains have been detected in Italy (up to the end of November 2009) since the first case of the novel A/H1N1v virus was identified in the country (May 2009).

Nel periodo 1995-2006, nel corso di programmi di sorveglianza virologica in Italia centrale e del Nord, sono stati isolati ceppi di influenza aviaria di sottotipo H1N1 da uccelli acquatici selvatici e domestici. 13 di questi ceppi sono stati caratterizzati geneticamente, e confrontati con ceppi dello stesso sottotipo isolati da suini di allevamenti italiani. a) definire le caratteristiche molecolari ed evolutive della popolazione di virus H1N1 aviari del lineaggio Eurasiatico; b) valutare le relazioni filogenetiche di questi ceppi con quelli di ceppi suini che circolano regolarmente negli allevamenti suini, e con virus umani; c) valutare le dinamiche di persistenza/variazione di virus aviari H1N1 nel serbatoio naturale in stagioni successive. Tamponi cloacali sono stati raccolti da uccelli selvatici e domestici, inoculati in uova embrionate di pollo e i liquidi allantoidei positivi per influenza sono stati tipizzati sierologicamente. L'RNA di 13 isolati aviari H1N1 e di 7 ceppi suini è stato estratto, sottoposto a trascrizione inversa, PCR e sequenziamento. Le sequenze degli 8 segmenti di ciascun virus sono state utilizzate per l'analisi filogenetica. L'analisi filogenetica del gene HA mostra che tutti i ceppi aviari italiani sono più simili ad alcuni ceppi del Giappone, formando due subclades minori, nettamente distinti dal lineaggio dei virus suini italiani. Quest'ultima evidenza si conferma anche nel resto del genoma. Inoltre, in ognuno dei geni interni, i virus si dividono in 3 o più clusters distinti e diversi tra loro. Conclusioni. Questo lavoro rappresenta la prima caratterizzazione estensiva di virus H1N1 aviari eurasiatici, importante anche perché permette di definire meglio i rapporti di derivazione del virus H1N1 umano di origine aviaria responsabile della pandemia del 1918. I dati dimostrano che l'HA dell'H1 aviaria forma un gruppo filogeneticamente molto ben 26 distinto sia dai ceppi aviari americani che dai ceppi suini e umani. Relativamente ai restanti geni, per effetto dello scambio di virus legato alle migrazioni degli uccelli selvatici, le costellazioni geniche di questi virus differiscono tra di loro sia da un anno all'altro, sia nello stesso anno. Infine, nessuna trasmissione interspecie sembra essersi verificata tra ceppi aviari e suini italiani.

Since September 2012, over 90 cases of respiratory disease caused by a novel coronavirus, now named Middle East respiratory syndrome coronavirus (MERS-CoV), have been reported in the Middle East and Europe. To ascertain the capabilities and testing experience of national reference laboratories across the World Health Organization (WHO) European Region to detect this virus, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe conducted a joint survey in November 2012 and a follow-up survey in June 2013. In 2013, 29 of 52 responding WHO European Region countries and 24 of 31 countries of the European Union/European Economic Area (EU/EEA) had laboratory capabilities to detect and confirm MERS-CoV cases, compared with 22 of 46 and 18 of 30 countries, respectively, in 2012. By June 2013, more than 2,300 patients had been tested in 23 countries in the WHO European Region with nine laboratory-confirmed MERS-CoV cases. These data indicate that the Region has developed significant capability to detect this emerging virus in accordance with WHO and ECDC guidance. However, not all countries had developed capabilities, and the needs to do so should be addressed. This includes enhancing collaborations between countries to ensure diagnostic capabilities for surveillance of MERS-CoV infections across the European Region., peer-reviewed

Tra gli uccelli acquatici gli Anseriformi rappresentano il principale serbatoio dei virus influenzali di tipo A e possono albergare la maggior parte dei sottotipi virali caratterizzati sino ad oggi da 16 emoagglutinine e 9 neuroaminidasi diverse. Occasionalmente virus influenzali aviari possono essere trasmessi ad altre specie, compresi i volatili domestici ed i mammiferi. Scopo del lavoro è stato quello di valutare sia la circolazione di virus influenzali tipo A in Anatidi selvatici sia i patterns di prevalenza correlati a stagionalità, sesso e classi di età degli animali campionati all’interno dell’Oasi WWF “Laguna di Orbetello” (GR) nel periodo ottobre 2005/settembre 2006. L’RNA virale è stato estratto da 146 tamponi cloacali prelevati da anatre di superficie catturate con gabbie ad invito. Mediante one-step RT-PCR diretta ad amplificare un tratto specifico del gene M, sono stati identificati i campioni contenenti virus influenzali. Tali campioni sono stati quindi inoculati, per l’isolamento virale, nella cavità allantoidea di uova embrionate di pollo all’undicesimo giorno di incubazione. I liquidi allantoidei ottenuti sono stati analizzati in prove di emoagglutinazione ed ELISA diretta verso la nucleoproteina virale e sono stati considerati positivi i liquidi allantoidei emoagglutinanti positivi ad entrambe queste metodiche. I test del Chi-quadrato ed Esatto di Fisher sono stati utilizzati al fine di evidenziare differenze statisticamente significative in funzione dell’età e sesso dei soggetti campionati e di tre periodi di campionamento (sovrapponibili rispettivamente alla migrazione di discesa, allo svernamento ed al periodo post-riproduttivo). La prevalenza totale è risultata del 26% in RT-PCR e del 9,6% nelle prove di isolamento su uova embrionate. Durante l’intero anno di campionamento nessuna differenza significativa è stata riscontrata tra le classi di età, di sesso e tra i diversi periodi di campionamento mentre dal confronto degli isolamenti ottenuti in adulti e giovani, durante la stagione di svernamento (gennaio/marzo 2006) emerge una differenza significativa (P

With regard to the role of wild ducks and coots as a source of viruses potentially pathogenic for domestic poultry we observed some differences: in coots, no evidence of H5 and H7 virus circulation was found either at serological or virological level whereas ducks serologically positive to H5 virus were detected every year. In the present study, the highest H5 antibody frequencies that were observed in DWMA during the first sampling period overlapped with the H5N2 Italian poultry epidemic (Donatelli et al., 2001). Moreover, even if no H5 viruses were isolated during the present study period, seroconversion observed in a mallard indicated that circulation of H5 subtype viruses occurred in the sampling area during the first sampling period (Table 2). Unlike H5 AIVs, no evidence of H7 virus circulation was found either at serological or virological level in ducks trapped in the area under study (Table 1). Thus it appears that no H7 viruses circulated among ducks sampled in the study area since 1992 (De Marco et al., 2004). In particular, duck sera resulted negative for H7 subtype both before and after the beginning of the catastrophic Italian poultry epidemic due to the H7N1 serovar (Capua & Alexander, 2004), suggesting a possible implication of other bird reservoirs. With regard to virus circulation, the H1N1 subtype appears to be endemic in Italian wetlands since 1992 (De Marco et al., 2004). H2N3 and H11N6 strains were isolated for the first time, even though ducks serologically positive to both subtypes had been found in previous years in the same study area (De Marco et al., 2004). The present results, together with data obtained before and after this study period in the same study area (De Marco et al., 2004, Campitelli et al., 2004b), suggest a perpetuation of H5 AIVs in the wild duck reservoir in Italy. This evidence is all the more relevant when we consider that the duck populations we analysed are representative of waterfowl populations that very likely make use of wetlands of Northern Italian regions (where H5N2, H7N1 and H7N3 poultry epidemics occurred in recent years) during their migrations northward in spring and southward in autumn (De Marco et al., 2000). The identification in 2001, in these wetlands, of H7N3 duck viruses almost identical to H7N3 strains affecting domestic poultry in Northern Italy in 2002-03 (Campitelli et al., 2004a) supports the existence of an epidemiological connection between these two areas and confirms the importance of maintaining an active influenza surveillance program in wild waterfowl reservoirs.

La diffusione dei focolai di influenza aviaria A/H5N1 e gli episodi di mortalità nell’uomo registrati nei paesi del Sud-Est asiatico, hanno determinato un crescente allarme nella comunità scientifica e nell’opinione pubblica a causa del potenziale pandemico rappresentato dalla evoluzione del virus. La tempestiva identificazione del virus nelle specie aviarie domestiche e selvatiche, attraverso l’impiego di strumenti diagnostici altamente sensibili, specifici e di rapida esecuzione, è essenziale per l’adozione rapida ed efficace delle misure sanitarie di controllo. La real-time RT-PCR, basata sull’impiego di sonde fluorescenti, rappresenta lo strumento diagnostico in grado di garantire l’identificazione dei virus influenzali, sia umani che aviari, presentando notevoli vantaggi in termini di rapidità, sensibilità e specificità rispetto alle metodiche classiche di isolamento del virus e alle altre metodiche molecolari. Nell’ottica di un continuo miglioramento dell’iter diagnostico per l’identificazione del virus dell’influenza aviaria, è stato sviluppato un saggio di real-time RT-PCR, impiegando una sonda fluorescente Minor Groove Binder, che presenta una serie di innovazioni rispetto alle piattaforme per real-time sinora disponibili per la diagnostica virologica. Il sistema diagnostico è stato calibrato e ottimizzato in termini di specificità, utilizzando un panel di virus influenzali aviari, di origine suina, equina, oltre che umana, dimostrando inoltre una sensibilità tale da renderlo applicabile per lo screening di campioni biologici prelevati dalle specie aviarie domestiche e selvatiche.

Phylogenetic analyses of the HA and neuraminidase (NA) genes of representative B strains, isolated throughout Italy during 2001–04, showed that during the first influenza season the NA genes, as well as the HAgenes, separated into the two distinct clades, the Yamagata- and Victoria lineages, and showed no evidence of genetic reassortment. On the contrary, all the B viruses isolated in the 2002–03 and most of those isolated in the 2003–04 epidemic season were ‘‘Victoria HA–Yamagata NA’’ reassortants similar to those isolated in other parts of the world, showing that these reassortants became established in the human population.

BACKGROUND: The objective of this study was to evaluate, within the Italian National Influenza Epidemiological and Virological Surveillance, the rate of vaccination coverage, the incidence of Influenza Like-Illness (ILI), the incidence of Acute Respiratory Illness (ARI), and to identify the virus strains circulating in Apulia from 1999 to 2003. METHODS: Vaccination coverage rates were calculated based on the number of doses administered to individuals > 65 years of age. Every week, sentinel physicians reported ILI and ARI cases having occurred among their patients. Voluntary general practitioners (GPs) and paediatricians (Ps) collected oropharyngeal swab samples from patients suspected with ILI. Influenza viruses were isolated and identified by cell culture (MDCK cells) and RT-PCR. Virological surveillance was carried out by the ISS, in collaboration with a network of peripheral laboratories. RESULTS: In Apulia, vaccination coverage progressively increased to 68.6% during the 2002-2003 season. The analysis of ILI cases showed higher incidence rates during the 1999-2000 and 2002-2003 seasons. ARI rates appeared to have a more constant trend. ILI and ARI incidence rates were higher in the 0-14 year age group. CONCLUSION: The increase in vaccination coverage rates and implementation of the network of clinical, and epidemiological and virological surveillance are fundamental for the control and prevention of influenza