Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

First detection of Hedgehog coronavirus 1 in Poland.

Hedgehogs are common in the majority of European countries and are known to host various pathogens, including viruses. The recent discovery of MERS-related coronaviruses (CoVs) in hedgehogs from Germany, France, the UK, China, and Italy suggests that hedgehogs may represent a wild reservoir of betacoronaviruses. This study reports the first detection and characterization of novel betacoronovirus, subgenus Merbecovirus in wild hedgehogs in Poland. The CoV RNA was detected in 10 out of 40 hedgehogs' rectal swabs and in 1 out of 18 samples of the lung. No viral RNA was identified in the duodenum and kidney. There was no significant relationship between clinical status, gender, hedgehogs' age, and coronaviral RNA detection. Phylogenetic analysis showed that CoVs detected in our study grouped together with other representatives of Hedgehog coronavirus 1 species identified in Western Europe. Our findings provide further evidence that hedgehogs are a natural reservoir of Merbecovirus. Considering the high mutation rate of CoVs and their potential for crossing interspecies barriers, the proper management of hedgehogs admitted to wildlife rehabilitation centres is needed. It cannot be excluded that merbecovirus strains detected in hedgehogs may recombine with other CoVs leading to new viruses with potential for interspecies transmission.

The unresolved question on COVID-19 virus origin: The three cards game?

The ongoing discussion about the real origin of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) feeds acrimonious debates. Where did SARS-CoV-2 come from? Was SARS-CoV-2 transmitted in the wild from an animal to a person before exploding in Wuhan or was it an engineered virus that escaped from research or a laboratory in Wuhan? Right now, we still don't know enough whether SARS-CoV-2 is human-made or not, and lab-leak theories remain essentially speculative. Many recent studies have pointed out several plausible scenarios. Anyhow, currently, even if suspicions by some about the possibility of lab-leak hypothesis still remain, the consensus view is that the pandemic probably started from a natural source and, to determine the real origin of the SARS-CoV-2 virus, further research is needed.

Utilizing the VirIdAl Pipeline to Search for Viruses in the Metagenomic Data of Bat Samples.

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.

Metagenomic identification of a new sarbecovirus from horseshoe bats in Europe.

The source of the COVID-19 pandemic is unknown, but the natural host of the progenitor sarbecovirus is thought to be Asian horseshoe (rhinolophid) bats. We identified and sequenced a novel sarbecovirus (RhGB01) from a British horseshoe bat, at the western extreme of the rhinolophid range. Our results extend both the geographic and species ranges of sarbecoviruses and suggest their presence throughout the horseshoe bat distribution. Within the spike protein receptor binding domain, but excluding the receptor binding motif, RhGB01 has a 77% (SARS-CoV-2) and 81% (SARS-CoV) amino acid homology. While apparently lacking hACE2 binding ability, and hence unlikely to be zoonotic without mutation, RhGB01 presents opportunity for SARS-CoV-2 and other sarbecovirus homologous recombination. Our findings highlight that the natural distribution of sarbecoviruses and opportunities for recombination through intermediate host co-infection are underestimated. Preventing transmission of SARS-CoV-2 to bats is critical with the current global mass vaccination campaign against this virus.

Etiology and clinical characteristics of SARS-CoV-2 and other human coronaviruses among children in Zhejiang Province, China 2017-2019.

BACKGROUND: A novel coronavirus (SARS-CoV-2) emerging has put global public health institutes on high alert. Little is known about the epidemiology and clinical characteristics of human coronaviruses infections in relation to infections with other respiratory viruses. METHODS: From February 2017 to December 2019, 3660 respiratory samples submitted to Zhejiang Children Hospital with acute respiratory symptoms were tested for four human coronaviruses RNA by a novel two-tube multiplex reverse transcription polymerase chain reaction assays. Samples were also screened for the occurrence of SARS-CoV-2 by reverse transcription-PCR analysis. RESULTS: Coronavirus RNAs were detected in 144 (3.93%) specimens: HCoV-HKU1 in 38 specimens, HCoV-NL63 in 62 specimens, HCoV-OC43 in 38 specimens and HCoV-229E in 8 specimens. Genomes for SARS-CoV-2 were absent in all specimens by RT-PCR analysis during the study period. The majority of HCoV infections occurred during fall months. No significant differences in gender, sample type, year were seen across species. 37.5 to 52.6% of coronaviruses detected were in specimens testing positive for other respiratory viruses. Phylogenic analysis identified that Zhejiang coronaviruses belong to multiple lineages of the coronaviruses circulating in other countries and areas. CONCLUSION: Common HCoVs may have annual peaks of circulation in fall months in the Zhejiang province, China. Genetic relatedness to the coronaviruses in other regions suggests further surveillance on human coronaviruses in clinical samples are clearly needed to understand their patterns of activity and role in the emergence of novel coronaviruses.

Mito: "tendríamos que preguntarnos si la COVID-19 realmente existe"

erie: Respuesta de la ciencia a los mitos más escuchados sobre vacunación y #COVID19​ Dr. Juan Arbiza. Investigador Grado 5 de Primer nivel del Programa de Posgrado en Ciencias Básicas (PEDECIBA) –UdelaR. Integra el Grupo Asesor Científico Honorario (GACH) de la Presidencia de la República. Profesor Titular de Virología con Dedicación exclusiva. Facultas de Ciencias. Investigador Nivel III del Sistema Nacional de Investigadores de Uruguay.

Mito: "la COVID-19​ es una creación de laboratorio para enriquecer a los que fabrican vacunas"

Serie: Respuesta de la ciencia a los mitos más escuchados sobre vacunación y #COVID19​ Dr. Juan Arbiza. Investigador Grado 5 de Primer nivel del Programa de Posgrado en Ciencias Básicas (PEDECIBA) –UdelaR. Integra el Grupo Asesor Científico Honorario (GACH) de la Presidencia de la República. Profesor Titular de Virología con Dedicación exclusiva. Facultas de Ciencias. Investigador Nivel III del Sistema Nacional de Investigadores de Uruguay.

Science in 5: pruebas de COVID-19

¿Qué tipo de pruebas de COVID-19 están disponibles? ¿Qué son los antígenos y los anticuerpos? ¿Qué pasa con la precisión de estas pruebas? El Dr. Hanan Balkhy de la OMS explica las pruebas de COVID-19 en este nuevo episodio.

Covid-19: monitoramento do vírus em esgotos pode colaborar na prevenção

O projeto de vigilância do novo coronavírus em esgotos de Niterói, na Região Metropolitana do Rio de Janeiro, teve resultados publicados no periódico Water Research, uma prestigiada revista científica internacional.

Animal Reservoirs and Hosts for Emerging Alphacoronaviruses and Betacoronaviruses.

The ongoing global pandemic caused by coronavirus disease has once again demonstrated the role of the family Coronaviridae in causing human disease outbreaks. Because severe acute respiratory syndrome coronavirus 2 was first detected in December 2019, information on its tropism, host range, and clinical manifestations in animals is limited. Given the limited information, data from other coronaviruses might be useful for informing scientific inquiry, risk assessment, and decision-making. We reviewed endemic and emerging infections of alphacoronaviruses and betacoronaviruses in wildlife, livestock, and companion animals and provide information on the receptor use, known hosts, and clinical signs associated with each host for 15 coronaviruses detected in humans and animals. This information can be used to guide implementation of a One Health approach that involves human health, animal health, environmental, and other relevant partners in developing strategies for preparedness, response, and control to current and future coronavirus disease threats.

Detection and differentiation of respiratory syncytial virus subgroups A and B with colorimetric toehold switch sensors in a paper-based cell-free system.

Respiratory syncytial virus (RSV) infection is the most common clinical infectious disease threatening the safety of human life. Herein, we provided a sensitive and specific method for detection and differentiation of RSV subgroups A (RSVA) and B (RSVB) with colorimetric toehold switch sensors in a paper-based cell-free system. In this method, we applied the toehold switch, an RNA-based riboswitch, to regulate the translation level of ß-galactosidase (lacZ) gene. In the presence of target trigger RNA, the toehold switch sensor was activated and the expressed LacZ hydrolyzed chromogenic substrates to produce a colorimetric result that can be observed directly with the naked eye in a cell-free system. In addition, nucleic acid sequence-based amplification (NASBA) was used to improve the sensitivity by amplifying target trigger RNAs. Under optimal conditions, our method produced a visible result for the detection of RSVA and RSVB with the detection limit of 52 aM and 91 aM, respectively. The cross-reaction of this method was validated with other closely related respiratory viruses, including human coronavirus HKU1 (HCoV-HKU1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Furthermore, we used the paper-based carrier material that allows stable storage of our detection elements and rapid detection outside laboratory. In conclusion, this method can sensitively and specifically differentiate RSVA and RSVB and generate a visible colorimetric result without specialized operators and sophisticated equipment. Based on these advantages above, this method serves as a simple and portable detector in resource-poor areas and point-of-care testing (POCT) scenarios.

Encefalopatías y encefalitis durante la infección aguda por SARS-CoV2. Registro de la Sociedad Española de Neurología SEN COVID-19 / Encephalopathy and encephalitis during acute SARS-CoV-2 infection. Spanish Society of Neurology COVID-19 Registry

OBJETIVOS: Desde el inicio de la pandemia por el virus SARS-CoV2 la Sociedad Española de Neurología (SEN) creó un registro de afectación neurológica para informar al neurólogo clínico. Las encefalopatías y encefalitis fueron una de las complicaciones más descritas. Analizamos las características de las mismas. PACIENTES Y MÉTODOS: Estudio descriptivo retrospectivo, observacional multicéntrico, de pacientes con sintomatología compatible con encefalitis o encefalopatía, introducidos en el Registro SEN COVID-19 desde el 17 de marzo hasta el 6 de junio de 2020. RESULTADOS: Se han registrado 232 casos con síntomas neurológicos, 51 casos de encefalopatía/encefalitis (21,9%). Ningún paciente era trabajador sanitario. Los síndromes más frecuentes fueron: cuadro confusional leve-moderado (33%) y encefalopatía grave o coma (9,8%). El tiempo medio entre el inicio de la infección y la clínica neurológica fue de 8,02 días. Punción lumbar en el 60,8% de pacientes; solo hubo un caso con PCR positiva. Resonancia craneal en el 47% de los pacientes (alterada en el 7,8% de ellos). Se realizó electroencefalograma en el 41,3% de los casos (alterado en el 61,9% de los mismos). CONCLUSIONES: Las encefalopatías y encefalitis son dos de las complicaciones más frecuentes descritas en el SEN COVID-19. Más de un tercio de los pacientes presentó un cuadro de síndrome confusional leve o moderado. El tiempo medio de aparición de la sintomatología neurológica desde el inicio de la infección fue de 8 días (hasta 24 h antes en mujeres que en hombres). El electroencefalograma fue la prueba más sensible en estos pacientes, encontrando muy pocos casos con alteraciones en las pruebas de neuroimagen. Todos los pacientes que recibieron tratamiento con bolos de corticoides o inmunoglobulinas tuvieron una evolución favorable

OBJECTIVES: Since the beginning of the COVID-19 pandemic, the Spanish Society of Neurology has run a registry of patients with neurological involvement for the purpose of informing clinical neurologists. Encephalopathy and encephalitis were among the most frequently reported complications. In this study, we analyse the characteristics of these complications. PATIENTS AND METHODS: We conducted a retrospective, descriptive, observational, multicentre study of patients with symptoms compatible with encephalitis or encephalopathy, entered in the Spanish Society of Neurology's COVID-19 Registry from 17 March to 6 June 2020. RESULTS: A total of 232 patients with neurological symptoms were registered, including 51 cases of encephalopathy or encephalitis (21.9%). None of these patients were healthcare professionals. The most frequent syndromes were mild or moderate confusion (33%) and severe encephalopathy or coma (9.8%). The mean time between onset of infection and onset of neurological symptoms was 8.02 days. Lumbar puncture was performed in 60.8% of patients, with positive PCR results for SARS-CoV-2 in only one case. Brain MRI studies were performed in 47% of patients, with alterations detected in 7.8% of these. EEG studies were performed in 41.3% of cases, detecting alterations in 61.9%. CONCLUSIONS: Encephalopathy and encephalitis are among the complications most frequently reported in the registry. More than one-third of patients presented mild or moderate confusional syndrome. The mean time from onset of infection to onset of neurological symptoms was 8 days (up to 24 hours earlier in women than in men). EEG was the most sensitive test in these patients, with very few cases presenting alterations in neuroimaging studies. All patients treated with boluses of corticosteroids or immunoglobulins progressed favourably

Orientaciones para la selección de muestras de SARS-CoV-2 para caracterización y vigilancia genómica / Guidance for the selection of SARS-CoV-2 samples for genomic characterization and surveillance

La secuenciación genómica ha sido una herramienta esencial para generar datos virológicos, impulsar la respuesta del laboratorio y comprender mejor los patrones evolutivos y de dispersión del SARS-CoV-2. Además de la caracterización de los patrones de circulación global, la detección temprana de las variantes del SARS-CoV-2 dentro de cada país es fundamental para complementar la vigilancia epidemiológica y virológica.