Recombinant GII.P16 genotype challenges RT-PCR-based typing in region A of norovirus genome.

OBJECTIVES: In latest years GII.4[P16] and GII.2[P16] noroviruses have become predominant in some temporal/geographical settings. In parallel with the emergence of the GII.P16 polymerase type, norovirus surveillance activity in Italy experienced increasing difficulties in generating sequence data on the RNA polymerase genomic region A, using the widely adopted JV12A/JV13B primer set. Two sets of modified primers (Deg1 and Deg2) were tested in order to improve amplification and typing of the polymerase gene. METHODS: Amplification and typing performance of region A primers was assessed in RT-PCR on 452 GII norovirus positive samples obtained from 2194 stool samples collected in 2016-2019 from children hospitalized with acute gastroenteritis. RESULTS: The use of Deg1 increased the rate of samples types in region A from 49.5% to 81.4% and from 21.9% to 69.7% in 2016 and 2017, respectively. The rate of Deg1 typed samples remained high in 2018 (90.1%), but sharply decreased to 11.8% in 2019. The second primers set, Deg2, was able to increase to 64.9% the rate of 2019 samples typed in region A, while typing efficiently 73.2%, 69%, and 86.4% of samples collected in 2016, 2017 and 2018, respectively. CONCLUSIONS: The plasticity of norovirus genomes requires continuous updates of the primers used for strain characterization.

Phylodynamic Analysis and Implication of HCV Genotype 4 Variability on Antiviral Drug Response and T-Cell Recognition.

Therapies for HCV care could change the prevalence and the geographic distribution of genotypes due to differences in Sustained Virologic Response (SVR). In this scenario, uncommon genotypes/subtypes, such as genotype 4, could spread from high-risk groups, replacing genotypes eradicated by antiviral drugs. Genotype eradication is also strongly influenced by the CD8+ T cell response. In this study, the genetic variability in HCV genotype 4 strains obtained from a cohort of 67 patients naïve to DAA therapy was evaluated. We found that the presence of resistance-associated substitutions (RAS) was able to affect drug responses. Next, using a prediction tool, viral mutations were identified by their ability, or lack thereof, to reduce the binding affinity with HLA, which affects T cell recognition. The Bayesian coalescent analysis suggested two different circulation clusters, one in risk groups (IDUs and MSM) and the other due to migration flows, dated to 1940 and 1915, respectively. Most of the RAS overlapped with HLA and a lack of binding mutations was observed in 96% of strains. This study describes the introduction of HCV genotype 4 in a region of the Mediterranean basin and evaluates how HCV genotype 4's genetic variability could affect the response of antiviral drugs and CD8+ T cell recognition.

Sentinel hospital-based surveillance for norovirus infection in children with gastroenteritis between 2015 and 2016 in Italy.

Noroviruses are one of the leading causes of gastro-enteric diseases worldwide in all age groups. Novel epidemic noroviruses with GII.P16 polymerase and GII.2 or GII.4 capsid type have emerged worldwide in late 2015 and in 2016. We performed a molecular epidemiological study of the noroviruses circulating in Italy to investigate the emergence of new norovirus strains. Sentinel hospital-based surveillance, in three different Italian regions, revealed increased prevalence of norovirus infection in children (<15 years) in 2016 (14.4% versus 9.8% in 2015) and the emergence of GII.P16 strains in late 2016, which accounted for 23.0% of norovirus infections. The majority of the strains with a GII.P16 polymerase showed a GII.2 capsid genotype (79.5%). Also, a marked circulation of strains with a GII.17 capsid (14.0%) was observed, chiefly in early 2016. The emergence and global spread of non-GII.4 noroviruses pose challenges for the development of vaccine strategies.

Performance evaluation of a newly developed molecular assay for the accurate diagnosis of gastroenteritis associated with norovirus of genogroup II.

The performance of a newly proposed fully automated cassette-based sample-to-results solution for norovirus (NoV) detection, InGenius Norovirus ELITe MGB®, was evaluated. A total of 120 selected archival stool samples from children hospitalized for acute gastroenteritis were used to compare the results to a reference real-time RT-PCR. The InGenius NoV assay showed optimal diagnostic accuracy (sensitivity, 100%; specificity, 95.7%) and was able to correctly detect the entire wide panel of epidemiologically relevant genotypes tested. These preliminary results suggest that the InGenius NoV assay can be recommended as a valuable method for accurate diagnosis of NoV GII infection in epidemic and sporadic gastroenteritis.

Is Italian population protected from Poliovirus? Results of a seroprevalence survey in Florence, Italy.

OBJECTIVES: Periodical assessments of population susceptibility to polioviruses (PV) is essential for evaluating population protection and planning appropriate vaccination strategies. The aim of the current work was to assess serological protective titers against all three polioviruses in the general population of Florence. METHODS: A convenience sample of 328 sera, collected in 2009 in Florence (Central Italy) was analyzed. Samples were considered protective if neutralizing antibodies were detected at dilutions ≥1:8, according to the WHO protocols. RESULTS: The immune coverage was 75.3%, 69.2% and 46% for PV1, PV2 and PV3, respectively. The protective titers of neutralizing antibodies were generally higher in children up to 14 years of age, with 74.4% (PV1), 75.6% (PV2) and 56.7% (PV3) of seroprevalence. From the age of 11 years, most of the study subjects were seronegative for PV3. CONCLUSIONS: In a polio-free country with strong migration pressures, such as Italy, our results bring clear support to the recent recommendation of Italian health authorities to introduce a fifth dose of IPV vaccine in adolescence all over the country.

Molecular evolutionary analysis of type-1 human astroviruses identifies putative sites under selection pressure on the capsid protein.

Human astroviruses (HAstV) are important enteric pathogens that can be classified into eight sero/genotypes (HAstV-1 to -8). Although the various HAstV types show global spread, type-1 strains tend to be predominant. Molecular analysis of the genomic region encoding the capsid protein (ORF2) has revealed discrete sequence variation, with different lineages within each HAstV type and at least three major lineages have been identified within HAstV-1. Longitudinal epidemiological surveillance has revealed temporal shift of the various HAstV-1 lineages. Metadata analysis of HAstV-1 sequences available in the databases also revealed temporal shifts of the circulation of HAstV-1 lineages, suggesting possible antigenic-related mechanisms of selection at the sub-genotype level. By comparison of HAstV-1 capsid sequences, lineage-defining residues under positive selection were identified. Structural analysis of HAstV-1 capsid allowed identifying at least six residues exposed on the virion surface. Two residues were located in the VP34 (shell region) whilst four residues were mapped in the VP25/27 (protruding region) of HAstV capsid protein, in proximity of the putative receptor binding S site. These findings suggest that mechanisms similar to those observed and/or hypothesized for other enteric viruses are also shaping the evolution of HAstVs, with intra-typic diversification being a possible mechanism to decrease the antigenic pressure to which these viruses are exposed.

Characterization of measles virus strains circulating in Southern Italy (Palermo area, Sicily) between 2010 and 2011.

Measles virus (MV) was classified in 24 genotypes that show a distinct geographic distribution. Genotypes contain multiple distinct lineages. In 2011 large outbreaks of measles occurred in Italy and in many European countries. Aims of this study are to analyze the intra-genotype variability and to follow the importation and the spread of new MV strains in Sicily. A fragment of 450 bps of MV C-terminal nucleoprotein was sequenced from sera of 73 Sicilian patients with symptomatic measles infections, occurred between 2010 and 2011. Five MV strains were D4 genotype and 68 were D8 genotype. The MV/D4 sequences were related to MV/D4-Enfield variant. Two lineages of MV/D8 genotypes, related to MV/D8-Villupuram variant and to a strain found in Birmingham in 2006 respectively, were identified. This is the first study that reports the co-circulation of different MV genotypes and lineages in Sicily suggesting multiple origins of the outbreak that occurred during 2010 and 2011 years.

Impact of HBV genotypes A and D genetic variability on infection evolution.

HBV is characterized by a high genetic variability, which is the basis of its classification into eight genotypes (A-H). HBV infection is associated with different outcomes, from self-limiting acute hepatitis to active chronic hepatitis, asymptomatic carriage, and occult infection. The aim of this study was to analyze the genetic variability of HBV genotypes A and D isolates from 79 cases of self-limiting acute hepatitis and chronic hepatitis, in order to identify HBV variants associated with resolution or chronicity of acute HBV infection. The entire preS-S sequence and a fragment of 346 bp of the preC-C region, containing Enhancer II and Basal Core Promoter sequences, were analyzed. A phylogenetic analysis of preS/S region showed that the 45.45% (15/33) of isolates from acute hepatitis cases were genotype A compared to 8.69% (4/46) of chronic hepatitis cases. (p = 0.0002). Mutations associated with immune-escape (T131N, D144A/E, G145K), amino acid polymorphisms in "a determinant" domain of S protein and mutations/deletions in preC/C region were found in isolates from acute and chronic hepatitis B cases. In this study mutations/deletions in preS-S and preC-C regions, usually associated with fulminant acute hepatitis, advanced forms of liver disease and increased risk for HCC, were identified in HBV strains of genotype A and D obtained both from patients with self-limiting acute HBV infection and from persistent infected patients. This founding probably is due to the natural viral evolution under host immune response and to the circulation of a wide variety of HBV strains in our geographic area because of the ancient introduction of genotype D and the migrant fluxes from North Africa. Moreover, the analysis of circulation of new HBV antigenic variants is fundamental for the epidemiological surveys and for the evaluation of the impact of viral evolution on vaccine prophylaxis strategies.

HCV-1b intra-subtype variability: Impact on genetic barrier to protease inhibitors.

Due to error-prone RNA polymerase and the lack of proofreading mechanisms, to the spread worldwide and probable long-term presence in human population, HCV showed a high degree of inter- and intra-subtype genetic variability. Protease inhibitors (PIs), a new class of drugs, have been designed specifically on the HCV genotype 1 NS3 protease three-dimensional structure. The viral genetic barrier limits the efficacy of PIs, and fourteen loci in the HCV NS3 gene are involved in resistance to PIs. A sensitive method (15UI/ml) for study the HCV genetic profile of 125 strains from patients naïve to PIs, was developed through the use of new degenerate primers for subtype 1b. We observed the presence of naturally resistance-associated variants in 14% of the HCV strains (V36L, F43S, T54S, I153V, R155Q, D168A/G). T54S was the most common mutation (4%) detected. We investigated, through minimal score (m.s.) calculating, how the HCV intra-subtype 1b variability modifies the genetic barrier to PIs. For >60% of strains a single transition (m.s. of 1) was required for selection of low to medium resistance mutations, while more than one transition/transversion (m.s. ⩾2.5) or one transition plus one transversion (m.s. ⩾3.5) was necessary for most of the high level PI-resistant-associated mutations, except for A156V, for which a single transition was sufficient (m.s. of 1). However, the presence at locus 36 of the amino acid polymorphism S36 in one case and the wild type V36 in 6 isolates, encoded by unusual GTA or GTG codons, might determined a higher probability of V36L/M mutations because of the reduction of the genetic barrier. Instead, the presence of the CGA and CGT codons in the 155(th) position increases the genetic barrier for R155M or R155Q/M. The large intra-subtype variability, suggests that a routine baseline resistance test must be used before PIs-treatment.

Phylogenetic analysis of isolates from new cases of HBV infection in Southern Italy.

The level of endemicity of hepatitis B virus (HBV) infections in Italy is low and genotype D infections predominant. New HBV strains may however be introduced as a result of movements of people from regions of high endemicity. The aim of the present study was to determine whether strains from new cases of acute hepatitis B detected in southern Italy were due to endemic or new HBV strains. We studied 34 isolates from patients with acute hepatitis B infection, and 35 from chronic hepatitis B patients. A phylogenetic analysis of preS/S region was done by comparing the sequences from the acute and chronic cases with references sequences. The study showed that 44% of strain from acute hepatitis B patients were of genotype A, 53% of genotype D, and 3% of genotype E. The molecular analysis of isolates from acute hepatitis B patients from Sicily showed a change in the local epidemiology of this infection, with an increase in HBV/A infections and a clustering effect for HBV D2, possibly correlated to immigration. The introduction of new genotypes , could have an effect on HBV-correlated diseases due to the different association between genotype, liver disease and response to antiviral therapy.