Giovanni Ianiro

Rotavirus Genotypes in Sewage Treatment Plants and in Children Hospitalized with Acute Diarrhea in Italy in 2010 and 2011

ABSTRACT Although the molecular surveillance network RotaNet-Italy provides useful nationwide data on rotaviruses causing severe acute gastroenteritis in children in Italy, scarce information is available on rotavirus circulation in the general Italian population, including adults with mild or asymptomatic infection. We investigated the genotypes of rotaviruses present in urban wastewaters and compared them with those of viral strains from clinical pediatric cases. During 2010 and 2011, 285 sewage samples from 4 Italian cities were tested by reverse transcription-PCRs (RT-PCRs) specific for rotavirus VP7 and VP4 genes. Rotavirus was detected in 172 (60.4%) samples, 26 of which contained multiple rotavirus G (VP7 gene) genotypes, for a total of 198 G types. Thirty-two samples also contained multiple P (VP4 gene) genotypes, yielding 204 P types in 172 samples. Genotype G1 accounted for 65.6% of rotaviruses typed, followed by genotypes G2 (20.2%), G9 (7.6%), G4 (4.6%), G6 (1.0%), G3 (0.5%), and G26 (0.5%). VP4 genotype P[8] accounted for 75.0% of strains, genotype P[4] accounted for 23.0% of strains, and the uncommon genotypes P[6], P[9], P[14], and P[19] accounted for 2.0% of strains altogether. These rotavirus genotypes were also found in pediatric patients hospitalized in the same areas and years but in different proportions. Specifically, genotypes G2, G9, and P[4] were more prevalent in sewage samples than among samples from patients, which suggests either a larger circulation of the latter strains through the general population not requiring medical care or their greater survival in wastewaters. A high level of nucleotide identity in the G1, G2, and G6 VP7 sequences was observed between strains from the environment and those from patients.

Genetic diversity of G9P[8] rotavirus strains circulating in Italy in 2007 and 2010 as determined by whole genome sequencing

This study reports the molecular characterization of G9P[8] rotavirus strains from children with acute diarrhea identified in different cities of Italy, in 2007 and 2010. Seventeen samples exhibited a G9P[8] genotype by RT-PCR and semi-nested PCR. Preliminary sequence analysis of the VP7 and VP8(*) encoding genes revealed nucleotide identities ranging between 96% and 100%. Full genome sequencing of four G9P[8] strains selected in different cities or years showed that the investigated Italian strains possessed a complete Wa-like genotype constellation. However, phylogenetic analyses assigned strains to different clusters reflecting point mutations and possibly earlier reassortment between Wa-like RVA strains. Deduced amino acid sequence of the VP7 and VP4 genes for the G9P[8] strains revealed at least five substitutions in relevant antigenic sites of both proteins.

Unexpected spreading of G12P[8] rotavirus strains among young children in a small area of central Italy

Rotavirus gastroenteritis is associated mainly with the five genotypes G1,3,4,9P[8] and G2P[4] that are common worldwide, but emerging strains including G6, G8, and G12 are also reported sporadically. G12P[8] rotavirus was observed unexpectedly to spread in a limited area of Italy during the rotavirus surveillance season 2012–2013. All strains were genotyped for VP7 and VP4 and subjected to phylogenetic analysis. Amino acid sequences of antigenic regions were compared with vaccine and field strains. G12P[8] strains were detected in the stools of 52 of 69 (75%) children infected with rotavirus in the central Italian region of Umbria. All G12 strains belonged to lineage III, and presented the P[8] genotype. Sequence analysis showed close nucleotide identity of both VP4 and VP7 genes among Umbria G12P[8] strains. The VP7 gene was also similar to other G12 strains circulating in different years and countries, and the VP4 gene was closely related to other local and global P[8] strains possessing different G‐types. Overall findings suggest either the introduction and evolution of a G12 VP7 gene into the local Wa‐like rotavirus population or the spreading of a strain novel for the area. Comparison of the VP8* and VP7 antigenic regions showed high conservation between the amino acid sequences of Umbria G12P[8] strains, and revealed various substitutions in the VP8* antigenic regions between the Italian G12P[8] strains and RotaTeq™ and Rotarix™ vaccine strains. The sudden and unexpected emergence of G12P[8] rotavirus confirms that these strains have the potential to become a sixth common genotype across the world. J. Med. Virol. 87:1292–1302, 2015.

Molecular characterization of human G8P[4] rotavirus strains in Italy: proposal of a more complete subclassification of the G8 genotype in three major lineages.

In 2011, two children with acute rotavirus gastroenteritis were hospitalized in Sardinia, Italy. Two RVA strains with G8P[4] genotype were detected in their stools, and were named SS56/2011 and SS65/2011. The aim of the study was to characterize these two rare strains, collected within a national RVA gastroenteritis surveillance program. Eight of the 11 RVA genes were sequenced and phylogenetic analysis performed. VP7 amino acid sequence was also analyzed. Sequencing of genes encoding the VP4, VP6, VP7, and NSP1-5 proteins classified both strains as G8-P[4]-I2-A2-N2-T2-E2-H2, not detected previously in Italy. Phylogenetic analysis revealed that most genes of Italian RVA strains were closely similar to typical DS-1 like strains circulating worldwide, whereas the VP7 gene was strictly related to G8 strains firstly reported in Africa. This finding of G8P[4] RVA strains with a DS-1 like genomic constellation also in a southern European country further confirms the wide circulation of this uncommon genotype in the world. Comparison of the deduced amino acid sequence of the VP7 capsid protein of the Italian G8P[4] RVA strains with sequences reported previously suggests that the G8 genotype should be divided into three major lineages.

Full-length genomic analysis of porcine rotavirus strains isolated from pigs with diarrhea in Northern Italy

Group A rotaviruses (RVA) cause acute dehydrating diarrhea in young of man and many animal species, including pigs. Swine RVA has an important economic impact on the farming industry, and pigs represent a potential reservoir for zoonotic transmission of RVA to humans. To investigate the genetic diversity of porcine RVA strains in Italy and identify their possible zoonotic characteristics, 25 RVA-positive feces were collected from diarrheic pigs in Northern Italy, in 2009-2010; all viral strains were characterized by G and P genotyping RT-PCR. Three samples were selected for full genome sequencing. Sequencing of the NSP3 genes of all samples was also performed. Rotavirus diagnosis was carried out by ELISA and electron microscopy. RT-PCR and Sanger sequencing were performed in a one-tube format, using primer sets specific for each of the 11 genome segments. Analysis of the G (VP7) and P (VP4) genotypes showed that all strains identified were typical porcine RVAs (G4, G5, G9; P[6], P[13], P[23]). Full-length genome sequencing was performed on selected G9 isolates. Most segments belonged to the genotype constellation 1 (Wa-like), which is shared by most human RVA strains, but gene types such as I5 (VP6) and A8 (NSP1), which are typical of porcine and rare among human RVAs, were also detected. We identified RVA strains showing the T7 genotype, an NSP3 gene type that was previously reported in unusual strains of possible porcine or bovine origin from children with diarrhea. Recent reports suggested that G9 RVA may have been introduced from swine to human populations involving gene reassortment events. The observation that some of the RVA genotypes from swine in Italy were similar to viruses characterized in children underlines the importance of animal RVA surveillance, to clarify and monitor the role of animals as genetic reservoirs of emerging RVA strains pathogenic for humans.

Detection of unusual G6 rotavirus strains in Italian children with diarrhoea during the 2011 surveillance season.

Two rare G6 rotavirus A (RVA) strains, designated as RVA/human‐wt/ITA/CEC06/2011/G6P[6] and RVA/human‐wt/ITA/PG05/2011/G6P[9], were identified in stool specimens from children hospitalized in Central Italy. After PCR genotyping, the samples CEC06 and PG05 gave G‐UD‐P[6] and G‐UD‐P[9] genotypes, respectively. To determine the G‐type and to characterize further the two strains, sequencing of 8 of the 11 genomic segments was performed. CEC06 and PG05 strains were found to possess unusual genotype constellations: G6‐P[6]‐I2‐A2‐N2‐T2‐E2‐H2 and G6‐P[9]‐I2‐A3‐N2‐T3‐E3‐H3, respectively. This study reports the first detection of rare G6P[6] and G6P[9] RVA strains in peninsular Italy. Phylogenetic analysis of VP4 (VP8*), VP7, VP6, and NSP1‐5 showed no evidence of zoonosis or inter‐species reassortment, revealing for both strains constellations previously associated to human cases. J Med. Virol. 85:1860–1869, 2013.

Full-Genome Characterization of a G8P[8] Rotavirus That Emerged among Children with Diarrhea in Croatia in 2006

ABSTRACT The whole genome of a G8P[8] rotavirus from the 2006 epidemic in Croatia was sequenced and showed a Wa-like genotype constellation. Its VP7 gene clustered with DS-1-like G8 African rotaviruses and a G8P[4] German strain. Remaining genes clustered with contemporary Belgian G1P[8] rotaviruses, suggesting reassortment between human G8 and G1P[8] rotaviruses in Croatia or other European countries.

Prevalence of Foodborne Viruses in Mussels in Southern Italy

In this study, the prevalence of various enteric viruses in Mytilus galloprovincialis (Mediterranean mussel) belonging to class A and class B mollusc-harvesting areas in the Campania region in southern Italy was evaluated. One hundred and eight mussels were analysed using real-time reverse transcription PCR during a 2-year collection period (2014–2015) to detect the following viruses: human norovirus (genogroups I and II), rotavirus, astrovirus, sapovirus, aichivirus, hepatitis A virus and hepatitis E virus. Overall, 50.93% of mussels were contaminated by at least one of the tested viruses. Of these virus-positive mussels, 63.63% were contaminated by two or more viruses. In 2014, only three of the eight investigated viruses were detected: astrovirus, sapovirus and aichivirus, whereas in 2015, seven of the eight viruses were detected (only hepatitis E virus was not identified). Astrovirus was the most frequently detected virus in both sampling periods. In 2014, sapovirus was detected at the same frequency as astrovirus (16.00%), followed by aichivirus (8%). In 2015, astrovirus (32.53%) was most frequently detected, followed by norovirus GII (26.50%), sapovirus (18.07%), hepatitis A virus (16.87%), rotavirus (16.87%), aichivirus (13.25%) and norovirus GI (12.05%).This study describes, for the first time, the presence of aichivirus and sapovirus in mussels in Italy.

Molecular analysis of group A rotaviruses detected in adults and adolescents with severe acute gastroenteritis in Italy in 2012

Hospital‐based surveillance of acute gastroenteritis caused by rotavirus has produced ample knowledge on the infection in children, whereas little is known on rotavirus infection among adults. The Italian surveillance program RotaNet‐Italia collected 1,595 samples from patients admitted to hospital with gastroenteritis in 2012. All patients presented with vomiting, diarrhea, fever, and/or abdominal pain. Forty‐two samples obtained by the RotaNet‐Italia (2.6%) were from adolescents or adults (10–89 years). The study compared the genotypes and gene sequences of the rotavirus strains identified in adults with strains obtained from children worldwide. All 42 Italian strains were genotyped by the EuroRotaNet RT‐nested‐PCR protocols, and 12 rotaviruses from patients >13‐year‐old were subjected to nucleotide sequencing of their VP7 and/or VP4 genes. All strains analyzed belonged to the common human genotypes G1P[8], G2P[4], G4P[8], and G9P[8], except an uncommon G3P[19] genotype detected in a single patient. Phylogenetic analysis of the 12 strains showed that within each genotype they clustered in RVA lineages reported worldwide. The amino acid sequences of the VP7 and the VP8* hypervariable regions were highly conserved between the RVA strains collected from adults and children, in each lineage. Genotyping, phylogenetic analysis, and the study of viral epitopes revealed that rotaviruses circulating in adults in Italy are closely similar to the strains circulating in children, with high nucleotide identity particularly with strains reported in Europe and Asia. The circulation of the same rotavirus strains in both children and adults suggests that adults may contribute to sustain the circulation of rotaviruses through the population. J. Med. Virol. 86:1073–1082, 2014.

Detection of Rare G3P[19] Group A Rotavirus in Human Patient, Italy

Infection with a rare G3P[19] rotavirus A strain was identified in an immunosuppressed patient in Italy. The strain showed a P[19] viral protein 4 gene and a complete AU-1–like genomic constellation. Phylogenetic analyses showed high nucleotide identity between this strain and G3P[19] rotavirus A strains from Asia, indicating possible reassortment events.