Epidemiology Update of Hepatitis E Virus (HEV) in Uruguay: Subtyping, Environmental Surveillance and Zoonotic Transmission.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease of increasing concern in developed regions. HEV genotype 3 (HEV-3) is mainly transmitted through consumption of contaminated food in high-income countries and is classified into at least 13 subtypes (3a-3n), based on p-distance values from complete genomes. In Latin America, HEV epidemiology studies are very scant. Our group has previously detected HEV3 in clinical cases, swine, wild boars, captive white-collared peccaries, and spotted deer from Uruguay. Herein, we aimed to provide novel insights and an updated overview of the molecular epidemiology of zoonotic HEV in Uruguay, including data from wastewater-based surveillance studies. A thorough analysis of HEV whole genomes and partial ORF2 sequences from Uruguayan human and domestic pig strains showed that they formed a separate monophyletic cluster with high nucleotide identity and exhibited p-distance values over the established cut-off (0.093) compared with reference subtypes' sequences. Furthermore, we found an overall prevalence of 10.87% (10/92) in wastewater, where two samples revealed a close relationship with humans, and animal reservoirs/hosts isolates from Uruguay. In conclusion, a single, new HEV-3 subtype currently circulates in different epidemiological settings in Uruguay, and we propose its designation as 3o along with its reference sequence.

Emergence and Spread of a B.1.1.28-Derived P.6 Lineage with Q675H and Q677H Spike Mutations in Uruguay.

Uruguay controlled the viral dissemination during the first nine months of the SARS-CoV-2 pandemic. Unfortunately, towards the end of 2020, the number of daily new cases exponentially increased. Herein, we analyzed the country-wide genetic diversity of SARS-CoV-2 between November 2020 and April 2021. We identified that the most prevalent viral variant during the first epidemic wave in Uruguay (December 2020-February 2021) was a B.1.1.28 sublineage carrying Spike mutations Q675H + Q677H, now designated as P.6, followed by lineages P.2 and P.7. P.6 probably arose around November 2020, in Montevideo, Uruguay's capital department, and rapidly spread to other departments, with evidence of further local transmission clusters; it also spread sporadically to the USA and Spain. The more efficient dissemination of lineage P.6 with respect to P.2 and P.7 and the presence of mutations (Q675H and Q677H) in the proximity of the key cleavage site at the S1/S2 boundary suggest that P.6 may be more transmissible than other lineages co-circulating in Uruguay. Although P.6 was replaced by the variant of concern (VOC) P.1 as the predominant lineage in Uruguay since April 2021, the monitoring of the concurrent emergence of Q675H + Q677H in VOCs should be of worldwide interest.

Real-Time Genomic Surveillance for SARS-CoV-2 Variants of Concern, Uruguay.

We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.

First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011-2012 and 2017-2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011-2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.

Detection, Quantification, and Microbial Risk Assessment of Group A Rotavirus in Rivers from Uruguay.

The aim of this study was to detect, quantify, and assess the risk of infection and illness for Group A Rotavirus (RVA) in the watersheds of the Santa Lucia and Uruguay rivers in Uruguay. Monthly sampling was carried out for one year in six sites in the watershed of the Santa Lucía River and four in the Uruguay River. All the collection sites are used for recreational activities. Viral concentration was performed with the adsorption-elution method, and detection and quantification of RVA was carried out by TaqMan quantitative PCR (qPCR). Quantitative microbial risk assessment was applied to estimate the daily and annual risk of RVA infection, as well as the daily risk of illness considering direct exposure through recreational activity. RVA was detected in 42% (20/48) of the analyzed samples in the Uruguay River and 40% (29/72) in the Santa Lucía River. The virus was present in all the analyzed points in both watersheds. A pattern of seasonality, characterized by a higher detection frequency of the virus during coldest month of the year, was observed in both basins. The mean concentration for RVA was 1.3 × 105 genomic copies/L. The microbiological risk assessment shows that Santa Lucía watershed presented the highest daily risk of infection (6.41E-01) and illness (3.20E-01) estimated for the point downstream of Florida City; meanwhile for Uruguay River, the highest probabilities of infection (6.82E-01) and illness (3.41E-01) were estimated for the collection site for drinking water intake in Salto city. These results suggest that RVA contamination of these important rivers negatively impact on their microbiological quality since they are used for recreation and drinking water intake, demonstrating that the disposal of waste from cities located in their riverside confers a constant threat of infection for the general population, especially for children.

Microbial Source Tracking Analysis Using Viral Indicators in Santa Lucía and Uruguay Rivers, Uruguay.

The aim of this study was to determine the origin (human, bovine or porcine) and the concentration of the fecal sources of contamination in waters from Santa Lucía basin and Uruguay River in Uruguay by using host-specific viral markers (adenoviruses and polyomaviruses) as microbial source tracking (MST). Between June 2015 and May 2016, monthly collections of surface water samples were performed in six sites in Santa Lucía basin and four sites in Uruguay River (n = 120 samples). Viral concentration was carried out using an absorption-elution method. Detection and quantification of human and porcine adenovirus (HAdV and PAdV, respectively) and human and bovine polyomavirus (HPyV and BoPyV, respectively) were performed by quantitative PCR (qPCR). To evaluate the infectivity of circulating HAdV, an integrated cell culture-qPCR (ICC-qPCR) was used. A logistic regression analysis was carried out to estimate the influence of environmental variables on the virus presence in surface waters. Overall, HAdV was the prevalent (18%; 21/120) followed by BoPyV (11%; 13/120) and HPyV (3%; 3/120), whereas PAdV was not detected in this study. The mean concentration ranged from 1.5 × 104 genomic copies/L (gc/L) for HAdV to 1.8 × 102 gc/L for HPyV. Infective HAdVs were observed in two out of ten analyzed samples. A significant effect of environmental temperature (p = 0.001) and river (p = 0.012) on the presence of human viruses was found. These results suggest that fecal contamination could affect the water quality of these rivers, showing deficiencies in the procedure of sewage discharge from regional cities, livestock and dairy farms.

Human Bocavirus: Detection, Quantification and Molecular Characterization in Sewage and Surface Waters in Uruguay.

Human bocavirus (HBoV) infections are related to respiratory and gastroenteric diseases. The aim of this study was to investigate the presence of HBoV in both sewage and surface waters in Uruguay. Sixty-eight sewage samples from the cities of Salto, Paysandú, Bella Unión, Fray Bentos, Treinta y Tres and Melo and 36 surface water samples from the cities of Salto, Florida and Santa Lucía were studied. HBoV was screened by multiplex qPCR for the detection of the four subtypes, followed by monoplex qPCRs for the independent quantification of each subtype. A qualitative PCR followed by DNA sequencing and phylogenetic analysis was carried out for molecular characterization of HBoV strains. HBoV was present in a high frequency (69%) in sewage and only one positive sample (3%) was found in surface water. Concerning sewage samples, HBoV1 was detected in 11 (23%) out of the 47 positives samples, with a mean concentration of 8.2 × 104 genomic copies/Liter (gc/L), HBoV3 was detected in 35 (74%) of the positive samples with a mean concentration of 4.1 × 106 gc/L and subtypes 2 and/or 4 were detected in 39 (83%) of the positive samples with a mean concentration of 7.8 × 106 gc/L. After the phylogenetic analysis performed by a Bayesian approach, the four HBoV subtypes were confirmed. This is the first study determining a high frequency of HBoV and the presence of the four HBoV subtypes in aquatic matrices in Latin America, mainly in sewage. Although HBoV was scarcely detected in surface water, a waterborne transmission is likely to occur if people enter in contact with polluted surface waters for recreational activities such as fishing or swimming since an elevated frequency of HBoV was detected in raw sewage which is usually directly discharged into surface waters.