The Brazilian Cerrado fauna shows very wide diversity and can be a potential viral reservoir. Therefore, the animal's susceptibility to some virus can serve as early warning signs of potential human ...virus diseases. Moreover, the wild animal virome of this biome is unknown. Based on this scenario, high-throughput sequencing contributes a robust tool for the identification of known and unknown virus species in this environment. In the present study, faeces samples from cerrado birds (
,
, and
) and mammals (
,
, and
) were collected at the Veterinary Hospital, University of Brasília. Viral nucleic acid was extracted, submitted to random amplification, and sequenced by Illumina HiSeq platform. The reads were de novo assembled, and the identities of the contigs were evaluated by Blastn and tblastx searches. Most viral contigs analyzed were closely related to bacteriophages. Novel archaeal viruses of the
family were detected. Moreover, sequences of members of
,
,
,
, and
families were identified. Complete and nearly complete genomes of known anelloviruses, circoviruses, and parvoviruses were obtained, as well as putative novel species. We demonstrate that the metagenomics approach applied in this work was effective for identification of known and putative new viruses in faeces samples from Brazilian Cerrado fauna.
Pseudomonas aeruginosa is an opportunistic pathogen and one of the leading causes of nosocomial infections. Moreover, the species can cause severe infections in cystic fibrosis patients, in burnt ...victims and cause disease in domestic animals. The control of these infections is often difficult due to its vast repertoire of mechanisms for antibiotic resistance. Phage therapy investigation with P. aeruginosa bacteriophages has aimed mainly the control of human diseases. In the present work, we have isolated and characterized a new bacteriophage, named Pseudomonas phage BrSP1, and investigated its host range against 36 P. aeruginosa strains isolated from diseased animals and against P. aeruginosa ATCC strain 27853.
We have isolated a Pseudomonas aeruginosa phage from sewage. We named this virus Pseudomonas phage BrSP1. Our electron microscopy analysis showed that phage BrSP1 had a long tail structure found in members of the order Caudovirales. "In vitro" biological assays demonstrated that phage BrSP1 was capable of maintaining the P. aeruginosa population at low levels for up to 12 h post-infection. However, bacterial growth resumed afterward and reached levels similar to non-treated samples at 24 h post-infection. Host range analysis showed that 51.4% of the bacterial strains investigated were susceptible to phage BrSP1 and efficiency of plating (EOP) investigation indicated that EOP values in the strains tested varied from 0.02 to 1.72. Analysis of the phage genome revealed that it was a double-stranded DNA virus with 66,189 bp, highly similar to the genomes of members of the genus Pbunavirus, a group of viruses also known as PB1-like viruses.
The results of our "in vitro" bioassays and of our host range analysis suggested that Pseudomonas phage BrSP1 could be included in a phage cocktail to treat veterinary infections. Our EOP investigation confirmed that EOP values differ considerably among different bacterial strains. Comparisons of complete genome sequences indicated that phage BrSP1 is a novel species of the genus Pbunavirus. The complete genome of phage BrSP1 provides additional data that may help the broader understanding of pbunaviruses genome evolution.
Viruses of four families of arthropod-specific, large dsDNA viruses (the nuclear arthropod large DNA viruses, or NALDVs) possess homologs of genes encoding conserved components involved in the ...baculovirus primary infection mechanism. The presence of such homologs encoding
per os
infectivity factors (
pif
genes), along with their absence from other viruses and the occurrence of other shared characteristics, suggests a common origin for the viruses of these families. Therefore, the class
Naldaviricetes
was recently established, accommodating these four families. In addition, within this class, the ICTV approved the creation of the order
Lefavirales
for three of these families, whose members carry homologs of the baculovirus genes that code for components of the viral RNA polymerase, which is responsible for late gene expression. We further established a system for the binomial naming of all virus species in the order
Lefavirales
, in accordance with a decision by the ICTV in 2019 to move towards a standardized nomenclature for all virus species. The binomial species names for members of the order
Lefavirales
consist of the name of the genus to which the species belongs (e.g.,
Alphabaculovirus
), followed by a single epithet that refers to the host species from which the virus was originally isolated. The common names of viruses and the abbreviations thereof will not change, as the format of virus names lies outside the remit of the ICTV.
The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form ...enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.
Dengue fever is caused by four distinct serotypes of the dengue virus (DENV1-4), and is estimated to affect over 500 million people every year. Presently, there are no vaccines or antiviral ...treatments for this disease. Among the possible targets to fight dengue fever is the viral NS3 protease (NS3PRO), which is in part responsible for viral processing and replication. It is now widely recognized that virtual screening campaigns should consider the flexibility of target protein by using multiple active conformational states. The flexibility of the DENV NS3PRO could explain the relatively low success of previous virtual screening studies. In this first work, we explore the DENV NS3PRO conformational states obtained from molecular dynamics (MD) simulations to take into account protease flexibility during the virtual screening/docking process. To do so, we built a full NS3PRO model by multiple template homology modeling. The model comprised the NS2B cofactor (essential to the NS3PRO activation), a glycine flexible link and the proteolytic domain. MD simulations had the purpose to sample, as closely as possible, the ligand binding site conformational landscape prior to inhibitor binding. The obtained conformational MD sample was clustered into four families that, together with principal component analysis of the trajectory, demonstrated protein flexibility. These results allowed the description of multiple binding modes for the Bz-Nle-Lys-Arg-Arg-H inhibitor, as verified by binding plots and pair interaction analysis. This study allowed us to tackle protein flexibility in our virtual screening campaign against the dengue virus NS3 protease.
The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we ...describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris−HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.
The genomes of two putative new RNA viruses (macula-like virus and bunya-like virus) were identified in total RNA extracted from dead eucalyptus snout beetles (
Gonipterus
spp.) from a laboratory ...colony. However, only bunya-like virus was detected in field-collected insects. The macula-like virus has a monopartite single-stranded RNA genome that contains three open reading frames (ORFs) encoding an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), protein with unknown function. The bunya-like virus genome was predicted to consist of two RNA segments: a large segment (L) encoding a single protein (RdRp) and a small segment (S) encoding a putative nucleocapsid protein.
Background: The correct understanding of the epidemiological dynamics of COVID-19, caused by the SARS-CoV-2, is essential for formulating public policies of disease containment. Methods: In this ...study, we constructed a picture of the epidemiological dynamics of COVID-19 in a Brazilian population of almost 17000 patients in 15 months. We specifically studied the fluctuations of COVID-19 cases and deaths due to COVID-19 over time according to host gender, age, viral load, and genetic variants. Results: As the main results, we observed that the numbers of COVID-19 cases and deaths due to COVID-19 fluctuated over time and that men were the most affected by deaths, as well as those of 60 or more years old. We also observed that individuals between 30- and 44-years old were the most affected by COVID-19 cases. In addition, the viral loads in the patients’ nasopharynx were higher in the early symptomatic period. We found that early pandemic SARS-CoV-2 lineages were replaced by the variant of concern (VOC) P.1 (Gamma) in the second half of the study period, which led to a significant increase in the number of deaths. Conclusions: The results presented in this study are helpful for future formulations of efficient public policies of COVID-19 containment.
Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial ...passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work,
Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in
C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the
gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.