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.

Coronavirus disease 2019 (COVID-19) surveillance, in Brazil, initiated shortly after its description, in China. Our aim was to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional pathogens in samples from the initial phase of the outbreak in Brazil, from late February to late March. From 707 samples analysed, 29 (4.1%) were SARS-CoV-2 positive. Fever and cough were their most prevalent symptoms. Co-detection of rhinovirus was observed in 2 (6.9%) cases. Additional pathogens were identified in 66.1% of the SARS-CoV-2 negative cases, mainly rhinovirus and influenza A(H1N1)pdm09. Thus, we emphasise the importance of differential diagnosis in COVID-19 suspected cases.

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.

As infecções causadas por patógenos intracelulares como os vírus, algumas bactérias e muitos parasitas terminam, em muitos casos, após ativação de respostas imunes de células T específicas, as quais reconhecem os antígenos estranhos e eliminam as células infectadas. As vacinas contra esses microorganismos infecciosos foram tradicionalmente desenvolvidas através da administração dos micróbios completos vivos ou atenuados. Hoje em dia, a pesquisa em vacinas de subunidade contendo os antígenos mais imunogênicos desses patógenos é a principal escolha para as doenças contra as quais não existem vacinas. Porém, quando as vacinas de subunidade são administradas seguindo os protocolos tradicionais de imunização, os níveis de imunidade celular induzidos são comumente baixos e incapazes de gerar proteção completa contra as doenças causadas pelos microorganismos intracelulares. Nesta revisão apresentamos uma alternativa promissora aos protocolos tradicionais, que é o uso como ferramentas de vacinação de vetores virais recombinantes que codificam vacinas de subunidade. Os vírus recombinantes têm várias características interessantes que os tornam extremamente eficientes na indução de respostas imunes mediadas por linfócitos T. Durante os últimos anos, observou-se que esta imunidade celular tem importância fundamental para a proteção contra duas doenças que são objetivos primordiais da Organização Mundial da Saúde: a malária e a AIDS. Por esses motivos esta revisão focaliza especialmente o desenvolvimento de novos protocolos de vacinação contra estas duas doenças.

Las infecciones por patógenos intracelulares como virus, algunas bacterias y numerosos parásitos son neutralizadas en la mayoría de los casos tras la activación de respuestas inmunitarias específicas mediadas por linfocitos T, que reconocen los antígenos extraños y eliminan las células infectadas. Las vacunas contra estos organismos infecciosos se han basado tradicionalmente en la administración de organismos enteros atenuados o inactivados. Actualmente la investigación se centra en el desarrollo de vacunas compuestas por subunidades que contengan los antígenos más inmunogénicos para cada patógeno particular. No obstante, si se administran vacunas de subunidades purificadas mediante los protocolos tradicionales de inmunización, los niveles inducidos de inmunidad celular son en su mayoría bajos e incapaces de generar una protección completa contra las enfermedades causadas por los microorganismos intracelulares. En esta revisión presentamos una prometedora alternativa a estos métodos tradicionales mediante el uso de virus recombinantes cuyo genoma codifica las subunidades de vacuna. Los virus recombinantes reúnen varias características que los hacen muy eficientes para inducir las respuestas inmunitarias mediadas por los linfocitos T. Recientemente se ha demostrado que la inmunidad celular es crucial en la protección contra la malaria y el SIDA, que son un objetivo prioritario de la Organización Mundial de la Salud en cuanto al desarrollo de vacunas. Por lo tanto, esta revisión se centra especialmente en el desarrollo de nuevos protocolos de vacunación contra estas dos enfermedades.