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.

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.

A multiplex-NGS approach to identifying respiratory RNA viruses during the COVID-19 pandemic.

A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts.

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.

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.

Resultados del tratamiento quirúrgico del cáncer de recto y ano en el Hospital Español / Results of the surgical treatment of rectal and anal cancer in the Hospital Español / Resultados do tratamento cirúrgico do câncer de reto e ânus no Hospital Español

La cirugía del cáncer de recto y ano se ha desarrollado considerablemente en las últimas décadas. En función de dichos avances, se ha observado una disminución en la morbimortalidad operatoria, así como también una mejoría en el pronóstico de estos pacientes. El objetivo del presente estudio es exponer y analizar los resultados del tratamiento quirúrgico del cáncer de recto y ano en un servicio universitario. Se realizó un estudio observacional, descriptivo y retrospectivo de todos los pacientes intervenidos por cáncer de recto y ano en el Hospital Español entre 2016 y 2020. Las variables registradas fueron: variables demográficas, clínico-oncológicas, relacionadas a la morbimortalidad operatoria y a la recidiva locorregional, y la sobrevida a 5 años. El procedimiento más realizado fue la resección anterior de recto (RAR) en 11 intervenciones (58%), mientras que las 8 restantes correspondieron a amputaciones abdominoperineales (AAP) (42%). Se diagnosticaron un total de 6 complicaciones intraoperatorias en 5 pacientes, siendo la perforación del tumor la más frecuente, y un total de 18 complicaciones postoperatorias en 11 pacientes, siendo la más frecuente la infección de la herida quirúrgica abdominal. La morbilidad operatoria mayor fue de 31,6% y la mortalidad operatoria a 90 días fue de 0%. La sobrevida global a 5 años fue de 63,2%. Los resultados quirúrgicos en la presente casuística fueron comparables con los de la bibliografía consultada. Destacamos la nula mortalidad a 90 días, con resultados oncológicos similares a los reportados en la literatura.

Rectal and anus surgery have been developed considerably in the last decades. Based on these advancements, it has been observed a decrease in the surgical morbidity and mortality, as well as an improved prognosis of these patients. The aim of the present study is to expose and analyze the results of the anus and rectal surgical treatment in a university service. An observational, descriptive and retrospective study was performed of all the intervened patients for rectum and anus cancer in the Hospital Español between 2016 and 2020. We recorded data about demographic, clinical-oncologic, related to the surgical morbidity and mortality, locoregional relapse and overall 5 year survival. The most performed procedure was the rectum anterior resection in 11 interventions (58%), while the 8 left corresponded to abdominoperineal resection (42%). There was a total of 6 intraoperative complications diagnosed in 5 patients, being the tumor perforation the most frequent one, and a total of 18 postoperative complications diagnosed in 11 patients, being the surgical wound infection the most frequent one. The serious surgical morbidity was 31,6%, while the surgical mortality rate at 90 days was 0%. Overall 5 year survival was 63,2%. The surgical results in the present study about the rectum and anal cancer were comparable with the results reported on the consulted bibliography. We highlight the null mortality within 90 days, with oncologic results similar to the ones reported in the literature.

A cirurgia do câncer retal e anal desenvolveu-se consideravelmente nas últimas décadas. Com base nesses avanços, observou-se diminuição da morbimortalidade operatória, bem como melhora no prognóstico desses pacientes. O objetivo deste estudo é apresentar e analisar os resultados do tratamento cirúrgico do câncer de reto e anal em um serviço universitário. Foi realizado um estudo observacional, descritivo e retrospectivo de todos os pacientes operados por câncer de reto e ânus no Hospital Espanhol entre 2016 e 2020. As variáveis ​​registradas foram: variáveis ​​demográficas, clínico-oncológicas, relacionadas à morbidade e mortalidade operatórias e recorrência locorregional. , e sobrevida em 5 anos. O procedimento mais realizado foi a ressecção anterior do reto (RAR) em 11 intervenções (58%) e as 8 restantes corresponderam a amputações abdominoperineais (AAP) (42%). Foram diagnosticadas 6 complicações intraoperatórias em 5 pacientes, sendo a perfuração tumoral a mais frequente, e um total de 18 complicações pós-operatórias em 11 pacientes, sendo a infecção da ferida operatória abdominal a mais frequente. A morbidade operatória maior foi de 31,6% e a mortalidade operatória em 90 dias foi de 0%. A sobrevida global em 5 anos foi de 63,2%. Os resultados cirúrgicos da presente casuística foram comparáveis ​​aos da bibliografia consultada. Destacamos a mortalidade nula em 90 dias, com resultados oncológicos semelhantes aos relatados na literatura.

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.

An update on genetic analysis of porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) in South America: identification of ORF5 sequences of lineage 1A, 1C and 1G.

Data regarding PRRSV-2 in South America are scant and a coordinated criterion for molecular characterization is needed. A phylogenetic analysis was performed using a dataset of 76 ORF5 sequences from South America, and results showed the identification of lineage 5 in the early 2000s and the predominance of lineage 1 at least since 2013. Lineage 1 sequences were further classified into sub-lineages according to a recent molecular characterization study of PRRSV-2 in United States. Our results revealed the recent identification in Uruguay of PRRSV-2 ORF5 sequences of lineage 1 sub-lineage C. Two additional sub-lineages were identified in South America, 1G in Chile and 1A in Peru. Continuous updating the molecular epidemiology of circulating viruses with coordinated investigations among countries is required to control and prevent the emergence of genetic variants of PRRSV-2.

Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes.

Deletions frequently occur in the six accessory genes of SARS-CoV-2, but most genomes with deletions are sporadic and have limited spreading capability. Here, we analyze deletions in the ORF7a of the N.7 lineage, a unique Uruguayan clade from the Brazilian B.1.1.33 lineage. Thirteen samples collected during the early SARS-CoV-2 wave in Uruguay had deletions in the ORF7a. Complete genomes were obtained by Illumina next-generation sequencing, and deletions were confirmed by Sanger sequencing and capillary electrophoresis. The N.7 lineage includes several individuals with a 12-nucleotide deletion that removes four amino acids of the ORF7a. Notably, four individuals underwent an additional 68-nucleotide novel deletion that locates 44 nucleotides downstream in the terminal region of the same ORF7a. The simultaneous occurrence of the 12 and 68-nucleotide deletions fuses the ORF7a and ORF7b, two contiguous accessory genes that encode transmembrane proteins with immune-modulation activity. The fused ORF retains the signal peptide and the complete Ig-like fold of the 7a protein and the transmembrane domain of the 7b protein, suggesting that the fused protein plays similar functions to original proteins in a single format. Our findings evidence the remarkable dynamics of SARS-CoV-2 and the possibility that single and consecutive deletions occur in accessory genes and promote changes in the genomic organization that help the virus explore genetic variations and select for new, higher fit changes.

Transmission cluster of COVID-19 cases from Uruguay: emergence and spreading of a novel SARS-CoV-2 ORF6 deletion.

BACKGROUND: Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES: We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS: Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS: We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS: Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

Complete Genome Sequence of Hepatitis E Virus Genotype 3 Obtained from a Chronically Infected Individual in Uruguay.

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide. We report the full-length genome sequence of an HEV-3 strain obtained from a chronically infected patient from Uruguay. This strain shared only 86% nucleotide sequence identity with the most closely related reference strain belonging to subtype 3m.

Genome Sequences of SARS-CoV-2 P.1 (Variant of Concern) and P.2 (Variant of Interest) Identified in Uruguay.

Two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants associated with increased transmission and immune evasion, P.1 and P.2, emerged in Brazil and spread throughout South America. Here, we report genomes corresponding to these variants that were recently detected in Uruguay. These P.1 and P.2 genomes share all substitutions that are characteristic of these variants.

A deletion in SARS-CoV-2 ORF7 identified in COVID-19 outbreak in Uruguay.

The analysis of genetic diversity in SARS-CoV-2 is the focus of several studies, providing insights into how the virus emerged and evolves. Most common changes in SARS-CoV-2 are single or point nucleotide substitutions; meanwhile, insertions and deletions (indels) have been identified as a less frequent source of viral genetic variability. Here, we report the emergence of a 12-nucleotide deletion in ORF7a, resulting in a 4-amino acid in-frame deletion. The Δ12 variant was identified in viruses from patients of a single outbreak and represents the first report of this deletion in South American isolates. Phylogenetic analysis revealed that Δ12 strains belong to the lineage B.1.1 and clustered separated from the remaining Uruguayan strains. The ∆12 variant was detected in 14 patients of this outbreak by NGS sequencing and/or two rapid and economic methodologies: Sanger amplicon sequencing and capillary electrophoresis. The presence of strong molecular markers as the deletion described here are useful for tracking outbreaks and reveal a significant aspect of the SARS-CoV-2 evolution on the robustness of the virus to keep its functionality regardless loss of genetic material.

Hepatitis E Virus (HEV) infection in captive white-collared peccaries (Pecari tajacu) from Uruguay.

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Clinical presentation of hepatitis E mainly occurs as an acute and self-limited disease, though chronic cases are now being commonly reported in immunocompromised individuals. In high-income developed areas and non-endemic regions, HEV is mainly transmitted by the zoonotic route through direct contact with infected animals or by consumption of contaminated meat products. Although pigs and wild boars are the main reservoirs of the disease, HEV can also infect deer, camels, and rats and seems to have an ever-expanding host range. Peccaries (Tayassuidae family, superfamily Suoidea), the 'new world pigs', share susceptibility to several pathogens with domestic pigs and wild boars. Herein, we performed a serological and molecular survey of two captive populations of white-collared peccaries (Pecari tajacu) from Uruguay, with the aim to assess the role of the species as an HEV reservoir. One-hundred and one serum samples were analysed for anti-HEV antibodies. Further evidences of active HEV infection were investigated in stool by RT-nested PCR. Animals from both wildlife reserves were exposed to HEV with an overall prevalence of 24.7%. Moreover, HEV RNA could be detected in peccaries' stool samples from one of the reserves. Phylogenetic analysis clustered the strains within HEV-3, closely related to both human and swine isolates. Our work provides the first evidences supporting the notion that white-collared peccaries are susceptible to HEV. However, these data should not be overinterpreted. Further research is needed concerning the role of peccaries in the transmission of HEV.

Transmission cluster of COVID-19 cases from Uruguay: emergence and spreading of a novel SARS-CoV-2 ORF6 deletion

BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

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.

Aislamiento de una cepa genotipo 3 del virus de la hepatitis E obtenida de heces humanas / Cell culture isolation of hepatitis e virus genotype 3 strain obtained from human feces

ABSTRACT Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/µL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/µL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

RESUMEN El virus de la hepatitis E (HEV) se considera como una de las principales causas de hepatitis viral aguda en el mundo; cada año ocurren aproximadamente 20 millones de infecciones y 57 000 muertes. Debido a la ausencia de un modelo de cultivo celular consenso, se sabe poco sobre el ciclo replicativo del virus. La línea celular A549 se considera susceptible al genotipo 3 de HEV, pero tanto la cepa viral como las condiciones del cultivo celular podrían afectar el aislamiento viral in vitro. Por tanto nos propusimos aislar in vitro una cepa genotipo 3 del HEV. Para ello, se inocularon células A549 con una cepa HEV-3 identificada previamente por caracterización genética, y se incubó durante dos horas a 37 °C. Cinco días después de la infección, las células se pasaron (subcultivaron) por primera vez, y se realizaron pases seriados cada cuatro días en promedio, durante 41 días. En cada pase se evalúo la replicación del HEV mediante RT-qPCR. La reinfección de la línea celular con progenie viral derivada de monocapas de A549 infectadas se evaluó mediante inmunofluorescencia y RT-qPCR. Se detectó ARN viral en cada pase a partir de monocapas, y el pico máximo se alcanzó a los 26 días post infección (2 x 106 copias/µL). En el ensayo de reinfección, se detectó antígeno de cápside perinuclearmente y formando focos, y se detectaron 1 x 104 copias/µL de RNA viral a las 96 horas post infección. El HEV recuperado de lisado de monocapas fue infeccioso. Este aislado viral ofrece una herramienta importante para estudiar aspectos desconocidos de la infección por HEV.

Locally Acquired Chronic Hepatitis E Followed by Epstein-Barr Virus Reactivation and Burkitt Lymphoma as a Suspected Extrahepatic Manifestation in a Liver Transplant Recipient.

BACKGROUND Hepatitis E virus (HEV) is a common cause of acute hepatitis in developing regions. In high-income countries, hepatitis E is an emergent zoonotic disease of increasing concern. Clinically, the infection is usually acute and self-limited in immunocompetent individuals, although rare chronic cases in immunocompromised patients have been reported. Both acute and chronic infections have been recently associated with several extrahepatic manifestations, including neurological and hematological disorders. CASE REPORT A case of autochthonous chronic HEV infection in a liver-transplanted man from a non-endemic country is presented. Phylogenetic analysis revealed a swine origin of the HEV human infection. Chronic hepatitis E was treated with a 9-week course of ribavirin, after which viral clearance was achieved. Subsequently, the patient developed a post-transplant lymphoproliferative disorder (PTLD) in the form of Burkitt lymphoma. At the time of lymphoma diagnosis, the patient had shown a strong reactivation of Epstein-Barr virus (EBV) infection. After additional antiviral ganciclovir therapy and chemotherapy, the patient had a complete recovery with no sequelae. CONCLUSIONS The differential diagnosis of persistently elevated transaminases in transplanted and/or immunocompromised patients should include testing for HEV by appropriate nucleic acid techniques (NATs). Cases of HEV infection with an atypical clinical outcome, such as the one presented herein, highlights the need for increased awareness of chronic hepatitis E and its association with a wide range of extrahepatic manifestations.

Evaluation of HBV-Like Circulation in Wild and Farm Animals from Brazil and Uruguay.

The origin of the hepatitis B virus is a subject of wide deliberation among researchers. As a result, increasing academic interest has focused on the spread of the virus in different animal species. However, the sources of viral infection for many of these animals are unknown since transmission may occur from animal to animal, human to human, animal to human, and human to animal. The aim of this study was to evaluate hepadnavirus circulation in wild and farm animals (including animals raised under wild or free conditions) from different sites in Brazil and Uruguay using serological and molecular tools. A total of 487 domestic wild and farm animals were screened for hepatitis B virus (HBV) serological markers and tested via quantitative and qualitative polymerase chain reaction (PCR) to detect viral DNA. We report evidence of HBsAg (surface antigen of HBV) and total anti-HBc (HBV core antigen) markers as well as low-copy hepadnavirus DNA among domestic and wild animals. According to our results, which were confirmed by partial genome sequencing, as the proximity between humans and animals increases, the potential for pathogen dispersal also increases. A wider knowledge and understanding of reverse zoonoses should be sought for an effective One Health response.

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.