High abundance of high-risk Human Papillomavirus genotypes in wastewater in Uruguay.

The aim of this study was to determine the frequency of Human Papillomavirus (HPV) genotypes in wastewater of Salto city, Uruguay, in order to obtain a general overview of the circulating genotypes in their population. HPV was detected in 34% (32/93) of the wastewater samples collected and analyzed during 2020/21 in Salto city, Uruguay. Thirty-three genotypes were observed, of which 16 presented read abundance higher than 1%, including both high-risk (HR) and low-risk (LR) genotypes. HR genotypes 31, 16, 58, 52, 33 and 59 were detected representing 40% (163,220 reads) of the total read abundance, with genotypes 31 (64,365), 16 (39,337) and 58 (36,332) being the most abundant. LR genotypes 72, 6, 11 and 40 were also detected in a high frequency, accounting for 37% (148,359) of the HPV reads. This study highlights the high frequency of HR genotypes of HPV, circulating in the population of Salto city which is a burden in public health mainly due to the devastating impact of cervical cancer in women.

Bovine Polyomavirus-1 (Epsilonpolyomavirus bovis): An Emerging Fetal Pathogen of Cattle That Causes Renal Lesions Resembling Polyomavirus-Associated Nephropathy of Humans.

Bovine polyomavirus-1 (BoPyV-1, Epsilonpolyomavirus bovis) is widespread in cattle and has been detected in commercialized beef at supermarkets in the USA and Germany. BoPyV-1 has been questioned as a probable zoonotic agent with documented increase in seropositivity in people exposed to cattle. However, to date, BoPyV-1 has not been causally associated with pathology or disease in any animal species, including humans. Here we describe and illustrate pathological findings in an aborted bovine fetus naturally infected with BoPyV-1, providing evidence of its pathogenicity and probable abortigenic potential. Our results indicate that: (i) BoPyV-1 can cause severe kidney lesions in cattle, including tubulointerstitial nephritis with cytopathic changes and necrosis in tubular epithelial cells, tubular and interstitial inflammation, and interstitial fibroplasia; (ii) lesions are at least partly attributable to active viral replication in renal tubular epithelial cells, which have abundant intranuclear viral inclusions; (iii) BoPyV-1 large T (LT) antigen, resulting from early viral gene expression, can be detected in infected renal tubular epithelial cells using a monoclonal antibody raised against Simian Virus-40 polyomavirus LT antigen; and (iv) there is productive BoPyV-1 replication and virion assembly in the nuclei of renal tubular epithelial cells, as demonstrated by the ultrastructural observation of abundant arrays of viral particles with typical polyomavirus morphology. Altogether, these lesions resemble the "cytopathic-inflammatory pathology pattern" proposed in the pathogenesis of Human polyomavirus-1-associated nephropathy in immunocompromised people and kidney allograft recipients. Additionally, we sequenced the complete genome of the BoPyV-1 infecting the fetus, which represents the first whole genome of a BoPyV-1 from the Southern Hemisphere. Lastly, the BoPyV-1 strain infecting this fetus was isolated, causing a cytopathic effect in Madin-Darby bovine kidney cells. We conclude that BoPyV-1 is pathogenic to the bovine fetus under natural circumstances. Further insights into the epidemiology, biology, clinical relevance, and zoonotic potential of BoPyV-1 are needed.

Encefalitis asociada a astrovirus bovino neurotrópico, ¿una enfermedad subdiagnosticada en Sudamérica? / Neurotropic bovine astrovirus-associated encephalitis: An underdiagnosed diseasein South America?

Resumen Describimos un caso de encefalitis asociada a infección por astrovirus bovino neu-rotrópico en una vaca lechera, raza Jersey, del departamento de San José, Uruguay. Esterepresenta el segundo caso reportado de esta condición en el hemisferio sur. La vaca, únicaafectada de un rodeo de 70 bovinos, manifestó signos clínicos neurológicos con curso de 2 días,luego de los que murió espontáneamente. El examen histopatológico reveló meningoencefalitislinfocítica, histiocítica y plasmacítica, con necrosis neuronal, sin cuerpos de inclusión. No sedetectaron en el cerebro otros agentes infecciosos, incluyendo el virus de la rabia (Lyssavirus),alfaherpesvirus bovino-1 y alfaherpesvirus bovino-5 (Varicellovirus), virus de la diarrea viralbovina (Pestivirus), virus del Nilo Occidental (Flavivirus), Listeria monocytogenes, Histophi-lus somni y otras bacterias. Dado que el descubrimiento de astrovirus neurotrópicos en variasespecies de mamíferos, incluidos humanos, es reciente, proponemos que los casos de encefalitis por astrovirus pudieron haber pasado inadvertidos en Sudamérica. Discutimos brevementeel diagnóstico patológico diferencial de encefalitis infecciosas en bovinos.

Abstract We describe a case of neurotropic bovine astrovirus-associated encephalitis in a Jer-sey dairy cow from the department of San José, Uruguay. This represents the second case of thiscondition reported in the Southern Hemisphere. The cow was the only one affected in a herd of70 cows, showing neurological signs with a 2-day clinical course, before dying spontaneously.Histopathological examination revealed lymphocytic, histiocytic, and plasmacytic meningoen-cephalitis with neuronal necrosis, without detectable inclusion bodies. Other infectious agents,including Rabies virus (Lyssavirus), Bovine alphaherpesvirus-1 and Bovine alphaherpesvirus-5(Varicellovirus), Bovine viral diarrhea virus (Pestivirus), West Nile virus (Flavivirus), Listeriamonocytogenes, Histophilus somni and other bacteria, were not detected in the brain. We pro-pose that given the recent discovery of neurotropic astroviruses in various mammalian species,including humans, cases of astrovirus encephalitis may have gone undetected in South America.We briefly discuss the differential pathologic diagnosis of infectious bovine encephalitis.

Causes of abortion in dairy cows in Uruguay / Causas de aborto em bovinos de leite no Uruguai

A case series study was conducted to determine the frequency of causes of abortion in dairy cattle in Uruguay. The sample size of 102 cases was composed of 53 fetuses, 35 fetuses with placentas, and 14 placentas without an associated fetus. All cases underwent gross and microscopic pathologic examinations as well as microbiological and serological testing. The etiology was determined in 54 (53%) of cases, 51 of which were caused by infectious agents. Within the observed 102 cases, 30 (29%) were caused by Neospora caninum, six (6%) by Coxiella burnetii and two (2%) by Campylobacter fetus subsp. venerealis. Bovine Parainfluenza-3 virus and Salmonella enterica serovar Newport caused one abortion each. Opportunistic bacteria (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes, and Providencia stuartii) were associated with 11 abortions. In two cases the fetal death was attributed to dystocia, and in one case the fetus had a congenital mesothelioma. Bovine viral diarrhea virus (BVDV) infection was identified in three fetuses; two of which were co-infected with and had typical lesions of N. caninum. No lesions were observed in the other fetus infected by BVDV. Leptospira interrogans was identified in one fetus without lesions. Despite the relatively low overall success rate in establishing an etiological diagnosis in cases of abortion in cattle, a systemic workup of bovine abortion is necessary to establish prevention and control strategies. This also facilitates monitoring and surveillance of reproductive diseases in dairy cattle, some of which represent a risk to public health.(AU)

Uma série de casos foi estudada para determinar a frequência de causas do aborto em bovinos leiteiros no Uruguai. A amostra, de 102 casos, foi composta por 53 fetos, 35 fetos com placentas e 14 placentas sem feto associado. Todos os casos foram submetidos a exames patológicos macroscópicos e microscópicos, além de testes microbiológicos e sorológicos. A etiologia foi determinada em 54 (53%) dos casos, 51 dos quais foram causados por agentes infecciosos. Nos 102 casos observados, 30 (29%) foram causados por Neospora caninum, seis (6%) por Coxiella burnetii e dois (2%) por Campylobacter fetus subsp. venerealis. O vírus da Parainfluenza-3 e Salmonella enterica serovar Newport causaram um aborto cada. Bactérias oportunistas (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes e Providencia stuartii) foram associadas a 11 abortos. Em dois casos, a morte fetal foi atribuída a distocia e, em um caso, o feto apresentava mesotelioma congênito. A infecção pelo vírus da diarreia viral bovina (BVDV) foi identificada em três fetos; dois dos quais foram co-infectados e apresentavam lesões típicas de N. caninum. Não foram observadas lesões no outro feto infectado pelo BVDV. Leptospira interrogans foi identificada em um feto sem lesões. Apesar da relativamente baixa taxa de sucesso no diagnóstico etiológico nos casos de aborto em bovinos, é necessário o diagnóstico sistemático dos abortos para estabelecer estratégias de prevenção e controle. Isso também facilita o monitoramento e a vigilância de doenças reprodutivas em bovinos leiteiros, algumas das quais representam um risco para a saúde pública.(AU)

An extensive field study reveals the circulation of new genetic variants of subtype 1a of bovine viral diarrhea virus in Uruguay.

Bovine viral diarrhea virus (BVDV) is a major pathogen worldwide, causing significant economic losses to the livestock sector. In Uruguay, BVDV seroprevalence at the farm level is >80%. In this work, 2546 serum, blood or tissue samples collected from animals suspected of being affected by BVD between 2015 and 2017 were analyzed by reverse transcription PCR and sequencing. Analysis of the BVDV genomic regions 5'UTR/Npro, Npro and E2 revealed that BVDV-1a, 1i and 2b circulate in the country, with BVDV-1a being the most prevalent subtype. Population dynamics studies revealed that BVDV-1a has been circulating in our herds since ~1990. This subtype began to spread and evolve, accumulating point mutations at a rate of 3.48 × 10-3 substitutions/site/year, acquiring specific genetic characteristics that gave rise to two local genetic lineages of BVDV-1a. These lineages are divergent from those circulating worldwide, as well as the vaccine strain currently used in Uruguay. The most notable differences between field and vaccine strains were found in the E2 glycoprotein, suggesting that the amino acid substitutions could result in failure of cross-protection/neutralization after vaccination. This is the first study that compares Uruguayan BVDV field and vaccine strains with other BVDV strains from throughout the world. The results obtained in this study will be very useful for developing a suitable immunization program for BVDV in Uruguay by identifying local field strains as candidates for vaccine development.

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.

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.

Phylodynamics of Merkel-cell polyomavirus and human polyomavirus 6: A long-term history with humans.

New human polyomaviruses have been described in the last years, including the Merkel-cell polyomavirus (MCPyV; Human polyomavirus 5) and the Human polyomavirus 6 (HPyV6). Although their infection is usually asymptomatic, in immunocompromised host can cause life-threatening pathologies, such as the Merkel cell carcinoma, an aggressive skin neoplasia associated to the MCPyV. Despite being prevalent viruses in population, epidemiological data from South America are scarce, as well as the characterization of the viral types circulating and their origin. The aims of this work were to describe MCPyV and HPyV6 from environmental samples with different geographical origin and to analyze their phylogenetic and evolutionary histories, particularly for MCPyV. Partial and complete genome sequences were obtained from sewage samples from Argentina, Uruguay and Spain. A total number of 87 sequences were obtained for MCPyV and 33 for HPyV6. Phylogenetic analysis showed that MCPyV sequences distributed according to their geographic origin in Europe/North America, Africa, Asia, South America and Oceania groups, suggesting that viral diversification might have followed human migrations across the globe. In particular, viruses from Argentina associated with Europe/North America and South America genotypes, whereas those from Uruguay and Spain also grouped with Africa genotype, reflecting the origin of the current population in each country, which could arrive not only during ancient human migration but also during recent migratory events. In addition, the South American group presented a high level of clusterization, showing internal clusters that could be related to specific locations, such as French Guiana and Brazil or the Southern region into South America, such as Argentina and Uruguay, suggesting a long term evolutionary process in the region. Additionally, in this work, we carried out the first analysis about the evolutionary history of MCPyV trough the integration of phylogenetic, epidemiological and historical data. Since a strong association is observed between the phylogenetic relationships and the origin of the sampled population, this analysis was based on the hypothesis of co-divergence between the virus and human populations. This analysis resulted in a substitution rate of 5.1 × 10-8 s/s/y (∼5.1% of divergence per million years) for the complete genome of MCPyV, which is in the range of those estimated for other double-stranded DNA viruses. Regarding HPyV6, a South American group with clusterization was observed (sequences from Uruguay). Meanwhile, sequences from Argentina grouped with European ones (France and Spain) and remained separated from those isolated in China, USA or Australia. The analysis of viruses from the environment allowed us to deep characterize prevalent infections in different geographic regions, reveling that viruses circulating in each population reflected its origin and that there are specific lineages associated with South America.

Leishmania repression of host translation through mTOR cleavage is required for parasite survival and infection.

The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.

Evidence of diversification of dengue virus type 3 genotype III in the South American region.

In order to gain insight into the genetic variability of dengue virus type 3 (DENV-3) genotype III isolated in the Latin American region, phylogenetic analysis were carried out using envelope (E) gene sequences from 57 DENV-3 genotype III strains isolated in 11 Latin American countries. At least six different genotype III clades were observed. Amino acids substitutions were found in domain III E protein neutralization epitopes and in surface-exposed domain II and III E protein amino acid sequences.

Evidence of intratypic recombination in natural populations of hepatitis C virus.

Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of subtypes have been reported. Genotype 1 is the most prevalent genotype found in South America. In the present study, three different genomic regions (5'UTR, core and NS5B) of four HCV strains isolated from Peruvian patients were sequenced in order to investigate the congruence of HCV genotyping for these three genomic regions. Phylogenetic analysis using 5'UTR-core sequences found strain PE22 to be related to subtype 1b. However, the same analysis using the NS5B region found it to be related to subtype 1a. To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossover event had taken place in the NS5B protein. We discuss the consequences of this observation on HCV genotype classification, laboratory diagnosis and treatment of HCV infection.

Molecular evolution of hepatitis A virus: a new classification based on the complete VP1 protein.

Hepatitis A virus (HAV) is a positive-stranded RNA virus in the genus Hepatovirus in the family Picornaviridae So far, analysis of the genetic variability of HAV has been based on two discrete regions, the VP1/2A junction and the VP1 N terminus. In this report, we determined the nucleotide and deduced amino acid sequences of the complete VP1 gene of 81 strains from France, Kosovo, Mexico, Argentina, Chile, and Uruguay and compared them with the sequences of seven strains of HAV isolated elsewhere. Overall strain variation in the complete VP1 gene was found to be as high as 23.7% at the nucleotide level and 10.5% at the amino acid level. Different phylogenetic methods revealed that HAV sequences form five distinct and well-supported genetic lineages. Within these lineages, HAV sequences clustered by geographical origin only for European strains. The analysis of the complete VP1 gene allowed insight into the mode of evolution of HAV and revealed the emergence of a novel variant with a 15-amino-acid deletion located on the VP1 region where neutralization escape mutations were found. This could be the first antigenic variant of HAV so far identified.

Caracterización molecular del virus de la hepatitis C en Montevideo-Uruguay / Molecular characterization of Hepatitis C virus in Montevideo-Uruguay

Introducción; las hepatitis causadas por el virus de la hepatitis C (VHC) se han transformado en uno de los principales problemas asociados a las infecciones emergentes. El VHC posee una alta variabilidad genética identificándose desde su descubrimiento a la fecha seis tipos principales y un número creciente de subtipos virales asociados a diferente respuesta al tratamiento y a la evolución natural de la enfermedad. Objetivo: contribuir al diagnóstico, al seguimiento de los infectados y al conocimiento de la epidemiología a través del análisis molecular determinando el genotipo y la carga viral. Material y método: se analizaron las muestras de 175 pacientes referidos entre marzo de 1997 y junio de 2001 para el diagnóstico de infección por VHC. En todos los casos se realizó determinación de anticuerpos por enzima inmuno análisis (ELISA) e inmunoblot y amplificación del genoma de VHC mediante técnicas de transcripción reversa y reacción en cadena de la polimerasa (RT-PCR). A una submuestra de pacientes PCR positivos se determinó el genotipo y carga viral. Resultados: se estableció viremia en 125 pacientes de un total de 175 (173 con serología positiva y dos casos con serología negativa para VHC). El análisis de los polimorfismos de restricción (RFLP's) y de la secuencia nucleotídica en 51 portadores crónicos mostró la siguiente distribución de genotipos y sus correspondientes subtipos: a) genotipo 1: 35 casos (subtipos 1a (17), 1b (16), variante (Mvnl y Mon2)); b) genotipo 2: 3 casos ((subtipos 2a (l) y 26 (2)) y c) genotipo 3:13 casos (subtipos 3a (13)). Conclusiones: en el presente trabajo se muestra la importancia de la implementación de técnicas moleculares aplicadas al diagnóstico y seguimiento de portadores de HVC, constituyendo una nueva herramienta, altamente precisa, sensible y específica. La gran capacidad analítica, a través de la secuenciación automática y el posterior análisis filogenético, nos ha permitido identificar en dos pacientes una nueva variante genética de HCV que no habría sido descrita, y que plantea nuevas interrogantes. Estas fueron publicadas e ingresadas al Gen-Bank con los nombres Mon 1 y Mon 2. Con respecto a la determinación de la carga viral no se evidenció ningún tipo de correlación entre el genotipo infectante y el valor de carga obtenido, siendo por ello la viremia genotipo independiente

Genetic variability of hepatitis A virus in South America reveals heterogeneity and co-circulation during epidemic outbreaks.

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. In order to gain insight into the genetic variability and mode of evolution of HAV in South America, an analysis was performed of sequence data obtained from the VP1 amino terminus and the VP1/2A region of HAV strains isolated over a short period of time in Uruguay, Argentina and Chile. Sequences obtained from 22 distinct HAV isolates were compared with published sequences from 21 different strains isolated all over the world. Phylogenetic analysis revealed that all strains isolated belong to a unique sub-genotype (IA). Strains isolated during an outbreak period showed a higher degree of heterogeneity than anticipated previously and the co-circulation of different isolates. The genetic variability among strains isolated in this region seems to be higher in comparison with strains isolated in other regions of the world.