Genetic variability of the measles virus hemagglutinin gene in B3 genotype strains circulating in Northern Italy.

Sequencing the whole measles virus hemagglutinin (H) gene, in conjunction with a 450-nucleotide region of the nucleoprotein gene (N-450), is helpful for the identification of new genotypes and as an auxiliary in outbreak characterization. In addition, it is essential to be able to predict the antigenic changes of the H protein to gain a better monitoring of the response to the vaccine. In this study, we obtained the full-length H gene sequences from 19 measles virus (MV) strains belonging to two B3 genotype variants circulating in Lombardy (Northern Italy) between July 2015 and February 2016 and evaluated the variability of the whole MV-H gene. Furthermore, we compared the obtained H amino acid sequences to all MV sequences available in the GenBank database (n = 1152 in total) and analyzed the amino acid substitutions in the H protein within clades where the Italian strains were included. We identified a higher variability in the H gene compared to the N-450 region and our results support previous studies, highlighting that the H gene is more informative for characterizing the MV B3 genotype than the N-450 sequence. Some of the amino acid substitutions were fixed in the viral population and, remarkably, some of the amino acid substitutions were typically present only in the Italian sequences. Accumulating further molecular information about MV-H gene will be necessary to enable in-depth analyses of the variability of this gene in the vaccinated population.

Phylogenetic characterization of Central/Southern European lineage 2 West Nile virus: analysis of human outbreaks in Italy and Greece, 2013-2014.

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.

Chikungunya infection in a human immunodeficiency virus-infected kidney transplant recipient returning to Italy from the Dominican Republic.

Since December 2013, chikungunya virus (CHIKV) spread in many countries of the Western Hemisphere, and during the last year some cases of infected European travelers, coming back from the Caribbean, have been reported. The risk of acquiring severe travel-related illness is higher in immunocompromised subjects, such as patients with human immunodeficiency virus (HIV) infection or solid organ transplant recipients. We reported the first case, to our knowledge, of CHIKV infection in an HIV-infected kidney transplant recipient.

Different drug-resistant influenza A(H3N2) variants in two immunocompromised patients treated with oseltamivir during the 2011-2012 influenza season in Italy.

BACKGROUND: Monitoring the emergence of drug-resistant influenza variants is crucial in influenza surveillance programs. OBJECTIVES: Influenza A kinetics and the emergence of drug-resistant strains in hospitalized patients treated with oseltamivir were investigated. STUDY DESIGN: Sequential samples from oseltamivir-treated and -untreated hospitalized patients in the period November 2011 through April 2012 were analyzed. NA gene was sequenced in samples from oseltamivir treated patients. Clonal analysis of the viral population was performed in patients unresponsive to treatment. Viral kinetics was determined in 24 (14 immunocompromised and 10 immunocompetent) A(H3N2)-positive patients treated and 24 (10 immunocompromised and 14 immunocompetent) untreated patients. RESULTS: Viral shedding was significantly reduced in treated vs untreated immunocompromised patients (7 vs 22 days, p<0.05, respectively). Viral load decreased significantly in immunocompromised and immunocompetent treated patients as compared with immunocompromised and immunocompetent untreated patients (0.73 and 0.93 vs 0.47 and 0.45 log10/day, p<0.05). In two (8.3%) treated patients with prolonged virus shedding, the oseltamivir resistance R292K mutation was revealed. In these patients, clonal analysis of the virus population showed the presence of additional oseltamivir-resistant mutants (E119V, N294S and deletion Del247-250). CONCLUSIONS: Oseltamivir resistance is reported for the first time in A(H3N2) virus strains during the 2011-2012 influenza season. Different drug-resistant viruses emerged in hospitalized immunocompromised patients showing prolonged virus shedding.

Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing.

This study was aimed at establishing the genetic heterogeneity of influenza virus haemagglutinin (HA) gene quasi-species and the polymorphisms at codon 222, by application of ultra-deep pyrosequencing (UDPS) to respiratory samples from patients hospitalized for influenza A(H1N1)pdm09 infection, presenting with severe or moderate-mild disease. HA diversity was significantly higher in samples collected from patients with severe manifestations than in those from patients with moderate-mild manifestations (p 0.02). D222 polymorphism was detected in 40.7% of patients by UDPS, and in only 7.1% by Sanger sequencing. D222E, D222G, D222N and D222A were observed in 37.0%, 11.1%, 7.4% and 3.7% of patients, respectively; 10.7% of samples harboured more than two variants. The relative frequency of each single variant showed a wide range of intrapatient variation. D222G/N/A were detected, as either minor or predominant variants, only in severe cases, whereas D222E was equally represented in severe and moderate-mild infections. Other amino acid variants were observed at different positions within the analysed HA fragment. Consistent with higher heterogeneity, non-D222 variants were more frequently detected in severe cases than in moderate-mild cases. In addition, seven non-D222 mutations carried by minority variants, not previously described, were observed.

Early co-circulation of different clades of influenza A/H1N1v pandemic virus in northern Italy.

INTRODUCTION: The spatial diffusion over time of pandemic influenza A/HINI virus (A/HIN1v) was surveyed in Northern Italy (nearly 10 million inhabitants)from April to December 2009, and the molecular characteristics of circulating viruses were analyzed to identify the appearance of drift variants. About 45% of analyzed samples were laboratory-confirmed cases of A/HINlv. Sporadic cases occurred until the middle of June 2009, then, case numbers began to increase delineating distinct epidemiological phases of viral circulation. METHODS: RNA was extracted using RNeasy Mini kit (QIAGEN GmbH, Germany). Virological diagnosis of A/HINlv infection was carried out by real-time RT-PCR assay. Sequence analysis of hemagglutinin (HA) gene was performed through a RT-PCR assay specific for a 995 bp fragment (nt. 64-1,058) in the HAl domain. The nucleotide sequences were obtained by automated DNA sequencing. The HAl sequences were aligned with other sequences collected from GenBank database by ClustalX software. The multiple sequence alignment was used to perform a basic phylogenetic analysis and a phylogenetic tree from HA sequences was constructed. RESULTS: The HA gene sequences ofA/HINlv analyzed segregated into three genetically distinct clades and were characterized by the appearance of amino acid variations that were progressively fixed in the field viral population under scrutiny. CONCLUSIONS: These data suggest an early co-circulation of genetically distinct A/HNINv variants and emphasize the importance of a close molecular surveillance to detect rapidly the spread of new viral variants and to define their epidemiological impact.

Severe outcome of influenza A/H1N1/09v infection associated with 222G/N polymorphisms in the haemagglutinin: a multicentre study.

In a multicentre study, influenza A/H1N1/09v 222G/N variants were more frequently detected in patients admitted to the intensive-care unit for invasive mechanical ventilation or extracorporeal membrane oxygenation (10/23; 43.5%) than in patients hospitalized in other units (2/27; 7.4%) and community patients (0/81; 0.0%) (p <0.01). A significantly higher virus load (p 0.02) in the lower vs the upper respiratory tract was observed. Predominance of 222G/N variants in the lower respiratory tract (40% of total virus population) vs the upper respiratory tract (10%) was shown by clonal analysis of haemagglutinin sequences in paired nasal swab and bronchoalveolar lavage samples. The time from illness onset to sampling was significantly longer in patients with severe infection vs community patients (p <0.001). It was concluded that the 222G/N variants showed increased virulence; mutant variants were probably selected in individual patients; and the longer duration of illness might have favoured the emergence of adaptive mutations through multiple replication cycles.

Correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients.

While human rhinoviruses (HRVs) are well accepted as a major cause of common cold syndromes (rhinitis), their role in the etiology of lower respiratory tract infections is still controversial, and their detection in asymptomatic patients is relatively common. The HRV pathogenic role in four groups of hospitalized patients (pediatric immunocompetent and immunocompromised patients, and adult immunocompetent and immunocompromised patients) was investigated by quantifying HRV load in nasopharyngeal aspirates or bronchoalveolar lavage samples by real-time reverse transcription PCR (RT-PCR). Real-time RT-PCR was performed in duplicate on all respiratory samples resulting positive by qualitative RT-PCR. In addition, molecular typing allowed detection of all known HRV species (A, B, and C). In immunocompetent pediatric patients HRVs were mostly associated with lower respiratory tract infections (in the absence of other viral agents) and wheezing, when viral load was > or =10(6) RNA copies/ml. In young immunocompromised patients (stem cell transplantation recipients), an inverse correlation between HRV persistence over time and time at which the infection occurred after transplantation was observed, whereas in adult immunocompromised patients (lung transplant recipients) HRVs could be detected at a medium-low level (<10(5) RNA copies/ml) in bronchoalveolar lavage samples taken routinely from asymptomatic patients. In conclusion, when detected at high viral load, HRVs may cause severe upper and lower respiratory tract infections, whereas when detected at a medium-low viral load, an event more frequent in immunocompromised subjects, they may represent only bystander viruses.

Clinical severity and molecular typing of human rhinovirus C strains during a fall outbreak affecting hospitalized patients.

BACKGROUND: The circulation rate and the clinical severity of infections caused by members of the new human rhinovirus C (HRV-C) species remain to be defined. OBJECTIVES: To investigate the epidemiologic and clinical impact of HRV-C strains in a fall outbreak interesting hospitalized patients. STUDY DESIGN: HRV species (A-C) were determined by phylogenetic analysis following amplification of two genome regions (5'NCR and VP4/VP2) by RT-PCR. HRV species were correlated with age, respiratory tract involvement, clinical symptoms, and HRV load in respiratory secretions. RESULTS: During the first week of the period October-November 2008, single HRV infections were associated with 95% of all respiratory syndromes affecting hospitalized patients. Then, HRV infections (single+coinfections) interested about 90% of positive samples until the end of October, when they declined in frequency until reaching about 30% at the end of November. Overall, 104 HRV strains were detected and, of these, 90 could be classified by phylogenetic analysis, as follows: 45 HRV-A, 12 HRV-B, 28 HRV-C, and 5 human enterovirus D strains. HRV-C identity was confirmed by detection of cis-acting replication elements (cre) in 23/23 strains. As for severity of respiratory syndromes, unlike HRV-A and HRV-B strains, HRV-C strains were responsible for a significantly higher rate (p<0.05) of lower respiratory tract infections in the pediatric as compared to adult patient population. CONCLUSIONS: HRV-C strains have been shown to circulate at a rate intermediate between HRV-A and HRV-B strains, showing a greater degree of clinical severity in the pediatric population.

Correlation of viral load as determined by real-time RT-PCR and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy.

BACKGROUND: In infants hospitalized for a lower respiratory tract infection (RTI) caused by respiratory syncytial virus (RSV), the correlation between viral load (VL) and patient clinical characteristics remains to be defined. OBJECTIVES: To define this correlation. STUDY DESIGN: prospective study of 47 infants admitted to hospital in the period November 2006-May 2007 with a diagnosis of lower RTI. Nasopharyngeal aspirates (NPAs) were taken at admission, discharge, and at post-discharge control visits. VL was quantified by real-time RT-PCR for RSV subgroups A and B. RESULTS: Patients with bronchiolitis were compared with young patients with lower RTI other than bronchiolitis. Patients with bronchiolitis had a significantly lower age than patients with other syndromes, and a significantly longer duration of symptoms. Duration of hospitalization was not different in the two groups of patients, and was not related to RSV subgroup or viral coinfection. A sustained decrease in VL was observed in the general patient population between admission, discharge and post-discharge follow-up visits. CONCLUSIONS: (i) patients with bronchiolitis were significantly younger than patients with other lower RTIs; (ii) symptom duration was significantly longer in patients with bronchiolitis; (iii) RSV VL significantly decreased between admission and discharge.

Changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons. Brief report.

From 2001 through 2004, 808 pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or RT-PCR during three consecutive winter-spring seasons. On the whole, 336 (42%) patients were detected as positive for one or more respiratory viruses. The most widely circulating virus was human respiratory syncytial virus (hRSV) infecting 50% of positive patients, followed by human metapneumovirus (hMPV) found in 13% of patients, and then by influenza virus type A, human parainfluenzaviruses and coinfections. Significant variations in the circulation rate of hRSV, hMPV and influenzavirus type A were observed during the individual seasons. In addition, the circulation rates of the different types of hMPV changed yearly. In 2001-2002 and 2002-2003 hMPV circulated at a significant lower proportion than hRSV, while in 2003-2004 the circulation rates of the two viruses were closer. In conclusion, the 4 hMPV subtypes circulated yearly in Northern Italy flanking hRSV as major respiratory pathogens in the infantile patient population.