Severe Streptococcus equi Subspecies zooepidemicus Outbreak from Unpasteurized Dairy Product Consumption, Italy.

During November 2021-May 2022, we identified 37 clinical cases of Streptococcus equi subspecies zooepidemicus infections in central Italy. Epidemiologic investigations and whole-genome sequencing showed unpasteurized fresh dairy products were the outbreak source. Early diagnosis by using sequencing technology prevented the spread of life-threatening S. equi subsp. zooepidemicus infections.

Species of mosquitoes present in Abruzzo and Molise and their possible role as vector of Usutu and West Nile viruses.

In 2019, entomological survey on mosquitoes was carried out in Abruzzo and Molise regions in central Italy to obtain data on local mosquito fauna. Collection sites were selected based on a previous ecoregion classification of the territory.  From 2019 to 2021 virological surveillance for West Nile virus (WNV) and Usutu virus (USUV) on mosquitoes was carried out in the same regions, selecting ecoregions where virus circulation and vector presence were more likely,  all mosquitoes were collected and identified, and the female mosquitoes were sorted in 3046 pools and tested for the presence of WNV and USUV by Real-time PCR. All pools tested negative for WND, while USUV was detected in 7 pools of Aedes caspius collected in Molise region, 17 pools of Culex pipiens s.l. (2 collected in Molise, 15 in Abruzzo), and 1 pool of Culiseta longiareolata collected in Molise. These results suggests the presence of an USUV enzootic cycle, maintained by Culex pipiens s.l. and Aedes caspius in both Italian regions, as well as providing a useful picture in terms of species presence and abundance for both regions. Ecoregions proved to be a very valuable tool in determining high risk areas for vector borne diseases.

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy.

In this study, starting from nucleic acids purified from the brain tissue, Nanopore technology was used to identify the etiological agent of severe neurological signs observed in a cow which was immediately slaughtered. Histological examination revealed acute non-suppurative encephalomyelitis affecting the brainstem, cerebrum, cerebellum, and medulla oblongata, while by using PCR-based assays, the nucleic acids of major agents for neurological signs were not detected. By using Nanopore technology, 151 sequence reads were assigned to Bovine Astrovirus (BoAstV). Real-time RT-PCR and in situ hybridization (ISH) confirmed the presence of viral RNA in the brain. Moreover, using the combination of fluorescent ISH and immunofluorescence (IF) techniques, it was possible to detect BoAstV RNA and antigens in the same cells, suggesting the active replication of the virus in infected neurons. The nearly whole genome of the occurring strain (BoAstV PE3373/2019/Italy), obtained by Illumina NextSeq 500, showed the highest nucleotide sequence identity (94.11%) with BoAstV CH13/NeuroS1 26,730 strain, an encephalitis-associated bovine astrovirus. Here, we provide further evidence of the role of AstV as a neurotropic agent. Considering that in a high proportion of non-suppurative encephalitis cases, which are mostly indicative of a viral infection, the etiologic agent remains unknown, our result underscores the value and versatility of Nanopore technology for a rapid diagnosis when the PCR-based algorithm gives negative results.

Virus like particles of GII.4 norovirus bind Toll Like Receptors 2 and 5.

Norovirus (NoV) is now recognized as a major cause of gastroenteritis outbreaks, worldwide. Norovirus replication mechanisms are still poorly understood, mainly because a reliable cell culture system is still lacking. The present study aims at understanding some aspects of the immune response against norovirus, and particularly the capacity of virus like particles (VLPs) from an Italian strain, belonging to the GII.4 genotype predominating worldwide, to interact with target cells via Toll Like Receptors (TLRs). The capacity of GII.4 NoV VLPs to interact and cause the activation of TLR2, 4 and 5 was studied in recombinant HEK cells. The results obtained show the ability of GII.4 NoV VLPs to induce activation of TLR2 and 5. The results on TLRs activation confirm that GII.4 NoV VLPs interact with TLR2 and 5, that may represent putative receptors and play a role in NoV infection of intestinal cells.

Detection of uncommon G3P[3] rotavirus A (RVA) strain in rat possessing a human RVA-like VP6 and a novel NSP2 genotype.

Rotavirus is one of the leading causes of acute gastroenteritis in infants and young children. RVAs infect not only humans but also a wide range of mammals including rats, which represent a reservoir of several other zoonotic pathogens. Due to the segmented nature of the RVA genome, animal RVA strains can easily adapt to the human host by reassortment with co-infecting human viruses. This study aims to detect and characterize RVA in the intestinal content of Italian sinantropic rats (Rattus rattus). Out of 40 samples examined following molecular approach, one resulted positive for RVA. The molecular characterization of VP1-4, 6 and 7, and NSP1-5 genes by sequencing revealed the genomic constellation G3-P[3]-I1-R11-C11-M10-A22-N18-T14-E18-H13. This uncommon genomic combination includes: the VP1-4,VP7, the NSP1, 3, 4 and 5 gene segments, closely related to those of RVA from rodents, the N18 novel genotype established for the NSP2 gene segment and the human Wa-like VP6 gene, suggesting interspecies reassortment.

Hospital-acquired rotavirus and norovirus acute gastroenteritis in a pediatric unit, in 2014-2015.

The occurrence of hospital-acquired acute gastroenteritis (AGE) is a major concern for public health. RotavirusA (RVA) and norovirus (NoV) are common causes of viral AGE in the pediatric population, and their role in nosocomial infections has been proven, remaining poorly investigated. To investigate RVA and NoV in hospital-acquired AGE, 55 stool samples from children with nosocomial AGE were collected between May 2014 and May 2015. To evaluate virus spreading routes, 51 environmental swabs were collected from staff and patients' rooms. Stools were tested for both RVA and NoV RNA by reverse-transcription-PCR. Nucleotide sequencing and phylogenetic analysis were performed to characterize the viruses. Forty-seven of 55 cases analyzed resulted positive for RVA. The predominant genotype was G4P[8] (18/55) followed by G1P[8] (14/55). Mixed RVA infections were also detected (7/55). Twenty-two samples were positive for NoV, and GII.4 was revealed to be the predominant genotype. Seventeen samples were positive for both RVA and NoV. This study aimed to evaluate the burden of norovirus and rotavirus nosocomial AGE, contributing to identify the environment source of infections and to activate effective strategies for intervention. The reduction in nosocomial AGE cases is an important aspect, considered the worsened disease course in transplant, cancer, and intensive care unit inpatients.

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.

Detection of Torque Teno Sus Virus in Pork Bile and Liver Sausages.

Torque teno viruses (TTV) are small DNA viruses widespread among humans and pigs. The clinical significance of TTV infections in either humans or pigs is uncertain. In fact, TTV viremia is highly prevalent in patients with different pathologies, but it can also be frequently observed in healthy subjects. Virus infection in pigs is considered a putative cofactor in several diseases; despite being detected frequently in healthy animals, its role still remains unknown. The present study aimed to investigate the presence of Torque teno sus virus (TTSuV) in 62 bile samples collected from pigs at slaughterhouse and in 36 fresh pork liver sausages bought at point of sale. Quantitative Real-Time PCR, confirmed that 19.4 and 58.3 % of bile and sausage samples tested positive for TTSuV, respectively. The mean viral load was established as 5.6 × 104 GE/µl for bile and 7.16 × 103 GE/g for sausages. TTSuV nucleotide sequence analysis confirmed a wide heterogeneity among the circulating TTSuV strains, which included both TTSuV1 and TTSuV2.

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.

Removal of enteric viruses and Escherichia coli from municipal treated effluent by zebra mussels.

Dreissena polymorpha is a widespread filter-feeder species, resistant to a broad range of environmental conditions and different types of pollutants,which has recently colonized Italian freshwaters. Although widely used to monitor pollution in freshwater environments, this species is also an important food source for some fish and water birds. It can also be used to concentrate or remove particulate organic matter to interrupt avian-to-human transmission of pollutants and control health risks for animals and humans. In this study, the accumulation/inactivation in D. polymorpha of human health-related spiked enteric viruses was described. The removal of endogenous Escherichia coli, the classical indicator of fecal contamination,was tested as well.Our preliminary lab-scale results demonstrate that zebra mussels can reduce significantly poliovirus titer after 24 h and rotavirus titer after 8 h. E. coli counts were also reduced in the presence of zebra mussels by about 1.5 log after 4 h and nearly completely after 24 h. The fate of the two enteric viruses after concentration by zebra mussels was also investigated after mechanical disruption of the tissues. To our knowledge, the accumulation from water and inactivation of human health-related enteric viruses by zebra mussels has never been reported.

Complete genome sequencing of a genotype 3 hepatitis E virus strain identified in a swine farm in Italy.

In this study, we investigated hepatitis E virus (HEV) infection in piglets sampled in two farms in southern Italy. The virus was detected in 11 out of 15 animals tested. Based on sequence analysis, the 6 Italian strains examined belonged to two clusters containing both swine and human strains of either genotype 3 subtype e or f from Europe and Japan. The two Italian strain clusters shared nucleotide identity of 81.8% and 87.5% in the ORF2 (capsid protein) and ORF1 (RdRp) diagnostic fragments, respectively, confirming the heterogeneity of genotype 3 viruses circulating in pigs in Italy. The complete genome of one genotype 3 subtype e strain and the full ORF2 and ORF3 coding regions of one of the genotype 3f strains, obtained in this study, were compared to other HEV sequences available on line (NCBI database). The results of analysis showed that porcine strains clustered together with human and swine strains detected in Europe. Most changes in the coding region corresponded to synonymous mutations, and only the ORF3 showed a positive selection. Further, analyses are needed to understand the clinical significance of HEV genotypes and subtypes.

Molecular characterization of avian rotaviruses circulating in Italian poultry flocks.

Avian rotaviruses are still largely undefined despite being widespread in several avian species and despite the economic impact of rotavirus (RV) enteritis in poultry flocks. In this study, the presence of different avian RV groups was investigated in commercial poultry flocks reared in Northern and Central Italy and with a history of enteric diseases. Faeces or intestinal contents from different avian species previously found to contain RV particles by electron microscopy (EM) were analysed by both RNA-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction specific for groups A, D, F and G RVs. Group D avian RV was detected in 107 of 117 samples tested (91.5%), whereas groups A, F and G avian RVs were present in 70 (59%), 61 (52.1%) and 31 (26.5%) samples, respectively. Multiple presence of different RV groups was detected in 83% of samples. This study provides novel data on the prevalence of genetically different avian RVs in Italian poultry flocks. This information is useful to elucidate the epidemiology of avian RVs circulating in Italy.

Detection and molecular characterization of zoonotic viruses in swine fecal samples in Italian pig herds.

Gastrointestinal disease is frequent in pigs, and among the different etiological agents involved, viruses are considered the leading cause of infection in this animal species. Furthermore, about half of the newly identified swine pathogens are viruses, many of which may be transmitted to humans by direct contact or by indirect transmission pathways. In this study, the prevalence of astrovirus (AstV), group A rotavirus (RVA), norovirus (NoV) and hepatitis E virus (HEV) infections in pigs was investigated. During 2012-2014, 242 fecal samples were collected from pigs at different production stages (5 to 220 days old) on eight swine farms located in northern, central and southern Italy. Seven out of eight farms analyzed were positive for AstV, which was detected in 163 out of 242 (67.4%) samples and was the most prevalent virus; 61 of the 163 AstV-positive animals (37.4%) had diarrhea. HEV was detected on six farms and in 45 (18.6%) of the 242 samples analyzed. Twenty-three HEV-infected pigs had diarrhea (51.1%). A lower prevalence was observed for RVA, which was found in 10 of the 242 samples (4.1%) from three positive farms, and diarrhea was present only in six infected pigs (60.0%). No swine samples were found to be positive for NoV. Genetic diversity and phylogenetic relationships of some strains representative of the different viruses detected were investigated, confirming a wide heterogeneity of viral strains circulating among pigs.

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.

Waterborne norovirus outbreak during a summer excursion in Northern Italy.

In September 2011, an acute gastroenteritis outbreak affected 33 children in Northern Italy. Patients had drunk river water during an excursion. Identical GI.4 norovirus genomes were detected from one patient's stools and from the river water. Improper discharge of human sewage into the river may have caused this waterborne outbreak.

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.

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.

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.

Infection of farmed pigs with porcine kobuviruses in Italy.

Two-hundred eight swine fecal samples from six Italian farms were tested using a kobuvirus-specific RT-PCR with primers that amplify a region within the 3D gene. All farms were kobuvirus positive, with prevalence rates ranging between 24 % and 84 %. Overall, 57.5 % of asymptomatic pigs and 49.7 % of animals with diarrhea were positive for kobuvirus. Sequence analysis showed a different predominant strain circulating on each farm and indicated that the strains detected were related to both European and Asiatic strains. A possible pathogenic role of kobuvirus should be investigated further, since infections with this virus occur frequently in pigs of different ages.