Molecular characterization of human Sapovirus in untreated sewage in Italy by amplicon-based Sanger and next-generation sequencing.

AIMS: Human Sapoviruses (HSaVs) are etiological agents of sporadic cases and outbreaks of acute gastroenteritis in humans of all ages. Evidence of worldwide distribution of HSaV has been documented; however, little is known about HSaV circulation in Italy. To study their occurrence and genetic diversity a nation-wide environmental surveillance was undertaken. METHODS AND RESULTS: One hundred and sixty-six raw sewage samples, collected from 16 wastewater treatment plants throughout Italy, were processed and analysed by a RT-nested PCR targeting the capsid region, followed by amplicon sequencing. HSaV was detected in 56 of 166 (33·7%) samples, characterized as genotypes GI.1 (n = 30 samples), GI.2 (n = 3), GI.3 (n = 2) and GII.1 (n = 1). Mixed electropherograms were detected in 20 samples. Next-generation sequencing (NGS)-based amplicon sequencing was performed on pools of PCR amplicons to detect viruses in mixed samples and less prevalent genotypes undetectable by conventional Sanger sequencing. NGS revealed three additional genotypes (GI.6, GII.6 and GV.1) beyond the four detected by Sanger sequencing. CONCLUSIONS: Our results show a significant circulation of HSaV in Italy with three genogroups (GI, GII and GV) and seven genotypes detected. The high detection rate in sewage samples suggests that HSaV infection in Italy could be underestimated or associated with asymptomatic or mild cases. SIGNIFICANCE AND IMPACT OF THE STUDY: The study detected HSaV in a relevant proportion of raw sewage samples, reflecting a considerable circulation of these viruses in the Italian population, pointing to the usefulness of including HSaV in testing patients with gastroenteritis. Furthermore, our results confirm that wastewater surveillance coupled with NGS is a powerful tool to study the molecular epidemiology of enteric viruses.

Correction to: Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing.

The original version of this article unfortunately contained a mistake. The presentation of Table 1 was incorrect. The corrected table is given below. The original article has been corrected.

Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing.

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing.

Human adenoviruses (HAdVs) are of major public health importance and are associated with a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract infections. To study the occurrence, prevalence and diversity of HAdV species and types circulating in Italy, we conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 22 wastewater treatment plants, covering 10 Italian regions, representative of northern, central, and southern Italy. A total of 141 raw sewage samples were collected from January to December 2013, and processed to detect and characterize by phylogenetic analysis a fragment of the hexon coding region of HAdVs. Nested PCR results showed the presence of HAdVs in 85 out of 141 samples (60% of samples). Fifty-nine samples were characterized by conventional Sanger sequencing as belonging to four HAdV species and four types: A (type 12, 5 samples), B (type 3, 8 samples), C (type 5, 1 sample) and F (type 41, 45 samples). The remaining 26 samples could not be characterized because of uninterpretable (mixed) electropherograms suggesting the presence of multiple species and/or types. Pools of characterized and uncharacterized PCR amplicons were further analyzed by next-generation sequencing (NGS). NGS results revealed a marked HAdV diversity with 16 additional types detected beyond the four types found by Sanger sequencing. Overall, 19 types were identified, belonging to HAdV species A-F: types 12 and 31 (species A), type 3 (species B), types 1, 2, and 5 (species C), types 9, 17, 24, 26, 37, 38, 42, 44, 48, and 70 (species D), type 4 (species E), and types 40 and 41(species F). An untypeable HAdV was also detected, showing similar percentages of identity with more than one prototype (types 15, 30, 56, and 59). Our findings documented the circulation of a wide variety of species and types in raw sewage, potentially able to affect other surface water environments and hence human health. Next-generation sequencing proved to be an effective strategy for HAdV genotyping in wastewater samples. It was able to detect a wide range of "less prevalent" types unidentified by conventional Sanger sequencing, confirming that studies based on conventional technologies may grossly underestimate the existence of some, possibly less common, types. Knowledge of the distribution of HAdV species and types would improve our understanding of waterborne HAdV-related health risks.

Outbreaks of Foot-and-Mouth Disease in Libya and Saudi Arabia During 2013 Due to an Exotic O/ME-SA/Ind-2001 Lineage Virus.

Foot-and-mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O-Ind-2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real-time RT-PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co-circulating in North Africa and western Eurasia.

Emergence of foot-and-mouth disease virus SAT 2 in Egypt during 2012.

The epidemiology of foot-and-mouth disease (FMD) in North Africa is complicated by the co-circulation of endemic FMD viruses (FMDV), as well as sporadic incursions of exotic viral strains from the Middle East and Sub-Saharan Africa. This report describes the molecular characterization of SAT 2 FMD viruses that have caused widespread field outbreaks of FMD in Egypt during February and March 2012. Phylogenetic analysis showed that viruses from these outbreaks fell into two distinct lineages within the SAT 2 topotype VII, which were distinct from a contemporary SAT 2 lineage of the same toptype from Libya. These were the first FMD outbreaks due to this serotype in Egypt since 1950 and required the development of a tailored real-time reverse-transcription PCR assay that can be used in the laboratory to distinguish FMD viruses of these lineages from other endemic FMD viruses that might be present in North Africa. These data highlight the ease by which FMDV can cross international boundaries and emphasize the importance of deploying systems to continuously monitor the global epidemiology of this disease.

Prevalence of border disease virus in Spanish lambs.

The prevalence of border disease virus (BDV) viraemia in Spanish lambs was determined from 2089 sera randomly collected at two slaughterhouses in 2001 and 2003, as well as in 126 sera obtained in 2004 from a fattening unit with an acute disease problem. BDV was detected with an indirect peroxidase monolayer assay (IPMA), and for the fattening unit sera also by an antigen ELISA. A subset of sera was additionally tested for BDV antibodies. The BDV prevalence in the slaughterhouse sera was 0.24%, whereas 7.1% of randomly selected and 38.6% of sera from clinically affected lambs in the fattening unit were virus positive. Pestivirus antibodies were found in 17.6% of the slaughterhouse sera and 28.6% of those from randomly selected lambs in the fattening unit. In total, 33 virus isolates and 3 antigen positive samples were identified. Genetic typing of 5'-UTR sequences classified all 36 pestiviruses as of BDV type 4. This shows that from a low BDV prevalence in apparently healthy lambs in the entire sheep population, clinical problems associated with BDV can develop when viraemic sheep are brought into intense rearing units.

Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula.

A selection of 10 pestiviruses isolated from sheep from the Iberian Peninsula from 2001 to 2004 was characterised at the molecular level. The 5' untranslated region (5'-UTR) and N(pro)-coding gene were amplified by the reverse transcription-polymerase chain reaction (RT-PCR) and sequenced directly from purified products. All isolates were also typed antigenically with a panel of monoclonal antibodies (mAbs) raised against representative isolates of the four recognised pestivirus species. The genetic typing placed all the isolates in a new tentative type 4 of border disease virus (BDV), which was closely related to a pestivirus recently found in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Overall, the genotyping indicated a relatively wide diversity of the BDV type 4, which was best defined on the basis of N(pro) sequences. Antigenically, the isolates were recognised by two pan-pestivirus specific anti-NS3 mAbs, but only by some of the anti-glycoprotein specific mAbs raised against BDV, indicating partial antigenic overlap with other BDV isolates.

Genetic typing and prevalence of Border disease virus (BDV) in small ruminant flocks in Spain.

Between 2001 and 2002, samples from 1,413 animals in 21 Spanish small ruminant flocks, most of them with animals showing clinical signs compatible with Border disease (BD), were screened for the presence of Pestivirus antigen and antibodies by an indirect peroxidase monolayer assay (IPMA) and the virus neutralization test (VNT), respectively. Although all flocks harboured seropositive animals, virus could only be isolated from animals in five of the flocks. Between 4 and 11 months later all animals older than 6 months in three of the flocks were resampled. At this time, 51-83% of them had neutralizing antibodies. The prevalence of persistently infected (PI) animals within two of the flocks was 0.3 and 0.6%, respectively. The third flock presumably had eliminated all the PI animals. Fourteen virus isolates were obtained. The 5' untranslated region (5'UTR) was amplified by RT-PCR and directly sequenced. Phylogenetic analyses classified them as a group of Border disease viruses (BDV), separated from BDV-1, but showing a relatively low bootstrap value. Three of the 14 isolates were in the same subgroup as a set of formerly characterised Spanish isolates from the Basque Country, which were allocated to subgroup BDV-C. In addition, they were in the group with an isolate from chamois, which is currently allocated in group BDV-4. Because of its close relation to the chamois isolate, these isolates were tentatively reallocated in a subgroup BDV-4a. The remaining isolates generated a new subgroup, related but not in the same cluster as the chamois isolate, and was therefore tentatively assigned to a new subgroup BDV-4b. Our results show that classification and nomenclature of BDV needs to be harmonised.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.