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.

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.

Acute and chronic bovine pulmonary edema and emphysema in Uruguay / Edema e enfisema pulmonar agudo e crônico em bovinos do Uruguai

An outbreak of pulmonary edema and emphysema with acute and chronic cases is reported in a farm in Uruguay. In a herd of 40 Hereford steers, 20 died. The deaths began four days after a change of paddock, from an old pasture of Avena sativa to a lush growing pasture of the same grass. Acutely affected animals showed severe dyspnea, sialorrhea, cough, and subcutaneous edema, and died within 72 hours. Chronically affected steers showed dyspnea, respiratory noises, weight loss, and intolerance to exercise. The deaths began four days after the change of paddock. Ten days after the first death, the steers were withdrawn from the pasture, but continued dying throughout the following 40 days. Twenty animals died and six were necropsied. Grossly, the lungs were diffusely armed and glistening, with reddish and crepitant cut surface, and presented alveolar septae sharply distended by edema and emphysema. There was subpleural emphysema with air blebs distributed across the pleural surface. Presence of Dictyocaulus viviparus was observed in three steers. In some animals, the trachea was diffusely reddish with presence of pink foam; in some others, there was bloody liquid in the tracheal lumen. Histologic examination showed severe diffuse alveolar and interstitial emphysema, hyaline membranes adhered to the alveolar wall, thickening of the interlobular septae with proliferation of type II pneumocytes, and moderate-to-severe multifocal histiocytic, neutrophilic and eosinophilic infiltrate. In the trachea, there was submucosal hemorrhage and moderate multifocal eosinophilic and lymphocytic infiltrate. The steers with chronic signs presented similar lung lesions, but multifocal pulmonary fibrosis and cardiac dilatation were also observed. The diagnosis of acute bovine pulmonary emphysema and edema (ABPE) was based on the occurrence of the disease after introduction of the herd in a lush green pasture, on the characteristic gross and histologic lesions, and on the absence of other toxic or infectious agents causing similar lesions. Cattle raisers should be alert to the risks of occurrence of this disease after the introduction of the herds into paddocks with green and lush pastures.(AU)

Descreve-se um surto de edema e enfisema pulmonar com casos agudos e crônicos em bovinos em uma criação semi-intensiva no Uruguai. De um lote de 40 novilhos da raça Hereford morreram 20. As mortes começaram quatro dias após uma mudança de alimentação, de uma pastagem mais velha de Avena sativa, para uma pastagem recentemente plantada de aveia que estava em brotação. Os animais afetados apresentaram sinais clínicos agudos de dispneia, sialorreia, tosse e alguns desenvolveram edema subcutâneo, morrendo em até 72 horas. Outros novilhos mais cronicamente afetados apresentaram dispneia, ruídos respiratórios, perda de peso e intolerância ao exercício. As mortes começaram quatro dias após a mudança de pastagens. Dez dias após a primeira morte, os novilhos foram retirados do pasto, mas morreram ainda durante 40 dias mais. Ao total, morreram vinte animais e seis foram necropsiados. Nas necropsias dos animais mortos na fase aguda os pulmões estavam difusamente armados e brilhosos e ao corte de coloração avermelhada e crepitante, com os septos alveolares acentuadamente distendidos por edema e enfisema. Havia enfisema subpleural caracterizado por bolhas de ar distribuídas pela superfície pleural. Em três bovinos havia ainda presença de Dictyocaulus viviparus. Alguns animais apresentaram a traqueia difusamente avermelhada com espuma de coloração rósea ou liquido sanguinolento livre na luz traqueal. Histologicamente havia edema e enfisema alveolar e intersticial difuso severo, membranas hialinas espessas aderidas à parede alveolar, espessamento dos septos interlobulares com proliferação de pneumócitos tipo II e infiltrado inflamatório histiocítico, neutrofílico e eosinofílico multifocal moderado a severo. Na traqueia havia hemorragias na submucosa e infiltrado eosinofílico e linfocítico multifocal. Os novilhos com sinais crônicos apresentaram lesões pulmonares semelhantes, entretanto, foram observadas também, fibrose pulmonar multifocal e dilatação cardíaca. O diagnóstico de EEPAB baseou-se na ocorrência da doença após a introdução do rebanho em uma pastagem viçosa em brotação, nas características macroscópicas e histológicas e na ausência de outros agentes tóxicos ou infecciosos que causam lesões semelhantes. Se alerta para os riscos da ocorrência desta enfermidade, quando houver mudanças de pastagens.(AU)

Challenges in research and management of hepatitis E virus infection in Cuba, Mexico, and Uruguay / Retos en la investigación y el tratamiento de la infección por el virus de la hepatitis E en Cuba, México y Uruguay / Desafios em pesquisa e tratamento da infecção pelo vírus da hepatite E em Cuba, ­México e ­Uruguai

ABSTRACT 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.

RESUMEN 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.

RESUMO 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.

Primer caso de falla hepática aguda por virus de la Hepatitis E en Uruguay / First case of Hepatitis E Virus- induced acute liver failure in Uruguay

Resumen: La infección por el Virus de la Hepatitis E (VHE) en individuos inmunocompetentes generalmente se presenta como hepatitis aguda autolimitada. En determinados escenarios clínicos (embarazadas y pacientes con enfermedad hepática crónica) puede manifestarse como falla hepática aguda. Se han descripto casos de hepatitis crónica en inmunocomprometidos. En Uruguay se han reportado 14 casos de hepatitis aguda autolimitada por VHE. En el presente artículo se describe el primer caso de falla hepática aguda por VHE en Uruguay.

Abstract: Hepatitis E Virus (HEV) typically causes an acute and self-limiting infection in immune-competent individuals, though acute liver failure is described in some settings (pregnancy, chronic liver disease). Chronic hepatitis has been described in immunosuppressed patients. Fourteen autochthonous cases of self-limiting acute hepatitis for HEV were reported in Uruguay. The first case of acute liver failure for HEV is described in the present article.

Serologic evidence of human metapneumovirus circulation in Uruguay

First identified in 2001, the human metapneumovirus (hMPV), is a respiratory tract pathogen that affects young children, elderly, and immunocompromised patients. The present work represents the first serologic study carried out in Uruguay. It was performed with the purpose of obtaining serological evidence of hMPV circulation in Uruguay and to contribute to the few serologic reports described until now. Sixty nine serum samples collected between 1998 and 2001 by vein puncture from patients without respiratory symptoms or underlying pathology aged 6 days to 60 years were examined using an indirect immunofluorescence assay (IFA). The global seropositivity rate of the samples was 80 percent (55/69). Rates of 60 percent (15/25) and 91 percent (40/44) were observed for the pediatric and adult cohorts, respectively. Results obtained from a longitudinal analysis of 6 children aged 6 days to 18 months are discussed. These results are a clear evidence of hMPV circulation in Uruguay, at least since 1998, and reinforce the previous data on worldwide circulation of this virus.