Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

Persistence of hepatitis E virus (HEV) subtypes 3c and 3e: Long-term cold storage and heat treatments.

Hepatitis E virus (HEV) is the causative agent of foodborne infections occurring in high income countries mainly by consumption of undercooked and raw pork products. The virus is zoonotic with pigs and wild boars as the main reservoirs. Several studies proved the presence of HEV-RNA in pork liver sausages, pâté and other pork by-products. However, the detection of HEV nucleic acids does not necessary correspond to infectious virus and information on the persistence of the virus in the food is still limited. To which extent and how long the virus can survive after conventional industrial and home-made conservation and cooking procedures is largely unknown. In the present study, we investigated the persistence of two subtypes of HEV-3, by measuring the viral RNA on cell supernatant of infected A549 cells, after long-term storage at +4 °C and -20 °C and after heating for short or long-time span. Results confirmed that either low temperature storage (+4 °C) or freezing (-20 °C) do not influence the survival of the virus, and only a moderate reduction of presence of its RNA after 12 weeks at +4 °C was observed. To the other side, heating at 56 °C for long time (1 h) or at higher temperatures (>65 °C) for shorter time inactivated the virus successfully.

Detection and whole genome sequencing of murine norovirus in animal facility in Italy.

Viruses belonging to the genus Norovirus (NoV) of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. NoVs are classified into 10 genogroups (GI-GX), and those belonging to the genogroup GV are able to infect several species of rodents. To evaluate the circulation of MNV among mice housed in an Italian facility, sampling was performed over two separate periods, in 2011, and 3 years later in 2014. During the two samplings, 75 fecal samples were collected from healthy mice housed in the animal facility and subjected to RT-PCR for viral detection. After the analysis, 41/75 animals (54.6%) resulted positive for the presence of MNV in feces. Nucleotide sequencing revealed the presence of two MNV variants co-circulating in both 2011 and 2014. One MNV strain was isolated on RAW264.7 cell line, and subjected to full genome sequencing. Our study showed that the murine noroviruses are widespread in the investigated animal facility, despite guidelines for animal care and maintenance. Full genome sequence analysis of the MNV strain described in this study showed a correlation with other strains circulating in Europe. Understanding the molecular epidemiology of this virus should give insight into its natural history and evolution in mice.

Hospital-acquired rotavirus acute gastroenteritis in 10 consecutive seasons in Umbria (Italy).

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young (aged <5 years) children. Several studies showed that RVA is one of the main cause of nosocomial gastroenteritis in hospitalized pediatric population worldwide, with an incidence ranging from 8 to 33 cases per 100 hospitalized children. Nosocomial infections, in which AGE symptoms develop at least 2 days after admission, may severely affect children already admitted to hospital for other causes. This study aimed to define the trends of the RVA genotypes through statistical analysis of the data obtained by the rotavirus surveillance in Umbria in 10 consecutive seasons, from 2007-2008 to 2016-2017, with update information on hospital-acquired RVA AGE. During RVA gastroenteritis surveillance in Umbria (Italy) in 2007 to 2017, a total of 741 RVA positive faecal samples were collected from children hospitalized with AGE, and RVA strains were genotyped following standard EuroRotaNet protocols. Of the 741 analyzed samples, 75 (10%) were reported to be hospital-acquired. Comparing the distributions of the RVA genotypes circulating in the community or associated with nosocomial infections, we observed a different distribution of genotypes circulating inside the hospital wards, with respect to those observed in the community except in 2010 to 2011, 2011 to 2012, and 2012 to 2013 when G1P[8], G4P[8] and the novel strain G12P[8] caused a large community- and hospital-acquired outbreak. Of the 741 analyzed samples, 75 (10%) were reported to be hospital-acquired. Comparing the distributions of the RVA genotypes circulating in the community or associated with nosocomial infections, we observed a different distribution of genotypes circulating inside the hospital wards, with respect to those observed in the community except in 2010 to 2011, 2011 to 2012, and 2012 to 2013 when G1P[8], G4P[8], and the novel strain G12P[8] caused a large community- and hospital-acquired outbreak. The information from this study will be useful to implement guidelines for preventing nosocomial RVA AGE, which should include an improved management of the hospitalized patients and an increase in vaccination coverage.

First detection of a reassortant G3P[8] rotavirus A strain in Italy: a case report in an 8-year-old child.

BACKGROUND: Acute gastroenteritis (AGE) due to group A rotavirus (RVA) agent is one of the major causes of hospitalization in paediatric age. The G3P[8] RVA genotype has been usually considered as one of the major human genotypes, largely circulating in Asia, but showing low detection rates in the European countries. In recent years, the G3P[8] RVAs emerged also in Europe as a predominant genotype and the viral strains detected revealed high similarities with equine-like G3P[8] RVA strains, resulting in a new variant circulating in humans and able to cause AGE in the paediatric population. CASE PRESENTATION: An 8-year-old boy was admitted to the Emergency Room because he had suffered from severe diarrhoea, vomiting, and high fever over the previous two days. Severe dehydration was evident based on low serum concentrations of potassium and sodium, low glycaemia, and pre-renal failure (creatinine 2.48 mg/dL, urea 133 mg/dL). Immunological tests were within normal range. Enzyme immunoassay for the detection of RV was positive, and a sample of faeces was collected in order to perform the molecular characterization of the viral strain. The phylogenetic trees revealed relatedness between the VP7 and VP4 genes of the G3P[8] RVA Italian strain (namely PG2) and those belonging to recent G3P[8] RVAs detected worldwide. The G3 VP7 belonged to the G3-I lineage and shared the highest nucleotide sequence identity (99.8%) with the equine-like G3 previously identified in other countries. The P [8] VP4 revealed a similar clustering pattern to that observed for the VP7. In addition, the molecular characterization of the 11 gene segments of strain PG2 revealed a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genomic constellation. CONCLUSIONS: This case shows the first detection in Italy of a reassortant G3P[8] RVA associated with a severe AGE, which is unusual in a school-age child without any known severe underlying problems. The findings reported in this paper highlight the importance of continuously monitoring the RVA strains circulating in paediatric age in order to detect novel viral variants able to spread in the general population.

Group A rotavirus surveillance before vaccine introduction in Italy, September 2014 to August 2017.

IntroductionGroup A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young children, causing ca 250,000 deaths worldwide, mainly in low-income countries. Two proteins, VP7 (glycoprotein, G genotype) and VP4 (protease-sensitive protein, P genotype), are the basis for the binary RVA nomenclature. Although 36 G types and 51 P types are presently known, most RVA infections in humans worldwide are related to five G/P combinations: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8].AimThis study aimed to characterise the RVA strains circulating in Italy in the pre-vaccination era, to define the trends of circulation of genotypes in the Italian paediatric population.MethodsBetween September 2014 and August 2017, after routine screening in hospital by commercial antigen detection kit, 2,202 rotavirus-positive samples were collected in Italy from children hospitalised with AGE; the viruses were genotyped following standard European protocols.ResultsThis 3-year study revealed an overall predominance of the G12P[8] genotype (544 of 2,202 cases; 24.70%), followed by G9P[8] (535/2,202; 24.30%), G1P[8] (459/2,202; 20.84%) and G4P[8] (371/2,202; 16.85%). G2P[4] and G3P[8] genotypes were detected at low rates (3.32% and 3.09%, respectively). Mixed infections accounted for 6.49% of cases (143/2,202), uncommon RVA strains for 0.41% of cases (9/2,202).ConclusionsThe emergence of G12P[8] rotavirus in Italy, as in other countries, marks this genotype as the sixth most common human genotype. Continuous surveillance of RVA strains and monitoring of circulating genotypes are important for a better understanding of rotavirus evolution and genotype distribution, particularly regarding strains that may emerge from reassortment events.

Phylogenetic analysis of two genotype 3 Hepatitis E viruses from wild boar, Italy.

The complete and near-complete genome sequences (7206 nt and 7229 nt) of two wild boar HEV strains detected in Southern Italy were obtained by the next generation sequencing. Phylogenetic analysis and p distance comparisons of one of the strains with HEV-3 reference subtype strains confirmed the detection of a subtype 3i (p distance = 0.110) strain in wild boar, never detected in Italy either in wild boar or pigs. The sequence of the second strain was not classifiable in any of the subtypes defined to date, showing a p distance > 0.138 and a low nucleotide identity with all HEV reference strains. The virus may represent a novel subtype, with a low relationship to other strains of genotype 3 detected in wild boar, pigs, or humans in Europe. This result suggests the circulation in Italy of an emerging or uncommon HEV strain. Sequencing followed by phylogenetic analyses of the complete HEV coding regions are important tools for understanding the evolutionary and epidemiological dynamics underlying the wide genetic diversity of HEV strains.

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.

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.

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.

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.

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.

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.

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.

Effects of natural treatments on the varroa mite infestation levels and overall health of honey bee (Apis mellifera) colonies.

The survival of the honey bee (Apis mellifera), which has a crucial role in pollination and ecosystem maintenance, is threatened by many pathogens, including parasites, bacteria, fungi and viruses. The ectoparasite Varroa destructor is considered the major cause of the worldwide decline in honey bee colony health. Although several synthetic acaricides are available to control Varroa infestations, resistant mites and side effects on bees have been documented. The development of natural alternatives for mite control is therefore encouraged. The study aims at exploring the effects of cinnamon and oregano essential oils (EOs) and of a mixed fruit cocktail juice on mite infestation levels and bee colony health. A multi-method study including hive inspection, mite count, molecular detection of fungal, bacterial and viral pathogens, analysis of defensin-1, hymenoptaecin and vitellogenin immune gene expression, colony density and honey production data, was conducted in a 20-hive experimental apiary. The colonies were divided into five groups: four treatment groups and one control group. The treatment groups were fed on a sugar syrup supplemented with cinnamon EO, oregano EO, a 1:1 mixture of both EOs, or a juice cocktail. An unsupplemented syrup was, instead, used to feed the control group. While V. destructor affected all the colonies throughout the study, no differences in mite infestation levels, population density and honey yield were observed between treatment and control groups. An overexpression of vitellogenin was instead found in all EO-treated groups, even though a significant difference was only found in the group treated with the 1:1 EO mixture. Viral (DWV, CBPV and BQCV), fungal (Nosema ceranae) and bacterial (Melissococcus plutonius) pathogens from both symptomatic and asymptomatic colonies were detected.

Investigation of Salmonella, hepatitis E virus (HEV) and viral indicators of fecal contamination in four Italian pig slaughterhouses, 2021-2022.

In the pork production chain, the control at slaughterhouse aims to ensure safe food thanks to proper hygienic conditions during all steps of the slaughtering. Salmonella is one of the main foodborne pathogens in the EU causing a great number of human cases, and pigs also contribute to its spreading. Pig is the main reservoir of the zoonotic hepatitis E virus (HEV) that can be present in liver, bile, feces and even rarely in blood and muscle. The aim of this study was to assess the presence of both Salmonella and HEV in several points of the slaughtering chain, including pig trucks. Other viruses hosted in the gut flora of pigs and shed in feces were also assayed (porcine adenovirus PAdV, rotavirus, norovirus, and mammalian orthoreovirus MRV). Torque teno sus virus (TTSuV) present in both feces, liver and blood was also considered. Four Italian pig abattoirs were sampled in 12 critical points, 5 of which were the outer surface of carcasses before processing. HEV and rotavirus (RVA) were not detected. Norovirus was detected once. Salmonella was detected in two of the 4 abattoirs: in the two lairage pens, in the site of evisceration and on one carcass, indicating the presence of Salmonella if carcass is improper handled. The sampling sites positive for Salmonella were also positive for PAdV. MRV was detected in 10 swabs, from only two abattoirs, mainly in outer surface of carcasses. TTSuV was also detected in all abattoirs. Our study has revealed a diverse group of viruses, each serving as indicator of either fecal (NoV, RVA, PAdV, MRV) or blood contamination (TTSuV). TTSuV could be relevant as blood contamination indicators, crucial for viruses with a viremic stage, such as HEV. The simultaneous presence of PAdV with Salmonella is relevant, suggesting PAdV as a promising indicator for fecal contamination for both bacterial and viruses. In conclusion, even in the absence of HEV, the widespread presence of Salmonella at various points in the chain, underscores the need for vigilant monitoring and mitigation strategies which could be achieved by testing not only bacteria indicators as expected by current regulation, but also some viruses (PAdV, TTSuV, MRV) which could represent other sources of fecal contamination.

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.

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.

Erinaceus coronavirus persistence in hedgehogs (Erinaceus europaeus) in a non-invasive, in vivo, experimental setting.

In the last 20 years, new zoonotic CoV strains have emerged (SARS-CoV, MERS-CoV, and SARS-CoV-2), and new species have also been reported in animals. In Europe, the Erinaceus coronavirus (EriCoV) was recently described in Erinaceus europaeus. However, information on the prevalence and duration of viral shedding is unknown. In this study, feces samples were collected from 102 European hedgehogs hosted in the Center for the Recovery of Wild Fauna in Rome and analyzed for the presence of EriCoV RNA by Reverse Transcription-PCR. In total, 45 animals (44.1%) resulted positive for EriCoV at the first sampling and 63 (61.7%) animals were positive at the follow-up, which was performed from the 3rd to the 86th day. The duration of fecal virus shedding showed a mean duration of 22.8 days and lasted up to 62 days. Eighteen hedgehogs showed intermittent viral shedding. Phylogenetic analysis showed a correlation with EriCoV strains reported in Germany, the United Kingdom, and northern Italy. None of the EriCoV sequences showed the CD200 ortholog insertion, previously observed in strains isolated in animals from northern Italy. Interestingly, all but one animal revealed the presence in their feces of the same EriCoV sequences, analyzing the short genomic region at 3' spike gene and 5' ORF3a 500bp fragment (100% nt.id.) in both first and follow-up samples. This result suggests that animals were infected with the same strain during their stay at the center. Our results confirm that EriCoV can persist in hedgehogs for a long period, underlining that hedgehogs are an important commensal reservoir for Merbecovirus. A long duration of viral shedding increases the likelihood that the virus will spread in the environment.