Poliovirus and Other Enteroviruses from Environmental Surveillance in Italy, 2009-2015.

Within the initiatives for poliomyelitis eradication by WHO, Italy activated an environmental surveillance (ES) in 2005. ES complements clinical Acute Flaccid Paralysis (AFP) surveillance for possible polio cases, detects poliovirus circulation in environmental sewage, and is used to monitor transmission in communities. In addition to polioviruses, the analyses comprised: (i) the monitoring of the presence of non-polio enteroviruses in sewage samples and (ii) the temporal and geographical distribution of the detected viruses. From 2009 to 2015, 2880 sewage samples were collected from eight cities participating in the surveillance. Overall, 1479 samples resulted positive for enteroviruses. No wild-type polioviruses were found, although four Sabin-like polioviruses were detected. The low degree of mutation found in the genomes of these four isolates suggests that these viruses have had a limited circulation in the population. All non-polio enteroviruses belonged to species B and the most frequent serotype was CV-B5, followed by CV-B4, E-11, E-6, E-7, CV-B3, and CV-B2. Variations in the frequency of different serotypes were also observed in different seasons and/or Italian areas. Environmental surveillance in Italy, as part of the 'WHO global polio eradication program', is a powerful tool to augment the polio surveillance and to investigate the silent circulation or the re-emergence of enteroviruses in the population.

Large-Scale Survey of Human Enteroviruses in Wastewater Treatment Plants of a Metropolitan Area of Southern Italy.

Human enteroviruses (HEVs) occur in high concentrations in wastewater and can contaminate receiving environmental waters, constituting a major cause of acute waterborne disease worldwide. In this study, we investigated the relative abundance, occurrence, and seasonal distribution of polio and other enteroviruses at three wastewater treatment plants (WWTPs) in Naples, Southern Italy, from January 2010 to December 2014. Influent and effluent samples from the three WWTPs were collected monthly. One hundred and sixty-one of the 731 wastewater samples collected (22.0%) before and after water treatment were CPE positive on RD cells; while no samples were positive on L20B cells from any WWTPs. Among the 140 non-polio enterovirus isolated from inlet sewage, 69.3% were Coxsackieviruses type B and 30.7% were Echoviruses. Among these, CVB3 and CVB5 were most prevalent, followed by CVB4 and Echo6. The twenty-one samples tested after treatment contained 6 CVB4, 5 CVB3, 3 Echo11, and 2 Echo6; while other serotypes were isolated less frequently. Data on viral detection in treated effluents of WWTPs confirmed the potential environmental contamination by HEVs and could be useful to establish standards for policies on wastewater management.

Acute flaccid paralysis surveillance in bosnia and herzegovina: Recent isolation of two sabin like type 2 poliovirus.

The WHO Regional Commission for the Certification of Poliomyelitis Eradication has recently indicated Bosnia and Herzegovina (B&H) as a high risk country for transmission, following importation, of wild poliovirus (WPV) or circulating vaccine-derived poliovirus (cVDPV). We analyzed data on Acute Flaccid Paralysis (AFP) surveillance between 2007 to 2016, and the trend of polio immunization coverage in B&H. The majority of AFP cases was recorded in 2016 suggesting an enhancement of the AFP surveillance activities. However, the decline in the immunization coverage, around 74%, and the isolation of two Sabin-like poliovirus type 2 strains, one of them close to a VDPV, require a particular attention in the area. Although B&H has successfully maintained its polio-free status since 2002 several challenges need to be addressed.

Surveillance of poliomyelitis in Northern Italy: Results of acute flaccid paralysis surveillance and environmental surveillance, 2012-2015.

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 children <15 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.

Molecular characterization of two rare human G8P[14] rotavirus strains, detected in Italy in 2012.

Since 2007, the Italian Rotavirus Surveillance Program (RotaNet-Italy) has monitored the diversity and distribution of genotypes identified in children hospitalized with rotavirus acute gastroenteritis. We report the genomic characterization of two rare human G8P[14] rotavirus strains, identified in two children hospitalized with acute gastroenteritis in the southern Italian region of Apulia during rotavirus strain surveillance in 2012. Both strains showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation (DS-1-like genomic background). Phylogenetic analysis of each genome segment revealed a mixed configuration of genes of animal and zoonotic human origin, indicating that genetic reassortment events generated these unusual human strains. Eight out of 11 genes (VP1, VP2, VP3, VP6, VP7, NSP3, NSP4 and NSP5) of the Italian G8P[14] strains exhibited close identity with a Spanish sheep strain, whereas the remaining genes (VP4, NSP1 and NSP2) were more closely related to human strains. The amino acid sequences of the antigenic regions of outer capsid proteins VP4 and VP7 were compared with vaccine and field strains, showing high conservation between the amino acid sequences of Apulia G8P[14] strains and human and animal strains bearing G8 and/or P[14] proteins, and revealing many substitutions with respect to the RotaTeq™ and Rotarix™ vaccine strains. Conversely, the amino acid analysis of the four antigenic sites of VP6 revealed a high degree of conservation between the two Apulia strains and the human and animal strains analyzed. These results reinforce the potential role of interspecies transmission and reassortment in generating novel rotavirus strains that might not be fully contrasted by current vaccines.

Genetic variability of VP7, VP4, VP6 and NSP4 genes of common human G1P[8] rotavirus strains circulating in Italy between 2010 and 2014.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young children worldwide. The RVA outer capsid layer is composed of the VP7 and VP4 proteins. The VP7 (G-type) and VP4 (P-type) genotypes are the basis for the binary RVA nomenclature. At least 27 G-types and 37 P-types of RVA are currently known, but most of human infections are related to the five major genotypes G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]. Every year G1P[8] strains cause approximately 50% of all symptomatic RVA infections reported in children in Italy. Fifteen G1P[8] RVA strains identified in different areas of Italy between 2010 and 2014 were selected. Strains were subjected to nucleotide sequencing of the VP7, VP4, VP6 and NSP4 genes to investigate their genetic variability with respect to geographic area and date of detection. Phylogenetic analyses showed that the 15 G1P[8] RVA strains belonged to two different lineages for both the VP7 and NSP4 genes, and showed some intra-lineage diversity in VP4 and VP6 genes. Similarities between strains correlated by either area or date of detection were also evaluated. The results obtained by phylogenetic analyses were confirmed analyzing the deduced amino acid sequences of the VP7, VP4, VP6 and NSP4 proteins of the G1P[8] RVA strains, detecting several substitutions in all proteins. The genetic variability observed between common G1P[8] RVAs highlights the constant evolution of the RVA genome through random point mutations (genetic drift) and intra-genotype reassortment (genetic shift). The evolution and diversity of the G1 RVA strains observed in this study can be related to the naturally acquired herd immunity, which represents the main mechanism of selective pressure in Italy, where mass anti-rotavirus vaccination was missing during the years of the study.

Genomic characterization of uncommon human G3P[6] rotavirus strains causing diarrhea in children in Italy in 2009.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis in young children, causing up to 450,000 deaths worldwide, mostly in developing countries. Most of RVA human infections in developed countries are related to five major G/P combinations: G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. During the surveillance activity of RotaNet-Italy, three uncommon G3P[6] RVA strains, designated as RVA/Human-wt/ITA/NA01/2009/G3P[6], RVA/Human-wt/ITA/NA06/2009/G3P[6], and RVA/Human-wt/ITA/NA19/2009/G3P[6], were identified in the stools of children with diarrhea hospitalized in Southern Italy in 2009. Samples NA01, NA06 and NA19 were characterized as genotype G3P[6]. To investigate the three strains further, partial sequencing of the eleven genomic segments was performed. RVA strains NA01, NA06 and NA19 were found to share the rare genotype constellation: G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2, which had not been reported previously in continental Italy. The phylogenetic analysis of the eleven genomic segments showed no evidence of zoonosis or inter-species reassortment at the origin of the Italian G3P[6] strains, indicating that they possessed DS-1-like genomic constellations similar to those detected previously in human cases in Africa and Europe. The analysis of the hypervariable regions of VP7 and VP4 (VP8*) revealed high amino acid identity between the Italian G3P[6] RVA strains involved in this study.

Evolution of human G4P[8] group A rotavirus strains circulating in Italy in 2013.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis in young (<5 years of age) children, causing up to 450.000 deaths worldwide, mostly in developing countries. VP7 (G-type) and VP4 (P-type) genotypes are the basis for the binary RVA classification. Although at least 27 G-types and 37 P-types of rotavirus are presently known, most RVA infections in humans worldwide are associated with the five major G/P combinations G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. During RVA gastroenteritis surveillance in Italy, a total of 1112 samples collected from children hospitalized with acute gastroenteritis in 2013 were RVA positive and were genotyped following standardized protocols from the EuroRotaNet. Most strains analyzed belonged to the five major human genotypes. Among these common strains, 22 G4P[8] RVA strains from different Italian regions were subjected to nucleotide sequencing of their VP4, VP6, VP7 and NSP4 genes to investigate their evolution. The phylogenetic analysis showed that the Italian strains belonged to lineage G4-I for VP7 and to lineage P[8]-III for VP4, in line with the modern G4P[8] RVA strains detected in children worldwide. The phylogenetic trees revealed high degrees of nucleotide identity between the RVA strains involved in this study and G4P[8] strains detected previously in Europe, Asia and Africa, but also demonstrated at least three separate evolution clusters within the same lineage. Based on the amino acid sequences deduced for their hypervariable regions, both the VP7 and VP8* proteins of the Italian G4P[8] RVA strains presented amino acid substitutions near known neutralizing epitopes.

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.

Genomic characterization and molecular investigation of VP7 epitopes of uncommon G10P[8] group A rotavirus strains detected in Italy in 2009.

Rotavirus strains with the uncommon genotype G10 have been detected sporadically in cases of acute gastroenteritis in humans and are thought to be transmitted zoonotically. During 2009, 10 G10P[8] rotavirus strains were detected in the stools of children hospitalized with acute diarrhoea in several paediatric hospitals in Italy. The phylogenetic analysis of the VP7 gene of the Italian G10P[8] strains analysed revealed nucleotide identities ranging from 94 to 99 %. Molecular characterization of the 11 genomic segments was performed for one of the G10 strains, which displayed a complete genomic constellation 1 for the non-G genes. The analysis of the deduced amino acid sequences of the G10 VP7 epitopes revealed low amino acid identity with common human strains of different G genotype and with the VP7 proteins included in both anti-rotavirus commercial vaccines (Rotarix and RotaTeq). Amongst the common G genotypes, the VP7 amino acid sequence of the G10 strains showed a high similarity with sequences from G9 strains. A hydrophobic cluster analysis (HCA) of the VP7 protein including aa 20-298 was performed for the G10 Italian sequences in comparison with the major human group A rotavirus G genotypes. The HCA analysis confirmed the findings obtained previously by amino acid analysis of the VP7 epitopes, detecting a genotype-specific pattern of hydrophobicity in the hypervariable regions of the major outer capsid protein.

Molecular characterization of group A rotavirus strains detected in children with diarrhea admitted to Nigerian hospitals in 2013.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in children worldwide and cause up to 455,000 deaths annually, mostly in developing countries. During 2013, 66 RVAs from children with AGE admitted to four Nigerian hospitals were investigated. The G3P[6], G1P[8] and G2P[4] genotypes predominated. The VP7 and/or VP4 genes of 18 G3P[6]/[8]/[4], six G2P[4], three G12P[8]/[4], and two G1P[8] RVA strains were sequenced. The G3P[6] strains belonged to lineage G3-III and were different from G3 strains widespread in Asia. Phylogenetic analysis revealed substantial sequence conservation, suggesting continuing evolution and genomic reassortment but no zoonotic RVA transmission from animals.

Rotavirus genotypes in sewage treatment plants and in children hospitalized with acute diarrhea in Italy in 2010 and 2011.

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.

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.

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.

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.

Full-genome characterization of a G8P[8] rotavirus that emerged among children with diarrhea in Croatia in 2006.

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.

Molecular characterization of rotavirus strains from children with diarrhea in Italy, 2007-2009.

The surveillance network RotaNet-Italia was established in 2007 in order to investigate the diversity of co-circulating rotavirus strains in Italy, and to provide a baseline for future assessment of possible effects of vaccine implementation in selecting novel versus common rotavirus strains. A total of 2,645 rotavirus strains from pediatric patients with acute diarrhea were collected over three consecutive seasons from September 2006 through August 2009, and partially characterized by standardized multiplex RT-PCR. Most of strains (89.1%) belonged to genotypes G1-G4, and G9, associated with either P[8] or P[4], commonly found in humans worldwide. However, in at least 2.0% of cases, viruses exhibited either a G or P type typical of animal viral strains, suggesting gene reassortment events between rotaviruses of different origin. Mixed infections with two or more rotavirus strains were observed frequently (7.6% of patients), and depended on the frequencies of co-circulating rotaviruses of one particular genotype. The numbers and genotypes of likely natural reassortants of common genotype rotaviruses were found to be correlated with the observed numbers and genotypes of mixed infections. Large variation in the relative frequency of different rotavirus genotypes was observed between different seasons and/or areas of Italy, suggesting independent evolution or differential introduction of viral strains with respect to both time and space.