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To assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy.12,393 respiratory specimens (nasopharyngeal swabs or broncho-alveolar lavages) collected from in/outpatients with influenza illness in the period spanning from January 1, 2022 (week 2022-01) to May 31, 2022 (week 2022-22) were analysed to identify IV genome and were molecularly characterized by 12 laboratories throughout Italy. A(H3N2) evolution was studied by conducting an in-depth phylogenetic analysis of the hemagglutinin (HA) gene sequences. The predicted vaccine efficacy (pVE) of vaccine strain against circulating A(H3N2) viruses was estimated using the sequence-based PThe overall IV-positive rate was 7.2% (894/12,393), all were type A IVs. Almost all influenza A viruses (846/894; 94.6%) were H3N2 that circulated in Italy with a clear epidemic trend, with 10% positivity rate threshold crossed for six consecutive weeks from week 2022-11 to week 2022-16. According to the phylogenetic analysis of a subset of A(H3N2) strains (n=161), the study HA sequences were distributed into five different genetic clusters, all of them belonging to the clade 3C.2a, sub-clade 3C.2a1 and the genetic subgroup 3C.2a1b.2a.2. The selective pressure analysis of A(H3N2) sequences showed evidence of diversifying selection particularly in the amino acid position 156. The comparison between the predicted amino acid sequence of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and the study strains revealed 65 mutations in 59 HA amino acid positions, including the substitution H156S and Y159N in antigenic site B, within major antigenic sites adjacent to the receptor-binding site, suggesting the presence of drifted strains. According to the sequence-based PAfter a long period of very low IV activity since public health control measures have been introduced to face COVID-19 pandemic, along came A(H3N2) with a new phylogenetic makeup. Although the delayed 2021-2022 influenza season in Italy was characterized by a significant reduction of the width of the epidemic curve and in the intensity of the influenza activity compared to historical data, a marked genetic diversity of the HA of circulating A(H3N2) strains was observed. The identification of the H156S and Y159N substitutions within the main antigenic sites of most HA sequences also suggested the circulation of drifted variants with respect to the 2021-2022 vaccine strain. Molecular surveillance plays a critical role in the influenza surveillance architecture and it has to be strengthened also at local level to timely assess vaccine effectiveness and detect novel strains with potential impact on public health.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was isolated from the oro/pharyngeal swabs of two Italian COVID-19 patients, physicians in a COVID-19 division hospital, with different courses of the disease. The complete genome sequences show that the two isolates belong to the B1.1 lineage, but contain a nucleotide mutation in the ORF6, leading to a stop codon and to the deletion of 6 amino acids in the C terminus. This deletion was unique, compared to the currently available sequences deposited in the GISAID and GenBank database. It did not affect the in vitro viral replication, neither the neutralizing activities of the patients' antibodies. Based on homology analysis with other Coronaviruses, the two isolated lacked the ORF6 aminoacidic portion responsible for the inhibition of the antiviral Interferon (IFN)-based host response. IFN seems to have a dual role of in SARS-CoV-2 infected patients: not only antiviral activity, but also a detrimental role in case of excessive production. A deletion in the SARS-CoV-2 ORF6 protein might have a specific, still unknown role in the viral pathogenesis.

BACKGROUND Besides seasonal influenza viruses (IV), several other pathogens-including respiratory syncytial virus (RSV)-are involved in clinically undistinguished influenza-like illnesses (ILIs). This study aimed at investigating the contribution of RSV in ILI cases in Lombardy (Northern Italy) during four consecutive winter seasons. MATERIALS AND METHODS In the framework of influenza surveillance, respiratory samples from ILI outpatients were collected from 2014-2015 to 2017-2018 season. IV-negative swabs were included in the study and analyzed to detect and molecularly characterize RSV-A and RSV-B. RESULTS A total of 12.9% (135/1047) of samples were positive to RSV that was mostly detected among children ≤5 years (51/183, 27.8%) and those aged 6 to 15 years (30/158, 18.9%), whereas elderly >65 years accounted for 12% of RSV cases (15/125). The median start of RSV epidemic was in the end of November, with a peak in mid-February and a width of nearly 4 months, almost overlapping seasonal influenza epidemic. RSV-A and RSV-B co-circulated in all considered seasons, with RSV-B predominating on RSV-A (63.6% vs 36.4%; P

An Italian 13-year-old boy immunosuppressed due to kidney transplant presented in November 2018 with acute flaccid paralysis with anterior horn cell involvement resembling the clinical, radiological, and laboratory features of poliomyelitis. Enterovirus was molecularly identified in cerebral spinal fluid and stool samples and the sequence analysis of the VP1 gene of enterovirus genome revealed the presence of Echovirus 30 both in CSF and in stool samples. Echovirus 30 is an emerging neurotropic virus able to cause outbreaks of aseptic meningitis and meningoencephalitis all over the world, but acute flaccid paralysis is not a classical manifestation. A 6-month follow-up revealed a poor outcome with severe motor deficits and only slight improvement in disability. Clinicians must be aware of the possible role of Echovirus 30 in acute flaccid paralysis and active surveillance should consider the possible influence of immunosuppression on the symptoms caused by the widening spectrum of enterovirus infections.

BackgroundSARS-CoV-2 variants of concern (VOC) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Some exhibit increased pathogenicity and transmissibility compared to the original wild type WUHAN (Hu-1). Broad-spectrum antivirals could complement and further enhance preventive benefits achieved through SARS-CoV-2 vaccination campaignsMethodsThe anti-coronavirus activity of Echinacea purpurea (Echinaforce® extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish) and B1.525 (eta), (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild-type SARS-CoV-2 (Hu-1) were analyzed. Molecular dynamics (MD) were applied to study interaction of Echinacea’s phytochemical markers with known pharmacological viral and host cell targets.ResultsEF extract broadly inhibited propagation of all investigated SARS-CoV-2 VOCs as well as entry of SARS-CoV-2 pseudoparticles at EC50’s ranging from 3.62 to 12.03 µg/ml. Preventive addition of 20 µg/ml EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) as well as with the common human strain OC43. MD analyses showed constant binding affinities to Hu-1, B1.1.7, B.1.351, P.1 and B1.617.2-typic S protein variants for alkylamides, caftaric acidand feruoyl-tartaric acid in EF extract. They further indicated that the EF extract could possibly interact with TMPRSS-2, a serine protease required for virus endocytosis.ConclusionsEF extract demonstrated stable antiviral activity across 6 tested VOCs, which is likely due to the constant affinity of the contained phytochemical marker substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain cell protective benefits of the extract by inhibition of endocytosis. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.

Here we described the virological and serological assessment of 23 COVID-19 patients hospitalized and followed up in Milan, Italy, during the first wave of COVID-19 pandemic. Nasopharyngeal (NPS), anal swabs, and blood samples were collected from 23 COVID-19 patients, at hospital admission, and periodically up to discharge, for a median time of 20 days (3–83 days). RNA was isolated and tested for SARS-CoV-2 by qRT-PCR; anti-SARS-CoV-2 IgM and IgG antibody titers were evaluated in serum samples by ELISA. SARS-CoV-2 genome was detected in the NPS swabs of the 23 patients, at the admission, and 8/19 (42.1%) were still positive at the discharge. Anal swabs were positive to SARS-CoV-2 RNA detection in 20/23 (86.9%) patients; 6/19 (31.6%) were still positive at discharge. The mean time of RNA negative conversion was 17 days (4–36 days) and 33 days (4–77 days), for NPS and anal swabs, respectively. SARS-CoV-2-RNA was detected in the blood of 6/23 (26.1%) patients. Thirteen/23 (56.5%) and 17/23 (73.9%) patients were seropositive for IgM and IgG, respectively, at the admission, and the median IgM and IgG levels significantly (p

Enteroviruses (EVs) and Hepatitis A Viruses (HAVs) are human pathogens with a wide spectrum of clinical manifestations. The monitoring of sewage samples enables to monitor the EVs and HAVs in circulation among the general population and recognize possible outbreaks. This study focused on the molecular characterization and phylogenetic analysis of the EVs and HAVs identified in 33 sewage samples collected every 15 days at the influent of a wastewater treatment plant located in Northern Italy from March to October 2016. According to the results of the molecular characterization, the most frequently identified viruses were Echovirus 6 (E-6), E-11 and HAV-IA. The phylogenetic analyses indicated the rapid genetic evolution of E-6 and E-1; noteworthy, most E-11 strains clustered with a strain isolated from a clinical sample collected in the same geographical area over the same period by our laboratory. Most of the HAV strains detected clustered with epidemic HAV-IA strains identified during the European hepatitis A outbreak that occurred in 2016-2017 affecting men who have sex with men (MSM). The detection of environmental HAV strains before and at the beginning of its spread amongst humans demonstrated that this outbreak could have been predicted by monitoring sewage samples. Moreover, conducting a genetic comparison between the HAV and EV strains identified in sewage and clinical samples may improve knowledge of viral epidemiology. EV and HAV molecular environmental surveillance may prove useful for identifying viral circulation and for issuing early warning alerts on possible outbreaks among the human population.

Enteroviruses (Enterovirus genus, Picornaviridae family) are distributed worldwide and are among the most common causes of human disease globally [...]

Background In the night of February 20, 2020, the first epidemic of the novel coronavirus disease (COVID-19) outside Asia was uncovered by the identification of its first patient in Lombardy region, Italy. In the following weeks, Lombardy experienced a sudden increase in the number of ascertained infections and strict measures were imposed to contain the epidemic spread. Methods We analyzed official records of cases occurred in Lombardy to characterize the epidemiology of SARS-CoV-2 during the early phase of the outbreak. A line list of laboratory-confirmed cases was set up and later retrospectively consolidated, using standardized interviews to ascertained cases and their close contacts. We provide estimates of the serial interval, of the basic reproduction number, and of the temporal variation of the net reproduction number of SARS-CoV-2. Results Epidemiological investigations detected over 500 cases (median age: 69, IQR: 57–78) before the first COVID-19 diagnosed patient (February 20, 2020), and suggested that SARS-CoV-2 was already circulating in at least 222 out of 1506 (14.7%) municipalities with sustained transmission across all the Lombardy provinces. We estimated the mean serial interval to be 6.6 days (95% CrI, 0.7–19). Our estimates of the basic reproduction number range from 2.6 in Pavia (95% CI, 2.1–3.2) to 3.3 in Milan (95% CI, 2.9–3.8). A decreasing trend in the net reproduction number was observed following the detection of the first case. Conclusions At the time of first case notification, COVID-19 was already widespread in the entire Lombardy region. This may explain the large number of critical cases experienced by this region in a very short timeframe. The slight decrease of the reproduction number observed in the early days after February 20, 2020 might be due to increased population awareness and early interventions implemented before the regional lockdown imposed on March 8, 2020.

Objectives: The authors conducted an in-depth phylogenetic analysis of the hemagglutinin (HA) gene of A(H3N2) identified during the 2016–2017 season to compare the circulating strains with both the...

A previously healthy 6-year-old boy was admitted to hospital with hypotonia and hyposthenia of lower limbs. Electromyography and slow motor nerve conduction velocity test identified a lower limb acute motor axonal neuropathy. Brain and spinal cord magnetic resonance imaging demonstrated multifocal cortical gray matter lesions in both cerebral hemispheres consistent with gray matter acute disseminated encephalitis otherwise with viral/Mycoplasma pneumoniae encephalitis, and signs of involvement of anterior nerve roots of the cauda equina consistent with Guillain-Barré syndrome. The patient resulted negative to routinely bacterial and viral investigations but positive to human parechovirus that sequence analyses confirmed as type 6. Intravenous immunoglobulins and methylprednisolone treatment were administered but did not relieve the symptoms of Guillain-Barré syndrome. The disease improved gradually over the next 3-month follow-up with a complete remission of both central and peripheral nervous system symptoms.

Aim: This paper discusses the unexpected findings from the RotaNet-Italy hospital-based surveillance study carried out in northern Italy. Materials & methods: From September 2015 to August 2016, 51 rotavirus-A (RVA) positive fecal samples were collected from children aged less than 15 years, who were hospitalized for acute gastroenteritis in Lombardy, northern Italy. Results: Molecular characterization revealed the predominance of the uncommon G12P[8] RVA strain, which was detected in 49% of cases. Phylogenetic analysis showed that these G12 strains clustered into lineage 3. Conclusion: To our knowledge, this is the first study on the G12P[8] genotype’s introduction in northern Italy. Our findings emphasize the importance of the surveillance of RVA gastroenteritis with the aim of obtaining new insight into the unusual newly emerging RVA strains.

Besides the influenza virus (IV), several other viruses are responsible for influenza-like illness (ILI). Although human parechoviruses (HPeVs) and enteroviruses (EVs) may impact on ILI, limited data on their epidemiological characteristics are available. During seven consecutive winter seasons (from 2010–2011 to 2016–2017), within the framework of an influenza surveillance system (InfluNet), 593 respiratory swabs were collected from children ≤5 years of age with ILIs. Molecular detection showed that 58.3 % of swabs were positive for at least one of the viruses under study: 46 % for IV, 13 % for EV and 5.4 % for HPeV. A single virus was identified in 51.3 % of samples while more than one virus was detected in 7 % of the samples. The risk of contracting IV was higher than the risk associated with EV, which in turn was higher than the risk of contracting HPeV. The risk of developing an IV infection was twofold greater in children >3 years than in those ≤3 years, who had higher risk of EV/HPeV infection. The frequency of EV/HPeV-positive swabs increased significantly during the 2016–2017 winter season compared to the previous six seasons. Sixteen EV genotypes were identified belonging to species A and B. HPeV-1 was the most frequently detected genotype, followed by -6 and -3. In this study, IV was mainly responsible for ILI, however EV and HPeV were also involved and particularly affected children ≤3 years of age. Influenza surveillance samples could provide us with valuable insight into the epidemiological features of viruses involved in ILI.

Between September and October 2018, an enterovirus D68 (EV-D68) outbreak occurred in patients hospitalised with severe acute respiratory infection in northern Italy; 21 laboratory-confirmed cases were reported. Phylogenetic analysis revealed that 16/20 of the EV-D68 sequences belonged to a divergent group within the sub-clade D1. Since its upsurge, EV-D68 has undergone rapid evolution with the emergence of new viral variants, emphasising the need for molecular surveillance that include outpatients with respiratory illness.

This study aimed at assessing the frequency and the distribution of influenza virus types/subtypes in 172 laboratory-confirmed influenza-positive patients admitted to intensive care units (ICUs) during the 2017&ndash, 2018 season in the Lombardy region (Northern Italy), and to investigate the presence of molecular pathogenicity markers. A total of 102/172 (59.3%) patients had influenza A infections (83 A/H1N1pdm09, 2 H3N2 and 17 were untyped), while the remaining 70/172 (40.7%) patients had influenza B infections. The 222G/N mutation in the hemagglutinin gene was identified in 33.3% (3/9) of A/H1N1pdm09 strains detected in the lower respiratory tract (LRT) samples and was also associated with more severe infections, whereas no peculiar mutations were observed for influenza B strains. A single-point evolution was observed in site 222 of A/H1N1pdm09 viruses, which might advantage viral evolution by favouring virus binding and replication in the lungs. Data from 17 paired upper respiratory tract (URT) and LRT samples showed that viral load in LRT samples was mostly higher than that detected in URT samples. Of note, influenza viruses were undetectable in 35% of paired URT samples. In conclusion, LRT samples appear to provide more accurate clinical information than URT samples, thus ensuring correct diagnosis and appropriate treatment of patients with severe respiratory infections requiring ICU admission.

Recent molecular diagnostic methods have significantly improved the diagnosis of viral pneumonia in intensive care units (ICUs). It has been observed that 222G/N changes in the HA gene of H1N1pdm09 are associated with increased lower respiratory tract (LRT) replication and worse clinical outcome. In the present study, the frequency of respiratory viruses was assessed in respiratory samples from 88 patients admitted to 16 ICUs during the 2014–2015 winter-spring season in Lombardy. Sixty-nine out of 88 (78.4%) patients were positive for a respiratory viral infection at admission. Of these, 57/69 (82.6%) were positive for influenza A (41 A/H1N1pdm09 and 15 A/H3N2), 8/69 (11.6%) for HRV, 2/69 (2.9%) for RSV and 2/69 (2.9%) for influenza B. Phylogenetic analysis of influenza A/H1N1pdm09 strains from 28/41 ICU-patients and 21 patients with mild respiratory syndrome not requiring hospitalization, showed the clear predominance of subgroup 6B strains. The median influenza A load in LRT samples of ICU patients was higher than that observed in the upper respiratory tract (URT) (p

Summary Influenza A virus (IAV) infection in swine plays an important role in the ecology of influenza viruses. The emergence of new IAVs comes through different mechanisms, with the genetic reassortment of genes between influenza viruses, also originating from different species, being common. We performed a genetic analysis on 179 IAV isolates from humans (n. 75) and pigs (n. 104) collected in Northern Italy between 2010 and 2015, to monitor the genetic exchange between human and swine IAVs. No cases of human infection with swine strains were noticed, but direct infections of swine with H1N1pdm09 strains were detected. Moreover, we pointed out a continuous circulation of H1N1pdm09 strains in swine populations evidenced by the introduction of internal genes of this subtype. These events contribute to generating new viral variants—possibly endowed with pandemic potential—and emphasize the importance of continuous surveillance at both animal and human level.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and young children. RSV is characterised by high variability, especially in the G glycoprotein, which may play a significant role in RSV pathogenicity by allowing immune evasion. To reconstruct the origin and phylodynamic history of RSV, we evaluated the genetic diversity and evolutionary dynamics of RSV A and RSV B isolated from children under 3 years old infected in Italy from 2006 to 2012. Phylogenetic analysis revealed that most of the RSV A sequences clustered with the NA1 genotype, and RSV B sequences were included in the Buenos Aires genotype. The mean evolutionary rates for RSV A and RSV B were estimated to be 2.1×10-3 substitutions (subs)/site/year and 3.03×10-3 subs/site/year, respectively. The time of most recent common ancestor for the tree root went back to the 1940s (95% highest posterior density-HPD: 1927-1951) for RSV A and the 1950s (95%HPD: 1951-1960) for RSV B. The RSV A Bayesian skyline plot (BSP) showed a decrease in transmission events ending in about 2005, when a sharp growth restored the original viral population size. RSV B BSP showed a similar trend. Site-specific selection analysis identified 10 codons under positive selection in RSV A sequences and only one site in RSV B sequences. Although RSV remains difficult to control due to its antigenic diversity, it is important to monitor changes in its coding sequences, to permit the identification of future epidemic strains and to implement vaccine and therapy strategies. © 2014 Elsevier B.V

Although in the last years poliovirus (PV) transmission has been reported at the lowest levels ever recorded, the spread of virus from endemic countries endures; the high levels of immigration flows across the Mediterranean Sea jeopardize Italy for PV reintroduction. The World Health Organization (WHO) strategic plan for global poliomyelitis (polio) eradication indicates the nationwide surveillance of Acute Flaccid Paralysis (AFP) as the gold standard for detecting cases of polio. In addition, the Environmental Surveillance (ES), seeking the presence of PV and Non-Polio Enterovirus (NPEV) in sewage, is recognized as a powerful tool to confirm PV circulation in absence of AFP cases, especially in polio-free countries. Here we report the results of AFP surveillance (AFPS) and ES in Lombardy (Northern Italy) from 2012 to 2015. Forty-eight AFP cases were identified during the study period. No AFP case was caused by PV infection. NPEVs were identified in 6.3% (3/48) of AFP cases. The annual AFP incidence rate was 0.87/100'000 children15 y in 2012, 1.42/100'000 in 2013, 1.02/100'000 in 2014, and 0.47/100'000 in 2015; according to WHO indicators, the sensitivity of AFPS was adequate in 2013 and 2014. Completeness of case investigation raised progressively during the study period to achieve the WHO standards in 2014 (92.3%) and 2015 (100%). Completeness of follow-up increased from 72.7% in 2012 to 100% in 2014. In the framework of the ES conducted in Milan, 268 wastewater samples were collected from 2012 to 2015 and no PVs were isolated. In contrast, NPEVs were detected in 65.3% (175/268) of samples. All NPEVs characterized belonged to enterovirus species B: echovirus type 11, 6 and 3 were the most frequently detected viruses, representing 29.1% (41/141), 20.6% (29/141) and 9.2% (13/141) of genotyped NPEVs, respectively. Keeping strong and encouraging both AFPS and ES is crucial to ensure that PV will not return unnoticed in Italy - as well as in other polio-free countries - and, as a final point, to achieve the global polio eradication goal.

The molecular characterization of circulating influenza A viruses is crucial to detect mutations potentially involved in increased virulence, drug resistance and immune escape. A molecular and phylogenetic analysis of A(H1N1)pdm09 neuraminidase (NA) gene sequences from different patient categories defined according to the severity of influenza infection were analyzed. A total of 126 influenza A(H1N1)pdm09 positive samples from patients with severe infections in comparison with those with moderate and mild infections was performed in Lombardy (Northern Italy, nearly 10 million inhabitants) during the 2010–2011 season. NA sequences included in this study segregated into five distinct clusters. Nineteen amino acid substitutions were detected exclusively in NA sequences of viruses identified in patients with severe or moderate influenza infection. Three of them (F74S, S79P, E287K) were observed in virus strains with the 222G/N hemagglutinin mutation. None of NA sequences under study had mutations related to the resistance to the NA inhibitors. Four out of 126 (3.2%) NA sequences from patients with severe infection lost a N-linked glycosylation site due to the change from N to K at residue 386. Two additional N-linked glycosylation sites in the NA stalk region (residues 42 and 44) were found in 12 (9.5%) NA sequences. Sporadic NA mutations were detected in NA viral sequences from critically ill patients, and no variants with reduced sensitivity to NA inhibitors were observed either in treated or untreated patients. J. Med. Virol. 85: 944–952, 2013. © 2013 Wiley Periodicals, Inc.

The activity and circulation of influenza viruses in Lombardy - Northern Italy - (a region with nearly 10 out of the 60 million inhabitants of Italy) were investigated during two consecutive seasons (2010-2011 and 2011-2012), as part of the Italian Influenza Surveillance Network. The molecular characteristics of the hemagglutinin (HA) sequence of circulating viruses were analyzed to investigate the emergence of influenza viral variants. In the surveyed area, the influenza activity of these two post-pandemic seasons was similar in terms of both time frame and impact. The timing of the influenza epidemics was similar to the timing seen prior to the emergence of the pandemic A(H1N1) virus in 2009. A(H1N1)pdm09 was the predominant virus circulating during the 2010-2011 post-pandemic season and then--unexpectedly--almost disappeared. The HA sequences of these A(H1N1)pdm09 viruses segregated in a different genetic group with respect to those identified during the 2009 pandemic, although they were still closely related to the vaccine viral strain A/California/07/2009. Influenza A(H3N2) viruses were the predominant viruses circulating during the 2011-2012 season, accounting for nearly 88% of influenza viruses identified. All HA sequences of the A(H3N2) viruses isolated in the 2011-2012 season fell into the A/Victoria/208/2009 genetic clade (although the A/Perth/16/2009 virus was the reference vaccine strain). B viruses presented with a mixed circulation of viral variants during these two seasons: viruses belonging to both B/Victoria and B/Yamagata lineages co-circulated in different proportions, with a notable rise in the proportion of B/Yamagata viruses (B/Wisconsin/1/2010-like) during the 2011-2012 epidemic. In conclusion, the continuous monitoring of the characteristics of circulating viruses is an essential tool for understanding the epidemiological and virological features of influenza viruses, for monitoring their matching with seasonal vaccine strains, and for tuning vaccination strategies.

Influenza A(H3N2) virus is once again the predominant strain after the 2009 pandemic. Its molecular epidemiology and phylogeny were investigated during the 2011-2012 season in Northern Italy. The epidemiological and virological influenza surveillance was carried out within the framework of the Italian Influenza Surveillance Network. The hemagglutinin (HA) gene of the A(H3N2) viruses detected was analyzed by means of a time-scaled phylogenetic approach. In Northern Italy, the 2011-2012 epidemic wave was sustained almost exclusively by influenza A(H3N2) viruses (87.2% of total influenza virus detections). The consultation rates for influenza-like illness (ILI) in the age group ≥65 years were 1.5 to 6-fold higher than those registered during the previous eight epidemics: A(H3N2) was the only virus identified in this group. The phylogenetic analysis of A(H3N2) viruses showed viruses belonging to the A/Victoria/208/2009 genetic clade, characterized by substitutions in HA antigenic sites with respect to the A/Perth/16/2009-like 2011-2012 vaccine strain. About one-third of analyzed sequences fell into group 6 and two thirds into group 3 (subdivided into 3A, 3B, and 3C). The time scale reconstruction of the phylogeny showed several independent introductions of A(H3N2) groups between summer and winter of 2011. However, the common origin of all the circulating A(H3N2) strains dated back to the 2009 pandemic period (November 2009). The time scale phylogenetic approach is of particular importance for the evaluation of the introduction and circulation of new variants in the area. Therefore, it should be implemented within the framework of influenza virological surveillance.

Background The pan-influenza A real-time RT-PCR detection assay developed by the Centers for Disease Control and Prevention (CDC) during the 2009 pandemic is widely utilized. A quantitative version of the assay may be useful to monitor influenza A infection and response to treatment. Objectives To prove in principle the possibility that a virtual quantification tool (VQT) would allow conversion of CDC real-time RT-PCR cycle threshold (Ct) values in virus RNA copy number. Study design A plasmid carrying the CDC real-time RT-PCR target region of the influenza A Matrix (M) gene was generated. In a multicenter study, a set of 5 ten-fold dilutions (equivalent to 1 × 10 2 to 1 × 10 6 copies/reaction) were prepared and distributed to the 4 participating virology laboratories and then amplified to generate a virtual quantification standard curve. Clinical samples ( n = 120) were quantified in parallel by interpolation with locally generated standard curves and using the VQT. Results A total of 40 standard curves were obtained by the participating centers (ten from each center). The intra- and inter-laboratory variability showed a coefficient of variation (CV) ≤5%. Influenza A virus quantification in 120 respiratory samples showed a significant correlation between interpolation with locally generated standard curves and the VQT ( R 2 = 0.9655). Bland Altman analysis showed that the majority (no. 111, 92.5%) of clinical samples had 10 variation. Conclusions VQT proofs the concept that qualitative results from real-time RT-PCR assays can be converted into quantitative determination of virus load in clinical samples without running standard curves in parallel.

The 2014/15 influenza season in Europe was characterised by the circulation of influenza A(H3N2) viruses with an antigenic and genetic mismatch from the vaccine strain A/Texas/50/2012(H3N2) recommended for the Northern hemisphere for the 2014/15 season. Italy, differently from other EU countries where most of the subtyped influenza A viruses were H3N2, experienced a 2014/15 season characterized by an extended circulation of two influenza viruses: A(H1N1)pdm09 and A(H3N2), that both contributed substantially to morbidity. Within the context of the existing National sentinel influenza surveillance system (InfluNet) a test-negative case-control study was established in order to produce vaccine effectiveness (VE) estimates. The point estimates VE were adjusted by age group (

Please cite this paper as: Zuccotti et al. (2011) Epidemiological and clinical features of respiratory viral infections in hospitalized children during the circulation of influenza virus A(H1N1) 2009. Influenza and Other Respiratory Viruses 5(6), e528–e534. Background Seasonal influenza viruses and respiratory syncytial virus (RSV) are primary causes of acute respiratory tract infections (ARTIs) in children. New respiratory viruses including human metapneumovirus (hMPV), human bocavirus (hBoV), and influenza 2009 A(H1N1) virus have a strong impact on the pediatric population. Objectives To evaluate epidemiological and clinical features of ARTIs in hospitalized children. Methods From December 1, 2008, to December 31, 2009, all children under age fifteen (n = 575) hospitalized for ARTIs were investigated for influenza A (subtype H1N1, H3N2, and 2009 H1N1) and B, RSV A and B, hMPV, and hBoV by PCR. Results Fifty-one percent of samples were positive for these respiratory viruses. The frequencies of virus detection were RSV 34·1%, hBoV 6·8%, hMPV 5%, seasonal influenza A 5%, and seasonal influenza B 0%. From April 2009, 11·6% of collected samples were influenza 2009 A(H1N1) positive. Respiratory syncytial virus activity peaked in January, hBoV in February, and hMPV in April. Seasonal influenza A was detected only between January and April 2009, while influenza 2009 A(H1N1) peaked in November. Respiratory syncytial virus and hMPV were mainly associated with lower respiratory tract infections (LRTIs) and with necessity of O2 administration. The 2009 pandemic influenza was more frequently detected in elder children (P

The discovery of human Metapneumovirus (hMPV) and human Bocavirus (hBoV) identified the etiological causes of several cases of acute respiratory tract infections in children. This report describes the molecular epidemiology of hMPV and hBoV infections observed following viral surveillance of children hospitalized for acute respiratory tract infections in Milan, Italy. Pharyngeal swabs were collected from 240 children ≤3 years of age (130 males, 110 females; median age, 5.0 months; IQR, 2.0–12.5 months) and tested for respiratory viruses, including hMPV and hBoV, by molecular methods. hMPV‐RNA and hBoV‐DNA positive samples were characterized molecularly and a phylogenetical analysis was performed. PCR analysis identified 131/240 (54.6%) samples positive for at least one virus. The frequency of hMPV and hBoV infections was similar (8.3% and 12.1%, respectively). Both infections were associated with lower respiratory tract infections: hMPV was present as a single infectious agent in 7.2% of children with bronchiolitis, hBoV was associated with 18.5% of pediatric pneumonias and identified frequently as a single etiological agent. Genetically distinct hMPV and hBoV strains were identified in children examined with respiratory tract infections. Phylogenetic analysis showed an increased prevalence of hMPV genotype A (A2b sublineage) compared to genotype B (80% vs. 20%, respectively) and of the hBoV genotype St2 compared to genotype St1 (71.4% vs. 28.6%, respectively). Interestingly, a shift in hMPV infections resulting from A2 strains has been observed in recent years. In addition, the occurrence of recombination events between two hBoV strains with a breakpoint located in the VP1/VP2 region was identified. J. Med. Virol. 83:156–164, 2011. © 2010 Wiley‐Liss, Inc.

During the 2006-2007 season, 19 HIV-uninfected and 33 HIV-infected children and adolescents with full immunovirologic response to HAART were immunized against influenza and subsequently followed up. One month post-immunization all subjects had protective antibodies titres which persisted for the whole influenza season. Seven vaccinees (four HIV-infected and three HIV-uninfected) were found to be infected by influenza viruses during the epidemic, but disease was lab-confirmed only in two HIV-infected subjects. Both presented a benign clinical course and were infected by an A/Brisbane/10/07-H3N2-like virus. These data indicate that HIV-infected subjects benefit from routine seasonal influenza vaccination.

To evaluate the long-lasting immunogenicity and reactogenicity of a virosomal influenza vaccine in subjects with type I diabetes, a trial was conducted during the 2007-2008 influenza season in Milan, Northern Italy. One hundred five subjects aged 9-30 years were randomized to receive by intramuscular injection vaccination by a single dose (0.5 ml) of either a virosomal (Inflexal V) (n=52) or a standard subunit (Influvac) (n=53) vaccine. Serum hemagglutinin inhibition antibody titres were determined against the three recommended influenza-like strains, A/H1N1, A/H3N2 and B, at pre-vaccination, and 1 and 6 months post-vaccination. Geometric mean titres were increased in the two groups 1 and 6 months post-vaccination (P0.001). One month post-vaccination both vaccines met the CPMP requirement for immunogenicity with high seroprotection rates (95%) for strains A/H1N1 and A/H3N2, and a seroprotection of 73% and 70% in the virosomal and subunit vaccine for strain B. Mean fold increase ranged 2.8 (A/H3N2)-6.2 (A/H1N1) in the virosomal group and 2.3 (A/H3N2)-4.8 (A/H1N1) in the subunit group. Immunogenicity declined 6 months post-vaccination in both groups, and the CPMP requirement for immunogenicity was satisfied only in the virosomal group. In subjects without pre-existing antibodies to strain B (titre10), the virosomal vaccine showed higher immune response than the subunit vaccine 6 months post-vaccination, with a geometric mean titre (95% CI) of 40.2 (30.7-54.6) vs. 21.2 (14.6-30.8). Reactogenicity was similar in the two vaccines. All reactions were transient and not severe. The results indicate that in older children and young adults with type I diabetes influenza vaccination with a virosomal or a standard subunit vaccine is safe and adequately immunogenic against the three influenza vaccine strains. In addition, the virosomal vaccine may show better long-lasting immune response than the standard subunit vaccine, especially in subjects without pre-existing antibodies to influenza strains.

Measles and rubella are infectious diseases and humans are the only reservoir of these infections. Effective vaccines are available with the potential for measles (MV) and rubella (RuV) virus eradication. According to the World Health Organisation guidelines, a national plan was approved in Italy in 2013 to achieve the MV/RuV elimination by 2015, and active MV/RuV integrated surveillance initiated. Towards this purpose, a regional laboratory centre was set up on 1 September 2013 in Lombardy, Northern Italy. This paper aimed at: (1) evaluating measles-mumps-rubella (MMR) vaccine coverage and MV/RuV notified cases retrospectively; and (2) presenting the results of MV/RuV integrated surveillance (laboratory confirmed and viral genetic profiles). The 95% target for MMR vaccine coverage was achieved in 2001, and coverage increased until 2007 (96.6%), but then a decreasing trend was observed. Since 2000 to 2014, 3026 rubella cases were notified, with nearly 58% of them in the 2002 epidemic. From 2009, less than 45 RuV cases per year were reported. From 2000 to 2014, 5024 measles cases were notified. Since 2008, three large outbreaks (in 2008, 2011, and 2013) were observed. From data obtained during our surveillance activity, there were no rubella cases, and 57.5% (46/80) collected samples were MV-positive by real-time RT-PCR. A fragment of the MV N gene was sequenced from 37 MV-positive samples; D8, D9, and B3 genotypes were detected. Data obtained retrospectively and from active surveillance underline the necessity to achieve and maintain high vaccination coverage and to improve surveillance and the effectiveness of healthcare actions.

Since 2009 pandemic, international health authorities recommended monitoring severe and complicated cases of respiratory disease, that is, severe acute respiratory infection (SARI) and acute respiratory distress syndrome (ARDS). We evaluated the proportion of SARI/ARDS cases and deaths due to influenza A(H1N1)pdm09 infection and the impact of other respiratory viruses during pandemic and postpandemic period (2009-2011) in northern Italy; additionally we searched for unknown viruses in those cases for which diagnosis remained negative. 206 respiratory samples were collected from SARI/ARDS cases and analyzed by real-time RT-PCR/PCR to investigate influenza viruses and other common respiratory pathogens; also, a virus discovery technique (VIDISCA-454) was applied on those samples tested negative to all pathogens. Influenza A(H1N1)pdm09 virus was detected in 58.3% of specimens, with a case fatality rate of 11.3%. The impact of other respiratory viruses was 19.4%, and the most commonly detected viruses were human rhinovirus/enterovirus and influenza A(H3N2). VIDISCA-454 enabled the identification of one previously undiagnosed measles infection. Nearly 22% of SARI/ARDS cases did not obtain a definite diagnosis. In clinical practice, great efforts should be dedicated to improving the diagnosis of severe respiratory disease; the introduction of innovative molecular technologies, as VIDISCA-454, will certainly help in reducing such "diagnostic gap." © 2014 Elena Pariani et al

The aim of this study was to reconstruct the evolutionary dynamics of the A(H1N1)pdm09 influenza virus in Italy during two epidemic seasons (2009/2010 and 2010/2011) in the light of the forces driving the evolution of the virus. Nearly six thousands respiratory specimens were collected from patients with influenza-like illness within the framework of the Italian Influenza Surveillance Network, and the A(H1N1)pdm09 hemagglutinin (HA) gene was amplified and directly sequenced from 227 of these. Phylodynamic and phylogeographical analyses were made using a Bayesian Markov Chain Monte Carlo method, and codon-specific positive selection acting on the HA coding sequence was evaluated. The global and local phylogenetic analyses showed that all of the Italian sequences sampled in the post-pandemic (2010/2011) season grouped into at least four highly significant Italian clades, whereas those of the pandemic season (2009/2010) were interspersed with isolates from other countries at the tree root. The time of the most recent common ancestor of the strains circulating in the pandemic season in Italy was estimated to be between the spring and summer of 2009, whereas the Italian clades of the post-pandemic season originated in the spring of 2010 and showed radiation in the summer/autumn of the same year; this was confirmed by a Bayesian skyline plot showing the biphasic growth of the effective number of infections. The local phylogeography analysis showed that the first season of infection originated in Northern Italian localities with high density populations, whereas the second involved less densely populated localities, in line with a gravity-like model of geographical dispersion. Two HA sites, codons 97 and 222, were under positive selection. In conclusion, the A(H1N1)pdm09 virus was introduced into Italy in the spring of 2009 by means of multiple importations. This was followed by repeated founder effects in the post-pandemic period that originated specific Italian clades.

Background: Since its appearance in 2009, the pandemic influenza A(H1N1) virus circulated worldwide causing several severe infections. Methods: Respiratory samples from patients with 2009 influenza A(H1N1) and acute respiratory distress attending 24 intensive care units (ICUs) as well as from patients with lower respiratory tract infections not requiring ICU admission and community upper respiratory tract infections in the Lombardy region (10 million inhabitants) of Italy during the 2010-2011 winter-spring season, were analyzed. Results: In patients with severe ILI, the viral load was higher in bronchoalveolar lavage (BAL) with respect to nasal swab (NS), (p

A cross-sectional study was carried out to improve the state of evidence regarding the spectrum of HPV types and HPV-16 LCR variants circulating among men and women infected with HIV-1 in Italy. This study, conducted in 518 HIV-positive subjects (346 males and 172 females), showed a high prevalence of HPV anal infections (88.7%) in men and of cervical infections (65.1%) in women. A wide spectrum of HPV genotypes has been observed, as both single and multiple infections. Low-risk HPV types 6, 11 and 61 were frequently detected. HPV-16 was the prevalent high-risk type. Fourteen different HPV-16 LCR variants were found. Ten belonged to the European lineage (78.7% were detected in Italian subjects and 21.3% in foreign-born, all homo/bisexual men), two to the Asiatic lineage and two to the African-2 lineage. This study underlines the great genotypic heterogeneity characterizing anal and cervical HPV infections and the marked polymorphism of the predominant HPV-16 in this high-risk population in Italy.

To assess whether the administration of a booster dose of influenza vaccine may enhance immune response in hemodialysis patients, 58 subjects were given two doses of the 2003/2004 season influenza vaccine, 1 month apart. ‘‘European Agency for the Evaluation of Medicinal Products’’ (EMEA) criteria were fully met in terms of percentage of response and of mean-fold increase of hemagglutination inhibiting (HI) antibody titer, but not in terms of seroprotection rates (HI antibody titers � 1:40). The second vaccine administration did not result in additional increase in seroprotection rate or in geometric mean titers. Protective immune response against the epidemic A/H3N2 Fujian-like strain, antigenically distant from that included in the vaccine (A/Panama/2007/99) was observed in94.7% of vaccineesprotected against the A/H3N2 vaccine strain 1 month after immunization. No adverse reactions were reported during follow-up. The study findings suggest that immune response to influenza vaccination may be suboptimal in hemodialysis patients and that the administration of an additional second dose of vaccine does not improve the humoral response. J. Med. Virol. 79:1176–1179

In order to evaluate the immunogenicity and the effect of a virosomal influenza vaccine on viral replication and T-cell activation in HIV-infected children receiving highly active antiretroviral therapy (HAART), 29 children infected with HIV-1 vertically (19 primed with a previous influenza vaccination and 10 who were not been immunized against influenza) were immunized with an intramuscular virosome-adjuvanted influenza vaccine. According to the European Agency for Evaluation of Medical Products (EMEA) criteria, the immunogenicity of the vaccine was adequate against all three influenza strains (A H1N1, A H3N2, and B) in the primed children, and against A H1N1 and A H3N2 in the unprimed children. After in vitro stimulation with vaccine antigens, the IFN-γ levels in the peripheral blood mononuclear cells cultures increased significantly from a baseline level of 103.0 ± 229.8 pg/ml to a 30-day level of 390.7 ± 606.3 pg/ml (P

Acute flaccid myelitis; Enterovirus D68; Surveillance Mielitis flàcida aguda; Enterovirus D68; Vigilància Mielitis flácida aguda; Enterovirus D68; Vigilancia We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.