Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells

BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Ampullae infection of the malpighian tubules of Bombyx mori by Alphabaculovirus / Infecção de células da ampola dos túbulos de malpighi de Bombyx mori pelo Alphabaculovirus

Alphabaculovirus is a genus of the entomopathogenic virus, whose species Bombyx mori nucleopolyhedrovirus (BmNPV) infects the silkworm, Bombyx mori, which is an important insect in the sericulture industry. A geographic isolate of BmNPV was identified in the state of Paraná, Brazil. It was infecting B. mori larvae and various organs and target tissues were identified, however, there was no information about the infection of Malpighian tubules (MT). The MT comprises the excretory system of B. mori and acts in the elimination of toxic substances and in hydroelectrolytic homeostasis. Thus, the present study examined the susceptibility and cytopathology of B. mori MT to BmNPV. To this end, hybrid fifth instar larvae were inoculated with a virus suspension at different days post-inoculation (dpi). MT segments were collected and divided into the ampullae, proximal, medial and distal regions. These were processed for light microscopy and transmission electron analysis. The MT regions revealed differences in susceptibility to BmNPV and the ampullae in its transition area was infected from the sixth dpi; the other regions did not reveal any evidence of infection. The transition area of the ampullae has not been previously described in Lepidoptera and its cytopathology revealed a hypertrophic nucleus with viroplasm, followed by the formation and development of viral polyhedra, which are common characteristics of infections by Alphabaculovirus. Thus, infection of the ampullae of the MT of B. mori by BmNPV, together with other known targets, compromises the metabolic balance of the insect, which results in consequences for silk production and damage to the sericulture sector.

Alphabaculovirus é um gênero de vírus entomopatogênico, cuja espécie Bombyx mori nucleopolyhedrovirus (BmNPV) infecta o bicho-da-seda, Bombyx mori, inseto importante na indústria sericícola. Um isolado geográfico do BmNPV foi identificado no estado do Paraná, Brasil, infectando lagartas de B. mori e vários órgãos e tecidos alvos foram identificados; entretanto, não há informações sobre a infecção do túbulo de Malpighi (TM). O TM compõe o sistema excretor de B. mori, atuando na eliminação de substâncias tóxicas e na homeostase hidroeletrolítica. Assim, o presente estudo, analisou a susceptibilidade e a citopatologia do TM de B. mori ao BmNPV. Para tanto, lagartas híbridas de 5° instar foram inoculadas com uma suspensão viral e em diferentes dias pós-inoculação (dpi), segmentos do TM foram coletados e subdivididos nas regiões da ampola, proximal, média e distal; sendo processados para análises em microscopias de luz e eletrônica de transmissão. As regiões do TM revelaram diferenças na susceptibilidade ao BmNPV e a ampola, na sua área de transição, foi infectada a partir do 6° dpi, já as demais regiões não revelaram quaisquer indícios de infecção. A área de transição da ampola, ainda não havia sido descrita em lepidópteros e sua citopatologia revelou núcleo hipertrófico com viroplasma, seguido da formação e desenvolvimento dos poliedros virais, características comuns das infecções pelo Alphabaculovirus. Assim, a infecção da ampola do TM de B. mori ao BmNPV, somada a de outros alvos conhecidos, compromete o equilíbrio metabólico do inseto, com consequências na produção de seda e prejuízos ao setor sericícola.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR / Detecção rápida do Coronavírus Bovino (BCoV) por meio de uma semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

O Coronavírus bovino (BCoV) pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal ...