In vitro antiviral activity of favipiravir and its 6- and 3-O-substituted derivatives against coronavirus: Acetylation leads to improvement of antiviral activity.

Favipiravir is currently approved for the treatment of the influenza virus and has shown encouraging results in terms of antiviral capacity in clinical studies against severe acute respiratory syndrome coronavirus 2. Favipiravir is a prodrug, where its favipiravir-ribofuranosyl-5B-triphosphate metabolite is capable of blocking RNA replication of the virus. However, the antiviral efficiency of favipiravir is limited by two factors: (i) low accumulation in plasma and rapid excretion/elimination post-administration and (ii) low conversion rate into the active metabolite. To tackle these problems, herein, we have designed new favipiravir analogues focusing on the replacement of the fluorine atom at the 6-position by halogen or hydrogen atoms and 3-O-functionalization with labile groups. The first type of functionalization seeks to increase the antiviral activity because of the better ability of the keto-tautomer as a function of the halogen, and it is hypothesized that the keto-tautomer tends to promote the formation of the ribofuranosyl-5B-triphosphate (RTP) metabolite. Meanwhile, the second type of functionalization seeks to promote lipophilicity and increase accumulation in cells. From the in vitro antiviral activity against two coronavirus models (bovine and human 229E), it was identified that the replacement did not improve the antiviral activity against both the models, which seems to be attributable to the low water solubility of these new 6-functionalized analogues. Meanwhile, with 3-O-functionalization, acetylation provided the most active compounds with higher half-maximal inhibitory concentration and selectivity than favipiravir, whereas benzylation/methanosulfonation yielded the least active compounds. In summary, acetylation is found to be a convenient functionalization to enhance the antiviral profile of favipiravir.

Detection and genome characterisation of SARS-CoV-2 P.6 lineage in dogs and cats living with Uruguayan COVID-19 patients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in domestic animals have occurred from the beginning of the pandemic to the present time. Therefore, from the perspective of One Health, investigating this topic is of global scientific and public interest. OBJECTIVES: The present study aimed to determine the presence of SARS-CoV-2 in domestic animals whose owners had coronavirus disease 2019 (COVID-19). METHODS: Nasopharyngeal and faecal samples were collected in Uruguay. Using quantitative polymerase chain reaction (qPCR), we analysed the presence of the SARS-CoV-2 genome. Complete genomes were obtained using ARTIC enrichment and Illumina sequencing. Sera samples were used for virus neutralisation assays. FINDINGS: SARS-CoV-2 was detected in an asymptomatic dog and a cat. Viral genomes were identical and belonged to the P.6 Uruguayan SARS-CoV-2 lineage. Only antiserum from the infected cat contained neutralising antibodies against the ancestral SARS-CoV-2 strain and showed cross-reactivity against the Delta but not against the B.A.1 Omicron variant. MAIN CONCLUSIONS: Domestic animals and the human SARS-CoV-2 P.6 variant comparison evidence a close relationship and gene flow between them. Different SARS-CoV-2 lineages infect dogs and cats, and no specific variants are adapted to domestic animals. This first record of SARS-CoV-2 in domestic animals from Uruguay supports regular surveillance of animals close to human hosts.

Structural aspects of hepatitis E virus.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Hepatitis E is an enterically transmitted zoonotic disease that causes large waterborne epidemic outbreaks in developing countries and has become an increasing public-health concern in industrialized countries. In this setting, the infection is usually acute and self-limiting in immunocompetent individuals, although chronic cases in immunocompromised patients have been reported, frequently associated with several extrahepatic manifestations. Moreover, extrahepatic manifestations have also been reported in immunocompetent individuals with acute HEV infection. HEV belongs to the alphavirus-like supergroup III of single-stranded positive-sense RNA viruses, and its genome contains three partially overlapping open reading frames (ORFs). ORF1 encodes a nonstructural protein with eight domains, most of which have not been extensively characterized: methyltransferase, Y domain, papain-like cysteine protease, hypervariable region, proline-rich region, X domain, Hel domain, and RNA-dependent RNA polymerase. ORF2 and ORF3 encode the capsid protein and a multifunctional protein believed to be involved in virion release, respectively. The novel ORF4 is only expressed in HEV genotype 1 under endoplasmic reticulum stress conditions, and its exact function has not yet been elucidated. Despite important advances in recent years, the biological and molecular processes underlying HEV replication remain poorly understood, primarily due to a lack of detailed information about the functions of the viral proteins and the mechanisms involved in host-pathogen interactions. This review summarizes the current knowledge concerning HEV proteins and their biological properties, providing updated detailed data describing their function and focusing in detail on their structural characteristics. Furthermore, we review some unclear aspects of the four proteins encoded by the ORFs, highlighting the current key information gaps and discussing potential novel experimental strategies for shedding light on those issues.

Serological and molecular prevalence of hepatitis E virus among blood donors from Uruguay.

Hepatitis E virus (HEV) infection is considered a neglected disease of major concern in developed countries. Clinically, HEV occurs as an acute and self-limited disease, though chronic cases mostly associated to HEV-3 are now being commonly reported in immunocompromised individuals and solid organ transplant recipients. Transmission of HEV through blood and derivatives have been increasingly described in the last years, highlighting the importance of including this agent on the screening programs. Since 2010 both acute and chronic hepatitis E cases have been frequently reported in Uruguay. However, updated prevalence data among different population groups are lacking and HEV is not currently screened in blood banks. Herein, we report a seroprevalence and molecular survey of HEV in 400 plasma samples from blood donors. Overall, our results showed an HEV seroprevalence rate of 10% (40/400); almost 10-fold higher than 20 years ago. Total anti-HEV immunoglobulin antibodies were found to increase with age. Moreover, we reported an RNA detection rate of at least 0.75%, and two strains were sequenced. Phylogenetic analysis grouped them with human and swine HEV-3 strains from Uruguay. Data presented here should prompt public health policies of HEV screening in blood banks to minimize the risk of transfusion-transmitted hepatitis E.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES: The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS: To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS: Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS: This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

Hepatitis E and chronic liver damage in apparently immunocompetent individuals: Now what?

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, accounting for 20 million infections per year and 70,000 deaths. In developed regions, sporadic locally acquired infections are most commonly caused by HEV3, and in this setting Hepatitis E is mainly asymptomatic. However, certain group of patients HEV infection may present as a fulminant disease or progressive fibrosis. Chronic HEV infection can occur in immunocompromised individuals, including transplant recipients. A high proportion of solid-organ transplant recipients exposed to HEV are at risk of developing a chronic infection, frequently associated to extrahepatic manifestations. However, clinical phenotype of sporadic cases of HEV infection is still poorly characterized. A recent work, focused on the retrospective study of HEV as a causative agent of viral hepatitis in adults form Mexico, pose novel challenges to understanding the HEV threat to human health. Main findings are brought into discussion herein, in light of the current knowledge concerning viral pathogenesis and host-pathogen interaction. The role of HEV infection in the development of chronic liver disease is also discussed. Hepatitis E is a cause of mortality and morbidity which negatively impacts the prognosis of patients with chronic liver disease. Recognition of HEV infection must be improved, by increasing awareness and knowledge of the clinical phenotype of the disease.

Fetal Pathology in an Aborted Holstein Fetus Infected With Bovine Parainfluenza Virus-3 Genotype A.

Bovine parainfluenza virus-3 (BPIV-3) is a recognized respiratory pathogen of cattle, and it has also been identified in aborted fetuses. However, little is known of this agent as a reproductive pathogen and detailed descriptions of fetal pathology on natural cases are lacking in the scientific literature. This article describes and illustrates lesions in a fetus spontaneously aborted by a first-calving Holstein heifer, naturally infected with BPIV-3 genotype A, broadening the current knowledge on fetal pathology by this virus. Fetal autopsy revealed diffusely reddened, rubbery and unexpanded lungs. Histologically, there was necrotizing bronchiolitis/alveolitis with intraluminal fibrin exudate and syncytial cells in the bronchiolar/alveolar spaces, and non-suppurative peribronchiolitis and perivascular interstitial pneumonia. In the small intestine there was multifocal necrotizing cryptitis and occasional necrotic syncytial enterocytes. Intralesional and extralesional BPIV-3 antigen was detected by immunohistochemistry in the lung and small intestine, and BPIV-3a was identified in fetal tissues by RT-PCR and sequencing.

Genotypes of human metapneumovirus circulating during 2010-2012 in children from Panama.

Human metapneumovirus (HMPV) is a common causative agent of severe respiratory tract infections in children under 5 years old, the elderly and immunocompromised patients, being responsible for 5-15% of all viral respiratory infections requiring hospitalization. Though HMPV was included in the surveillance program for respiratory viruses in 2010, its genotype distribution remains unknown. Herein, 45 positive samples to HMPV from children ≤5 years old were characterized by phylogenetic analysis based on N gene sequence. Results showed the co-circulation of four sub-lineages: A2a (8.8%), A2b (55.5%), B1 (15.6%), and B2 (20%), demonstrating the genetic heterogeneity of HMPV circulating in Panamá.

Hepatitis E virus in South America: The current scenario.

Hepatitis E virus (HEV) is one of the most frequent causes of acute viral hepatitis of enteric transmission worldwide. In South America the overall epidemiology has been little studied, and the burden of the disease remains largely unknown. A research of all scientific articles about HEV circulation in South America until November 2017 was carried out. Human seroprevalences of HEV varied according to the studied population: blood donors presented prevalence rates ranging from 1.8% to 9.8%, while reports from HIV-infected individuals, transplant recipients and patients on hemodialysis showed higher prevalence rates. Only 2 cases of chronic hepatitis in solid-organ transplant patients from Argentina and Brazil have been described. Detection of HEV in the swine population is widely prevalent in the region. Anti-HEV antibodies have also been recently documented in wild boars from Uruguay. Although scarce, studies focused on environmental and food HEV detection have shown viral presence in these kind of samples, highlighting possible transmission sources of HEV in the continent. HEV genotype 3 was the most frequently detected in the region, with HEV genotype 1 detected only in Venezuela and Uruguay. HEV is widely distributed throughout South America, producing sporadic cases of acute hepatitis, but as a possible agent of chronic hepatitis. Finding the virus in humans, animals, environmental samples and food, show that it can be transmitted through many sources, alerting local governments and health systems to improve diagnosis and for the implementation of preventive measures.

Hepatitis E Virus Genotype 1 and Hepatitis A Virus Dual Infection in Pediatric Patients with a Low Socioeconomic Status from Mexico.

OBJECTIVE: We aimed to detect and characterize hepatitis E virus (HEV) RNA in sera samples from a pediatric population infected with the hepatitis A virus (HAV) exhibiting acute hepatitis and to correlate the infection status with the clinical outcome. METHODS: Seventy-five ELISA-positive samples from children containing anti-HAV and anti-HEV IgM were used to amplify and characterize partial regions within HEV ORF2. A statistical comparison of clinical data between HEV IgM-positive/HEV RNA-positive patients and HEV IgM-positive/HEV RNA-negative patients was performed. RESULTS: Thirteen out of 75 IgM-positive samples provided amplification of discrete regions of the HEV genome. Nested RT-PCR-based detection and subsequent sequencing of 5 samples confirmed the identity of HEV genotype 1 (G1), which had not been previously reported in Mexico. Though not significant, a trend towards exacerbated clinical manifestations was found in HEV RNA-positive patients relative to HEV RNA-negative patients. CONCLUSIONS: An elevated rate of G1 RNA was detected. Hepatitis E seems to be a neglected disease in Mexico and epidemic strains of HEV are likely to play a role as causative agents of acute hepatitis in highly exposed children. Although HAV is endemic in Mexico, an HEV-RNA detection rate of 17% in co-infected samples shows the need for screening for HEV as a part of future vaccination strategies.

A case of incidental infection of Hepatitis E virus (HEV) genotype 1 in a domestic pig.

Hepatitis E virus (HEV) infection involving zoonotic genotypes is a public health problem in high-income and non-endemic developing countries. Herein we report the detection of a human genotype 1 (HEV-1) strain infecting a domestic pig, which is not considered a natural reservoir of this genotype. Viral load was quantified in stool by Real-Time qPCR and sequence analyses were performed. Infectivity of the HEV-1 strain was assesed by in vitro isolation in A549 cell line. Results suggest that certain epidemiological settings might favour accidental spillover infection and thus influence the host range restriction of HEV.

Challenges in research and management of hepatitis E virus infection in Cuba, Mexico, and Uruguay.

The symposium "Epidemiology of Hepatitis E virus (HEV) Infection and Associated Immune Response" was held at the Universidad de Guadalajara, Mexico, on 14 June 2017, to define the status of research on HEV infection in three countries in Latin America and the Caribbean (LAC)-Cuba, Mexico, and Uruguay-compared to the situation in Germany. Scientists identified specific research gaps in understanding HEV transmission and the resulting impact on development of disease in the three abovementioned LAC countries. Specific recommendations for implementing standardized serologic and molecular diagnostic methods and epidemiologic, basic, and applied research aimed to develop prevention and handling strategies for this infection, along with the associated comorbidities in the three LAC countries, were also discussed. Given similar demographic, sanitary, and economic conditions in other LAC countries that could predispose them to be at high risk for HEV transmission and infection, these research gaps and recommendations might apply to the entire LAC region. This report was -prepared by meeting participants based on 1) symposium presentations, 2) literature reviews, and 3) group discussions.

El 14 de junio del 2017 se realizó en la Universidad de Guadalajara (México) un simposio sobre las características epidemiológicas de la infección por el virus de la hepatitis E (VHE) y la respuesta inmunitaria asociada. El objetivo fue definir el estado de las investigaciones sobre la infección por el VHE en tres países de América Latina y el Caribe ­Cuba, México y Uruguay­ en comparación con la situación en Alemania. Los científicos señalaron que para comprender la transmisión del VHE y la consiguiente repercusión en el avance de la infección en estos tres países latinoamericanos aún faltan investigaciones sobre ciertos temas específicos. También analizaron recomendaciones concretas para poner en práctica métodos estandarizados de diagnóstico serológico y molecular, y realizar investigaciones epidemiológicas, básicas y aplicadas a fin de elaborar estrategias de prevención y tratamiento de esta infección y las comorbilidades asociadas en los tres países antes mencionados. Considerando que otros países de América Latina y el Caribe presentan condiciones demográficas, sanitarias y económicas similares que podrían implicar una predisposición a un riesgo alto de transmisión del VHE y de infección por este virus, este análisis sobre las brechas y recomendaciones en el ámbito de la investigación podría aplicarse en toda la subregión. El presente informe fue elaborado por los participantes del simposio sobre la base de: 1) presentaciones del simposio; 2) revisiones bibliográficas; y 3) debates en grupos.

O simpósio Epidemiologia da infecção pelo vírus da hepatite E (HEV) e ­resposta imune associada foi realizado na Universidade de Guadalajara, no México, em 14 de junho de 2017, para determinar a situação da pesquisa em HEV em três países da América Latina e Caribe (ALC) ­ Cuba, México e Uruguai ­ em comparação à Alemanha. Os especialistas identificaram lacunas específicas nas pesquisas no que se refere ao entendimento da transmissão do HEV e ao impacto resultante do surgimento da doença nos três países da ALC mencionados. Também foram debatidas recomendações aos três países da ALC, especificamente implementar métodos sorológicos e moleculares padronizados de diagnóstico e realizar pesquisa epidemiológica, básica e aplicada visando elaborar estratégias de prevenção e de enfrentamento da infecção e das comorbidades associadas. Diante da semelhança das condições demográficas, econômicas e de saúde que poderia predispor outros países da ALC a um maior risco de transmissão e infecção de HEV, as lacunas em pesquisa e recomendações provavelmente se aplicam à toda a Região da ALC. Este relatório foi preparado pelos participantes do encontro embasado nas apresentações do simpósio, revisão da literatura científica e discussões em grupo.

Computational analysis of a species D human adenovirus provides evidence of a novel virus.

A human adenovirus (HAdV) species D, was isolated from a hospitalised child with severe lower respiratory infection. It was initially detected in the nasopharyngeal aspirate of the child followed by conventional PCR amplification of the hexon, penton base, and fibre genes. Sanger DNA sequencing and phylogenetic analyses showed characteristics of a recombinant genome not described before. Next Generation Sequencing analysis was performed to reconstruct its complete DNA genome after viral isolation in adenocarcinoma human cell line (A549). A complete genomic sequence of 35.2 kb in length, with a G+C content of 57 % was obtained, related to HAdV-D29 (96 % identity). Imputed serology analysis demonstrated its novel type with a nucleotide sequence identity of 95.3 % (hexon loop 1) and 96 % (hexon loop 2) to HAdV-D9. The penton base gene showed a novel sequence, distantly related to HAdV-D44. The E3 and E4 regions evolved significantly from their ancestors. The fibre gene was almost identical to the knob region of HAdV-D15 but showed an unrelated shaft sequence. In conclusion the genomics of this novel HAdV, designated the HAdV-D83 [P83H9F15] prototype and bearing a new penton base gene, supports the importance of viral evolution to understand modified tissue tropism, enhanced transmission, or altered virulence.

Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.

Human papillomavirus genotypes distribution in cervical samples from Uruguayan women.

Persistent infection with high-risk human papillomavirus (HPV) causes cervical preneoplasic lesions and invasive cervical cancer. This study evaluated the prevalence and distribution of HPV genotypes in cervical exfoliated cells from Uruguayan women. Five hundred sixty-eight cervical specimens were examined by PCR using MY09/11 primer set, and were genotyped by restriction enzyme digestion (RFLP). Some of the samples which remained undetermined were reanalyzed by PGMY PCR combined with reverse line blot hybridization. Overall, about 42% of samples were positive for HPV; 96% in high-grade squamous intraepithelial lesion, 66% in low-grade squamous intraepithelial lesion, 15% in atypical squamous cells of undetermined significance, and 19% in samples negative for intraepithelial lesion or malignancy. HPV 16 was the most commonly found genotype, followed by HPV 68 and 58. Within low risk-HPV genotypes 6, 61, and 11 were the most frequent. This is the first cross-sectional study, accounting for prevalence and genotype distribution of HPV in Uruguayan women.

A combined approach of rolling-circle amplification-single site restriction endonuclease digestion followed by next generation sequencing to characterize the whole genome and intra-host variants of human Torque teno virus.

Torque Teno Virus (TTV) was initially associated with post-transfusion hepatitis, but growing evidence of its ubiquity in humans is compatible to no apparent clinical significance. TTV is a small non-enveloped virus with a circular single-negative-stranded DNA genome, belonging to the Anelloviridae family. Currently, TTVs are divided in seven phylogenetic groups and are further classified into 21 species. Studies about diversity of TTV in different conditions are receiving increasing interest and in this sense, sequencing of whole genomes for better genetic characterization becomes even more important. Since its discovery in 1997, few TTV complete genomes have been reported worldwide. This is probably due, among other reasons, to the great genetic heterogeneity among TTV strains that prevents its amplification and sequencing by conventional PCR and cloning methods. In addition, although metagenomics approach is useful in these cases, it remains a challenging tool for viromic analysis. With the aim of contributing to the expansion of the TTV whole genomes dataset and to study intra-host variants, we employed a methodology that combined a rolling-circle amplification approach followed by EcoRI digestion, generating a DNA fragment of ∼4Kb consistent with TTV genome length which was sequenced by Illumina next generation sequencing. A genogroup 3 full-length consensus TTV genome was obtained and co-infection with other species (at least those with a single EcoRI cleavage site) was not identified. Additionally, bioinformatics analysis allowed to identify the spectrum of TTV intra-host variants which provides evidence of a complex evolution dynamics of these DNA circular viruses, similarly to what occurs with RNA viruses.

Co-circulation of Hepatitis E Virus (HEV) Genotype 3 and Moose-HEV-Like Strains in Free-Ranging-Spotted Deer (Axis axis) in Uruguay.

Hepatitis E caused by hepatitis E virus (HEV) is considered an emerging foodborne zoonosis in industrialized, non-endemic countries. Domestic pigs and wild boars are considered the main reservoir of HEV. However, HEV can also infect an ever-expanding host range of animals, but they exact role in transmitting the virus to other species or humans is mostly unknown. In this work, we investigated the spread of HEV in free-living and captive spotted deer (Axis axis) from Uruguay in a 2-year period (2020-2022) and examined the role of this invasive species as a new potential reservoir of the virus. In addition, with the aim to gain new insights into viral ecology in the context of One Health, by using camera trapping, we identified and quantified temporal and spatial coexistence of spotted deer, wild boars, and cattle. In free-living animals, we detected an anti-HEV seropositivity of 11.1% (6/54). HEV infection and viral excretion in feces were assessed by RT-PCR. Thirteen of 19 samples (68.4%) had HEV RNA. Six samples were amplified using a broadly reactive RT-PCR and sequenced. No captive animal showed evidence of HEV infection. Additionally, HEV RNA was detected in a freshwater pond shared by these species. Phylogenetic and p-distance analysis revealed that zoonotic HEV genotype 3 strains circulate together with unclassified variants related to moose HEV whose potential risk of transmission to humans and other domestic and wild animals is unknown. The data presented here suggest that spotted deer (A. axis) may be a novel host for zoonotic HEV strains.

Modelling of Hepatitis E virus RNA-dependent RNA polymerase genotype 3 from a chronic patient and in silico interaction analysis by molecular docking with Ribavirin.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease, where chronic hepatitis E associated to solid organ transplant (SOT) recipients, related to genotype 3, is the clinical manifestation of major concern. In this setting, ribavirin (RBV) treatment is the only available therapy, though drug-resistant variants could emerge leading to a therapeutic failure. Crystallographic structures have not been reported for most of the HEV proteins, including the RNA-polymerase (RdRp). Therefore, the mechanism of action of RBV against HEV and the molecular interactions between this drug and RdRp are largely unknown. In this work, we aimed to model in silico the 3 D structure of a novel HEV3 RdRp (HEV_C1_Uy) from a chronically HEV infected-SOT recipient treated with RBV and to perform a molecular docking simulation between RBV triphosphate (RBVT), 7-methyl-guanosine-5'-triphosphate and the modelled protein. The models were generated using I-TASSER server and validated with multiple bioinformatics tools. The docking analysis were carried out with AutoDock Vina and LeDock software. We obtained a suitable model for HEV_C1_Uy (C-Score=-1.33, RMSD = 10.4 ± 4.6 Å). RBVT displayed a binding affinity of -7.6 ± 0.2 Kcal/mol by molecular docking, mediated by 6 hydrogen-bonds (Q195-O14, S198-O11, E257-O13, S260-O2, O3, S311-O11) between the finger's-palm-domains and a free binding energy of 31.26 ± 16.81 kcal/mol by molecular dynamics simulations. We identified the possible HEV RdRp interacting region for incoming nucleotides or analogs and provide novel insights that will contribute to better understand the molecular interactions of RBV and the enzyme and the mechanism of action of this antiviral drug.Communicated by Ramaswamy H. Sarma.

Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

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.