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.

Hand-foot-and-mouth disease in uruguay: Coxsackievirus A6 identified as causative of an outbreak in a rural childcare center.

To know the epidemiological context of hand-foot-and-mouth disease (HFMD) in a region of Uruguay and to identify the Enterovirus responsible for an outbreak in a rural childcare center in 2018. Swab samples from skin lesions and/or stools samples were collected from children suffering HFMD during an outbreak in a rural childcare center. Samples were subject to viral RNA extraction and reverse-transcription polymerase chain reaction towards VP1 coding segment, to identify the Enterovirus type by sequencing and phylogenetic analysis. Total of 149 cases of HFMD affecting 98 boys and 51 girls were reported in Salto Province-Uruguay in 2018. Total 60% of the cases were originated from outbreaks, which occurred in ten educative and childcare institutions from both urban and rural areas. Coxsackievirus-6 (CV-A6) was identified as responsible for one of the rural outbreaks. Uruguayan strains were more related to strains reported in Russia, Turkey, and Germany (2014-2017) than to strains reported in Brazil and Argentina from 2015 to 2016. This is the first report of CV-A6-associated HFMD in Uruguay, evidencing a wide geographic range of the virus in the Latin American region. Our report also warns about CV-A6-associated HFMD during winter, contrarily to most reports that register HFMD during summer and fall seasons.

Description of a human Bocavirus recombinant strain in the Americas.

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.

Epidemiology and genetic diversity of classic human astrovirus among hospitalized children with acute gastroenteritis in Uruguay.

Classic Human Astrovirus (Classic HAstV) are one of the most important causes of pediatric acute gastroenteritis (AGE), after rotaviruses and arguably caliciviruses. The aim of this study was to determine the molecular epidemiology of Classic HAstV from 175 clinical samples, being 153 stools and 22 vomits, collected from pediatric hospitalized patients with AGE in Salto city, Uruguay, from January 2011 to December 2012. Classic HAstV were detected and genotyped by using a qualitative Retro Transcription-Polimerase Chain Reaction (RT-PCR) directed to the Open Reading Frame-2 (ORF2) region C. Amplicons were sequenced and phylogenetic analyses were carried out in order to determine genotypes and lineages. Classic HAstV were detected in 18 out of 175 analyzed samples (10.3%) and 14 of them (78.0%) were successfully sequenced being 6 (42.8%) classified as HAstV-1 (1a lineage), 4 (28.6%) as HAstV-2 (2c lineage), and 4 (28.6%) as HAstV-3 (3c lineage). A higher detection of Classic HAstV infections was observed in autumn for both years of surveillance, and the majority of the positive cases were observed in 2011. The group of children between 2 and 5 years old presented the higher percentage of infections. To our knowledge, the present study represents the first report of astrovirus from acute gastroenteritis cases in Uruguay, evidencing its role as a relevant etiologic agent in severe cases of this disease.

Environmental Surveillance through Next-Generation Sequencing to Unveil the Diversity of Human Enteroviruses beyond the Reported Clinical Cases.

The knowledge about circulation of Human Enteroviruses (EVs) obtained through medical diagnosis in Argentina is scarce. Wastewater samples monthly collected in Córdoba, Argentina during 2011-2012, and then in 2017-2018 were retrospectively studied to assess the diversity of EVs in the community. Partial VP1 gene was amplified by PCR from wastewater concentrates, and amplicons were subject of next-generation sequencing and genetic analyses. There were 41 EVs detected, from which ~50% had not been previously reported in Argentina. Most of the characterized EVs (60%) were detected at both sampling periods, with similar values of intratype nucleotide diversity. Exceptions were enterovirus A71, coxsackievirus B4, echovirus 14, and echovirus 30, which diversified in 2017-2018. There was a predominance of types from EV-C in 2017-2018, evidencing a common circulation of these types throughout the year in the community. Interestingly, high genetic similarity was evidenced among environmental strains of echovirus 30 circulating in 2011-2012 and co-temporal isolates obtained from patients suffering aseptic meningitis in different locations of Argentina. This study provides an updated insight about EVs circulating in an important region of South America, and suggests a valuable role of wastewater-based epidemiology in predicting outbreaks before the onset of cases in the community.

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.

Recurrent Dissemination of SARS-CoV-2 Through the Uruguayan-Brazilian Border.

Uruguay is one of the few countries in the Americas that successfully contained the coronavirus disease 19 (COVID-19) epidemic during the first half of 2020. Nevertheless, the intensive human mobility across the dry border with Brazil is a major challenge for public health authorities. We aimed to investigate the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains detected in Uruguayan localities bordering Brazil as well as to measure the viral flux across this ∼1,100 km uninterrupted dry frontier. Using complete SARS-CoV-2 genomes from the Uruguayan-Brazilian bordering region and phylogeographic analyses, we inferred the virus dissemination frequency between Brazil and Uruguay and characterized local outbreak dynamics during the first months (May-July) of the pandemic. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 Brazilian lineages B.1.1.28 and B.1.1.33 into Uruguayan localities at the bordering region. The most probable sources of viral strains introduced to Uruguay were the Southeast Brazilian region and the state of Rio Grande do Sul. Some of the viral strains introduced in Uruguayan border localities between early May and mid-July were able to locally spread and originated the first outbreaks detected outside the metropolitan region. The viral lineages responsible for Uruguayan urban outbreaks were defined by a set of between four and 11 mutations (synonymous and non-synonymous) with respect to the ancestral B.1.1.28 and B.1.1.33 viruses that arose in Brazil, supporting the notion of a rapid genetic differentiation between SARS-CoV-2 subpopulations spreading in South America. Although Uruguayan borders have remained essentially closed to non-Uruguayan citizens, the inevitable flow of people across the dry border with Brazil allowed the repeated entry of the virus into Uruguay and the subsequent emergence of local outbreaks in Uruguayan border localities. Implementation of coordinated bi-national surveillance systems is crucial to achieve an efficient control of the SARS-CoV-2 spread across this kind of highly permeable borderland regions around the world.

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.

Coxsackievirus A10 causing hand-foot-and-mouth disease in Uruguay.

We report for the first time in South America an HFMD case associated with Coxsackievirus A10. The viral strain belongs to a lineage involved in important European outbreaks and probably entered Uruguay after 2017 with a Greek origin. These findings call for strengthening the regional surveillance of HFMD.

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.

An Environmental Surveillance in Uruguay Reveals the Presence of Highly Divergent Types of Human Enterovirus Species C and a High Frequency of Species A and B Types.

Information about Human Enterovirus circulation in Uruguay is scarce. The aim of this study was to generate the first description about their circulation in the country through the study of sewage samples collected before and after the switch from Oral Poliovirus Vaccine to Inactivated Poliovirus Vaccine. Viruses were concentrated by an adsorption-elution to a negatively charged membrane, and real-time quantitative PCR and qualitative PCR methods were used to detect, quantify, and characterize enteroviruses. Positive samples were inoculated in RD cells and two passages were performed. Additionally, RD+ samples were subsequently passed onto L20B cells. Human Enteroviruses were detected in 67.6% of the samples, with concentrations between 4.9 and 6.6 Log10 genomic copies per liter. 10% of positive samples replicated in RD cells, of which none in L20B cells. Molecular characterization of Human Enterovirus strains directly detected from sewage sample concentrates allowed the identification of highly divergent members of species C such as Enterovirus C99 and Coxsackievirus A13, as well as the frequent detection of species A and B members (particularly Coxsackievirus A16 and Echovirus 6, respectively). Other detected types were Coxsackievirus A2, A22, B1, B5, Echovirus 5, and 9. The characterization of viruses isolated in cell culture revealed the presence of Echovirus 6 and Coxsackievirus B3. Despite the absence of poliovirus, a wide circulation of different enterovirus types was evidenced in Uruguayan sewage samples, highlighting that the local populations are exposed to different kinds of diseases originated by several human enterovirus.

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.

Detection of Common, Emerging and Uncommon VP4, and VP7 Human Group A Rotavirus Genotypes from Urban Sewage Samples in Uruguay.

Environmental approach has proven to be a useful tool for epidemiological studies demonstrating through environmental studies the diversity of viruses circulating in a given population. The aim of this study was to perform a phylogenetic characterization of the group A rotavirus (RVA) glycoprotein (G)- and protease-sensitive (P)-genotypes obtained from sewage samples (n = 116) collected in six cities of Uruguay during March 2011 to April 2013. A worldwide standardized semi-nested multiplex RT-PCR (SNM RT-PCR) protocol directed against VP4 and VP7 genes were conducted for RVA detection and consensual DNA fragments were submitted to nucleotide sequencing. P and/or G genotype was successfully determined by phylogenetic analysis in 61% (n = 37) of the positive samples obtained by SNM RT-PCR (n = 61). The RVA genotypes were as follow: G1 (n = 2), G2 (n = 14), G3 (n = 5), G12 (n = 2), P[4] (n = 4), P[8] (n = 16), and P[3] (n = 2). Interestingly, through phylogenetic analysis, emerging, and uncommon human genotypes could be detected. Results obtained from the comparison of RVA genotypes detected in the current study and Uruguayan RVA strains previously described for contemporary clinical pediatric cases showed that monitoring sewage may be a good screening option for a rapid and economical overview of the circulating genotypes in the surrounding human population and a useful approximation to study RVA epidemiology in a future vaccine monitoring program. The present study represents the first report in Uruguay that describes the phylogenetic diversity of RVA from urban sewage samples.

Description of a human Bocavirus recombinant strain in the Americas

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.