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The genus Orthopoxvirus (OPXV) of the family Poxviridae comprises several viruses that are capable of infecting a wide range of hosts. One of the most widespread OPXVs is the Vaccinia virus (VACV), which circulates in zoonotic cycles in South America, especially in Brazil, infecting domestic and wild animals and humans and causing economic losses as well as impacting public health. Despite this, little is known about the presence and/or exposure of neotropical primates to orthopoxviruses in the country. In this study, we report the results of a search for evidence of OPVX infections in neotropical free-living primates in the state of Minas Gerais, southeast Brazil. The sera or liver tissues of 63 neotropical primates were examined through plaque reduction neutralization tests (PRNT) and real-time PCR. OPXV-specific neutralizing antibodies were detected in two sera (4.5%) from Callithrix penicillata , showing 55% and 85% reduction in plaque counts, evidencing their previous exposure to the virus. Both individuals were collected in urban areas. All real-time PCR assays were negative. This is the first time that evidence of OPXV exposure has been detected in C. penicillata , a species that usually lives at the interface between cities and forests, increasing risks of zoonotic transmissions through spillover/spillback events. In this way, studies on the circulation of OPXV in neotropical free-living primates are necessary, especially now, with the monkeypox virus being detected in new regions of the planet.

Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

Medically important arboviruses such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that the transmission of some viruses can be influenced by mosquito-specific and mosquito-borne viruses. Advancements in high-throughput sequencing (HTS) and bioinformatics have expanded our knowledge on the richness of viruses harbored by mosquitoes. HTS was used to characterize the presence of virus sequences in wild-caught adult Ae. aegypti from Tocantins (TO) state, Brazil. Samples of mosquitoes were collected in four cities of Tocantins state and submitted to RNA isolation, followed by sequencing at an Illumina HiSeq platform. Our results showed initially by Krona the presence of 3% of the sequenced reads belonging to the viral database. After further analysis, the virus sequences were found to have homology to two viral families found in insects Phenuiviridae and Metaviridae. Three possible viral strains including putative new viruses were detected and named Phasi Charoen-like phasivirus isolate To-1 (PCLV To-1), Aedes aegypti To virus 1 (AAToV1), and Aedes aegypti To virus 2 (AAToV2). The results presented in this work contribute to the growing knowledge about the diversity of viruses in mosquitoes and might be useful for future studies on the interaction between insect-specific viruses and arboviruses., (© 2021. Sociedade Brasileira de Microbiologia.)

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 × 10 6 GC/g, 2.03 × 10 6 GC/mL, and 1.36 × 10 5 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., Competing Interests: None., (© 2021 The Author(s).)

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., (© 2021. EcoHealth Alliance.)

We described the complete genome sequence of a novel baculovirus isolate of species Buzura suppressaria nucleopolyhedrovirus, called by isolate CNPSo-25. The occlusion bodies were found to be polyhedral in shape and to contain virions with singly embedded nucleocapsids. The size of the genome is 121,377 bp with a G+C content of 36.7%. We annotated 131 ORFs that cover 90.42% of the genome. Moreover, phylogenetic inference indicated that CNPSo-25 is a member of genus Alphabaculovirus that clustered together with two other Chinese isolates of the same species. We called the virus by Biston suppressaria nucleopolyhedrovirus isolate CNPSo-25 (BisuNPV-CNPSo-25), as Buzura was placed inside the lepidopteran genus Biston. As expected, we detected intra-population variability in the virus sample when the novel isolate was compared to the Chinese isolates: 292 single nucleotide variants were found in the genome, with 181 affecting the protein product. The closest representatives of other species to BisuNPV-CNPSo-25 was found to be Sucra jujuba nucleopolyhedrovirus and Hyposidra talaca nucleopolyhedrovirus, two other virus isolates of geometrid caterpillars. The study of baculovirus genomes is of importance for the development of tools for insect pest biological control and biotechnology.

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients' sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in cattle in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1, and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus-based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic p35 gene was constructed using the Bac-to-Bac™ system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the recombinant US3 protein in insect cells comparing with two other AcMNPV recombinants, one containing a functional copy of the AcMNPV anti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Brazil is one of the largest silk producers in the world. The domesticated silkworm (Bombyx mori) was formally introduced into the country in the twentieth century, and the state of Paraná is the main national producer. During larval stages, B. mori can be afflicted by many different infectious diseases, which lead to substantial losses in silk production. In this work, we describe the structure and complete genome sequence of the first non-Asian isolate of Bombyx mori nucleopolyhedrovirus (BmNPV), the most important silkworm pathogen. The BmNPV-Brazilian isolate is a nucleopolyhedrovirus with singly enveloped nucleocapsids within polyhedral occlusion bodies. Its genome has 126,861 bp with a G + C content of 40.4 %. Phylogenetic analysis clustered the virus with the Japanese strain (BmNPV-T3). As expected, we have detected intra-population variability in the virus sample. Variation along homologous regions (HRs) and bro genes was observed; there were seven HRs, deletion of bro-e, and division of bro-a into two ORFs. The study of baculoviruses allows for a better understanding of virus evolution providing insight for biological control of insect pests or protection against the pernicious disease caused by these viruses.