Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants.

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40-60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients' sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

First description of a multisystemic and lethal SARS-CoV-2 variant of concern P.1 (Gamma) infection in a FeLV-positive cat.

BACKGROUND: Phylogenetic studies indicate bats as original hosts of SARS-CoV-2. However, it remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. METHODS: In this study, we analyzed the first fatal case of a SARS-CoV-2 and FeLV co-infection in an eight-year-old male cat. We carried out a clinical evaluation and several laboratory analyses. RESULTS: As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to the animal's death. RT-qPCR analysis showed a SARS-CoV-2 as the causative agent. The virus was detected in several organs, indicating a multisystemic infection. The virus was found in a high load in the trachea, suggesting that the animal may have contribute to the transmission of the virus. The whole-genome sequencing revealed an infection by SARS-CoV-2 Gamma VOC (P.1), and any mutations indicating host adaptation were observed. CONCLUSION: Our data show that FeLV-positive cats are susceptible to SARS-CoV-2 infection and raise questions about the potential of immunocompromised FeLV-positive cats to act as a reservoir for SARS-CoV-2 new variants.

Genomic Epidemiology of SARS-CoV-2 in Tocantins State and the Diffusion of P.1.7 and AY.99.2 Lineages in Brazil.

Tocantins is a state in the cross-section between the Central-West, North and Northeast regions of Brazilian territory; it is a gathering point for travelers and transportation from the whole country. In this study, 9493 genome sequences, including 241 local SARS-CoV-2 samples (collected from 21 December 2020, to 16 December 2021, and sequenced in the MinION platform) were analyzed with the following aims: (i) identify the relative prevalence of SARS-CoV-2 lineages in the state of Tocantins; (ii) analyze them phylogenetically against global SARS-CoV-2 sequences; and (iii) hypothesize the viral dispersal routes of the two most abundant lineages found in our study using phylogenetic and phylogeographic approaches. The performed analysis demonstrated that the majority of the strains sequenced during the period belong to the Gamma P.1.7 (32.4%) lineage, followed by Delta AY.99.2 (27.8%), with the first detection of VOC Omicron. As expected, there was mainly a dispersion of P.1.7 from the state of São Paulo to Tocantins, with evidence of secondary spreads from Tocantins to Goiás, Mato Grosso, Amapá, and Pará. Rio de Janeiro was found to be the source of AY.99.2 and from then, multiple cluster transmission was observed across Brazilian states, especially São Paulo, Paraiba, Federal District, and Tocantins. These data show the importance of trade routes as pathways for the transportation of the virus from Southeast to Northern Brazil.

Genomic Epidemiology of SARS-CoV-2 in Tocantins State and the Diffusion of P.1.7 and AY.99.2 Lineages in Brazil

Tocantins is a state in the cross-section between the Central-West, North and Northeast regions of Brazilian territory;it is a gathering point for travelers and transportation from the whole country. In this study, 9493 genome sequences, including 241 local SARS-CoV-2 samples (collected from 21 December 2020, to 16 December 2021, and sequenced in the MinION platform) were analyzed with the following aims: (i) identify the relative prevalence of SARS-CoV-2 lineages in the state of Tocantins;(ii) analyze them phylogenetically against global SARS-CoV-2 sequences;and (iii) hypothesize the viral dispersal routes of the two most abundant lineages found in our study using phylogenetic and phylogeographic approaches. The performed analysis demonstrated that the majority of the strains sequenced during the period belong to the Gamma P.1.7 (32.4%) lineage, followed by Delta AY.99.2 (27.8%), with the first detection of VOC Omicron. As expected, there was mainly a dispersion of P.1.7 from the state of São Paulo to Tocantins, with evidence of secondary spreads from Tocantins to Goiás, Mato Grosso, Amapá, and Pará. Rio de Janeiro was found to be the source of AY.99.2 and from then, multiple cluster transmission was observed across Brazilian states, especially São Paulo, Paraiba, Federal District, and Tocantins. These data show the importance of trade routes as pathways for the transportation of the virus from Southeast to Northern Brazil.

SARS-CoV-2 loads in urine, sera and stool specimens in association with clinical features of COVID-19 patients.

Background: COVID-19 pandemic continues to be a priority in public health worldwide, and factors inherent to SARS-CoV-2 pathogenesis and genomic characteristics are under study. Investigations that evaluate possible risk factors for infection, clinical manifestations, and viral shedding in different specimens also need to clarify possible associations with COVID-19 prognosis and disease outcomes. Study design: In this study, we evaluated SARS-CoV-2 positivity and estimated viral loads by real-time RT-PCR in stool, sera, and urine samples from 35 patients, with a positive SARS-CoV-2 RNA molecular test in respiratory sample, attended at a University COVID-19 referral hospital in Goiania, Goias, Brazil. Whole-genome sequencing was also performed in samples with higher viral load. Results: The positivity index was 51.43%, 14.28%, and 5.71% in stool, sera, and urine specimens, respectively. The median viral load was 8.01 × 106 GC/g, 2.03 × 106 GC/mL, and 1.36 × 105 GC/mL in stool, sera, and urine, respectivelly. Of all patients, 88.57% had previous comorbidities, and 48.39% of them had detectable SARS-CoV-2 RNA in at least one type of clinical specimen evaluated by this study (stool, sera or urine). A higher viral load was observed in patients with more than two previous comorbidities and that were classified as severe or critical conditions. Samples with the highest viral loads were sequenced and characterized as B.1.1.33 variant. Conclusion: We conclude that SARS-CoV-2 RNA is present in more than one type of clinical specimen during the infection, and that the most critical patients had detectable viral RNA in more than one clinical specimen at the same time point.

No Evidence of SARS-CoV-2 Infection in Neotropical Primates Sampled During COVID-19 Pandemic in Minas Gerais and Rio Grande do Sul, Brazil.

In 2019, a new coronavirus disease (COVID-19) was detected in China. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was capable to infect domestic and captive mammals like cats, tigers and minks. Due to genetic similarities, concern about the infection of non-human primates (NHPs) and the establishment of a sylvatic cycle has grown in the Americas. In this study, neotropical primates (NP) were sampled in different areas from Brazil to investigate whether they were infected by SARS-CoV-2. A total of 89 samples from 51 NP of four species were examined. No positive samples were detected via RT-qPCR, regardless of the NHP species, tissue or habitat tested. This work provides the first report on the lack of evidence of the circulation of SARS-CoV-2 in NP. The expansion of wild animals sampling is necessary to understand their role in the epidemiology of SARS-CoV-2 and other potentially zoonotic pathogens in natural environments shared by humans.