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Virological Surveillance of Influenza in the eight epidemic seasons after the 2009 pandemic in Emilia-Romagna (Northern Italy)

Background and aim of the work: Influenza virological surveillance is essential for monitoring the evolution of influenza viruses (IVs) as well as for annual updating of the vaccine composition. The aim of this study is to analyse IVs circulation in Emilia-Romagna during the eight epidemic seasons after the 2009 pandemic and to evaluate their match with seasonal vaccine strains. Methods: A total of 7882 respiratory specimens from patients with influenza-like illness (ILI), were collected by regional sentinel practitioners and hospital physicians. Viral investigations were conducted by rRT-PCR assay. Genetic characterization was performed for a spatial-temporal representative number of influenza laboratory-confirmed specimens. Results: Influenza-positive samples per season ranged between 28.9% (2013-2014) and 66.8% (2012-2013). Co-circulation of IVs type A and type B was observed in all seasons, although with a different intensity. In all seasons, the highest number of positive samples was recorded in younger patients aged 5-14 years with relative frequencies ranging from 40% in the 2013-2014 season and 78% in the 2012-2013 season. Since the 2009 pandemic, A/H1N1pdm09 IVs circulating were closely related to the vaccine strain A/California/7/2009. Antigenic mismatch between vaccine strain and A/H3N2 IVs was observed in the 2011-2012 and 2014-2015 seasons. During 2015-2016, 2016-2017 and 2017-2018 seasons a complete or nearly complete mismatch between the predominant influenza B lineage of IVs type B circulating and vaccine B lineage occurred. Conclusions: This analysis confirms the importance of the virological surveillance and highlights the need of a continuous monitoring of IVs circulation, to improve the most appropriate vaccination strategies. (www.actabiomedica.it)


Introduction
Seasonal influenza is an acute, highly contagious viral respiratory infection of great importance from clinical and epidemiological point of view. Worldwide, the annual attack rate is estimated at 5-10% in adults and 20-30% in children with about 3 to 5 million case of severe illness and 290.000-650.000 deaths (1).
Influenza epidemiology mainly depends on the particular characteristics of influenza viruses (IVs), able to spread all over the world and rapidly evolve. Furthermore, a gradual and relatively continuous change in the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), allows them to escape the immunity that comes from prior infections or vaccination (2). Because of this, seasonal epidemics recur every year with different intensity and trend.
The impact of influenza also varies according to different age groups, in terms of morbidity, severe case illness and mortality among high-risk groups; the most affected patients are the elderly, young children, pregnant women and individuals with comorbidity (1,(3)(4)(5)(6)(7)(8)(9).
Periodically, in the range of 10-40 years, IVs type A caused pandemic events, due to the emergence of a new variant against which there is no pre-existing immunity in the population. The pandemic in 2009 was caused by a unique quadruple reassortant A/ H1N1 IV, including a complex combination of swine, avian and human IV genes (10). This new variant, A/ H1N1pdm09, has completely substituted the previous seasonal IVs of the same type and continues to circulate worldwide as seasonal IV, together with A/H3N2 subtype IVs and B type IVs. Moreover, a progressive diversification of B type IVs into two lineages, genetically and antigenically distinct, occurred starting from 1983 (11).
Within this context, influenza epidemiological and virological surveillance plays an essential role and it is carried out, at global level, by World Health Organization Global Influenza Surveillance and Response System (GISRS) (12) and at European level, by the European Centre for Disease Prevention and Control (ECDC) (13). The national influenza surveillance systems, together with the analysis of epidemiological features, have the specific goal to monitor the circulation of IVs, analyse antigenic, genetic and biological characteristics, also including the susceptibility to available antiviral drugs. Moreover, they work to recognize any new viral variants in order to implement the appropriate containment and prevention strategies in a timely manner (14). In Italy, these activities are carried out by influenza surveillance system, named InfluNet, coordinated by the National Influenza Centre at the Istituto Superiore di Sanità (NIC/ISS). Influnet is based on the collaboration of sentinel practitioners who, starting from the 46th week of each year until the 17th of the following year, perform respiratory samples from patients with a clinical presentation of influenza-like illness (ILI) (15)(16)(17). The regional influenza Laboratories perform isolations and antigenic characterizations of IVs during the epidemic season, to constantly monitor the different types/subtypes circulation and to evaluate the match between epidemic and vaccine strains. This is fundamental to formulate the vaccine composition of the following season, because the degree of match between circulating and vaccine strains is one of the factor, togheter with the characteristics of the person being vaccinated (such as their age and health), that contribute to the vaccine effectiveness (18)(19)(20)(21)(22)(23)(24).
Furthermore one of the main Public Health implication of the low vaccine effectiveness, togheter with the decrease and the poor vaccination uptake, even in the target population, is the real reduction of effectiveness of influenza vaccine on field. (19,(25)(26)(27)(28).
The aim of this paper is to describe the circulation of IVs in Emilia-Romagna and their matching with seasonal vaccine strains, during eight consecutive seasons (from 2010-2011 to 2017-2018) by analysis of virological surveillance data, performed by the regional influenza reference Laboratory of Parma (29)(30)(31).

Methods
Respiratory specimens from children and adults with a clinical presentation of ILI, were collected by regional sentinel physicians (general practitioners and pediatricians) and by hospital physicians of the care Units of Parma, Piacenza and Reggio Emilia hospital. Each clinical specimen was accompanied by a case report form filled in with epidemiological data. The "Virocult" diagnostic kit (MWE, England) and the commercial "UTM Viral Transport Media" kit (Copan, Brescia, Italy) were used to collect the clinical samples (32).
Viral isolation was performed in Madin Darby Canine Kidney cells (MDCK), and the presence of the virus was detected by a conventional haemagglutination assay using a 0,8% suspension in PBS of guinea pig red blood cells.
Viral nucleic acid was extracted from respiratory specimens and from viral cell culture supernatant, using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. A one-step Real Time retro-transcription PCR assay (rRT-PCR) was performed with Quantifast Pathogen+IC Kit, (Qiagen, Hilden, Germany) with specific primer/probe sets targeting the matrix region of A type IV and the nucleoprotein region of B type IV. For A type IV, samples were further subtyped using specific primer/probe sets for the HA gene to dis-criminate between A/H1N1pdm09 IVs and A/H3N2 IVs. The genetic lineage of confirmed B type IVs was determined by rRT-PCR. All assays were performed in compliance with institutional guidelines (33)(34).
A representative number of influenza laboratoryconfirmed specimens and viral isolates, were sent to the NIC/ISS for antigenic characterisations and phylogenetic analysis.

Results
During the study period, the reference Laboratory for influenza virological surveillance of Emilia-Romagna, analysed 7882 nasal or throat swabs, performed by regional sentinel practitioners and doctors working in hospital care Units of Parma, Piacenza and Reggio Emilia. The distribution of samples by season, prove-nience, age group, vaccination status, virological data, is reported in Table 1. Overall, the percentage of the samples range between 6.2% in 2013-2014 season and 18.6% in 2017-2018 season; 50.3% were outpatients and 49.7% were inpatients. In the first six seasons, the highest numbers of samples were collected in children ≤4 years of age and in school-aged children (5-14 years); in the two latest years, in young-adults (15-64 years) and in elderly ≥65 years of age. The percentage of influenza-positive samples per season ranged between 28.9% (2013-2014) and 66.8% (2012-2013). In the first three seasons after the 2009 pandemic, epidemics were particularly intense due to the highest number of specimens and influenza-positive samples, while in the following five seasons, the proportion of positive samples was lower (less than 50%) compared to the high number of samples (Table 1). In the surveyed area, epidemiological trends observed in the first six influenza The distribution of detected IVs is presented in Figure 1. During the study period, co-circulation of A type IVs and B type IVs was observed, although with a different intensity; in five seasons A type IVs pre-  Figure 2). During every season, a spatial-temporal representative number of influenza-positive samples was genetically characterized and the phylogenetic analysis was performed by the NIC/ISS (16).
The antigenic and molecular characteristics of IVs circulating were analysed, with particular attention to the match with seasonal vaccine strains (Table 3). In

Conclusions
This study provides the results of the virological surveillance in the eight epidemic seasons after the 2009 pandemic in Emilia-Romagna. The aim of this paper is to describe genetic and antigenic changes of IVs, and to evaluate their match with vaccine strains.
The circulation of IVs in Emilia-Romagna was similar to that of the other regions (36)(37)(38)(39). Type B IVs co-circulated with type A IVs in all seasons. With the exception of 2011-2012 season during which only A/ H3N2 subtype IVs circulated, in the others, both subtypes co-circulated, although with different intensity.
From a general point of view, yearly variations by distribution and frequency of viral types/subtypes were observed, as well as an alternation of the predominant type/subtype. The seasons with a modest circulation of a specific type/subtype, have been followed by seasons with its greater circulation and vice versa.
During all seasons, A/H1N1pdm09 IVs detected were closely related to the vaccine strain A/California/7/2009 and circulated intensely in 2010-2011 season, with the higher morbidity rates in schoolaged children (aged 5-14 years) ( Figure 2). Different considerations must be made for type A/H3N2 IVs and type B IVs. The continuous and rapid evolution of A/H3N2 IVs and the co-circulation of different A/ H3N2 IVs strains during the same season caused an incomplete match between the vaccine strains and seasonal A/H3N2 IVs, so a new vaccine strain was recommended in the vaccine formulation in four seasons (35).
Furthermore, because of the presence of two genetically and antigenically distinct type B IVs lineages, co-circulating in the same season with different intensity, it was very difficult to predict the type of virus that will circulate in the following season. During the study period, a complete mismatch between the type B IVs circulating and the vaccine strain, was observed in three consecutive seasons with a high number of cases and positive samples in children, young adults and elderly.
Overall, from this study we highlight that in none of the epidemic influenza season full match was achieved. Probably the Health Technology Assessment instruments, implemented with new studies on Artificial Intelligence, could help fill the information gap in the setting of the new influenza vaccine (48)(49)(50)(51)(52)(53)(54)(55).
This study also confirms the importance of the virological surveillance and the integration of epidemiological and virological data, and highlights the need of a continuous monitoring of types/subtypes of IVs circulation during epidemic season, to acquire useful informations for improve the most appropriate vaccination strategies.