Virome analysis of Desmodus rotundus tissue samples from the Amazon region.

BACKGROUND: Bats are renowned for harboring a high viral diversity, their characteristics contribute to emerging infectious diseases. However, environmental and anthropic factors also play a significant role in the emergence of zoonotic viruses. Metagenomic is an important tool for investigating the virome of bats and discovering new viruses. RESULTS: Twenty-four families of virus were detected in lung samples by sequencing and bioinfomatic analysis, the largest amount of reads was focused on the Retroviridae and contigs assembled to Desmodus rotundus endogenous retrovirus, which was feasible to acquire complete sequences. The reads were also abundant for phages. CONCLUSION: This lung virome of D. rotundus contributes valuable information regarding the viral diversity found in bats, which is useful for understanding the drivers of viral cycles and their ecology in this species. The identification and taxonomic categorization of viruses hosted by bats carry epidemiological significance due to the potential for viral adaptation to other animals and humans, which can have severe repercussions for public health. Furthermore, the characterization of endogenized viruses helps to understanding the host genome and the evolution of the species.

Correlation of cGAS, STING, INF-α and INF-ß gene expression with Zika virus kinetics in primary culture of microglia and neurons from BALB/c mice.

Pattern recognition receptors participate in the innate immune response. Among PRRs, the cGAS/STING pathway is known to detect cytosolic DNA and cyclic dinucleotides, but it's also important in RNA virus infection. We aimed to evaluate the gene expression of some important genes of cGAS/STING pathway and to correlate this expression with Zika virus kinetics in mice microglia and neurons. Cells were infected by MOI = 1.0. Indirect immunofluorescence, plaque titration of supernatant, extraction, and quantification of total intracellular RNA, RT-qPCR and Western blotting were performed. Plaque titration profile in microglia and neurons was similar, including higher titers of plaque forming units at 24, 48, 72 and 96 hpi, respectively. ZIKV kinetics evaluated by RT-qPCR was similar in both cells, with highest viral titers at 48, 72, 24 and 96 hpi, respectively. Expression profile of cGAS, STING, INF-α and INF-ß was quite different between the cells, including gene suppression, as observed for cGAS in neurons. Our results showed a differentiated expression profile of cGAS/STING pathway genes in mice microglia and neurons, which can be explained by the different mechanisms that ZIKV uses to bypass the immune response of these cells. Furthermore, each cell type responds differently to combat the viral infection.

Detection of dengue virus serotype 3 by reverse transcription - polymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas

The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1 percent) were positive for DENV3, providing a minimal infection rate of 2.1 percent of all analyzed infected female specimens of three different areas of the city.

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Para, Brazil.

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Para showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Pará, Brazil

Em muitos países, o Enterovírus 71 (EV-71) família Picornaviridae é associado a doença de pé-mão e boca e doenças neurológicas agudas enquanto que no Brasil esse vírus está mais associado às últimas. O objetivo desta pesquisa foi utilizar em estudos moleculares e soroepidemiológicos, o primeiro isolamento de EV-71 obtido na região norte do Brasil. No período de janeiro de 1998 a dezembro de 2000 foram coletadas 88 amostras (44 casos de PFA) de fezes das quais, duas (2,2%) foram positivas para EV-71 (73442/PA/99). A seqüência de nucleotídeos do gen que codifica a proteína VP1 mostrou que o isolado 73442/PA/99 foi similar às cepas de EV-71 pertencentes ao grupo B- mais próxima das norte americanas. Teste de neutralização com 389 amostras de soro colhidas no período de janeiro de 1998 a novembro de 2001, de indivíduos com idade de 0 a 15 anos residentes na cidade de Belém, Estado do Pará mostrou os seguintes resultados em relação ao isolado 73442/PA/99 e ao protótipo BrCr: 207 indivíduos (53,2%) tinham anticorpos neutralizantes para ambos os vírus, 167 (42,9%) não tinham anticorpos e 15 tinham anticorpos para um dos dois vírus. Somente 20,2% das crianças com idade de 0 a 3 anos tinham anticorpos neutralizantes para EV-71, indicando que essas crianças estavam mais suscetíveis à infecção. Tanto o estudo de soroprevalência quanto o de sequenciamento da VP1 foram importantes para demonstrar a propagação e o padrão molecular do EV-71 circulante na região norte do Brasil.

Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas.

The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1%) were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.

Multiplex nested PCR for Brazilian Alphavirus diagnosis.

A reverse-transcriptase PCR (RT-PCR) and a multiplex nested PCR were developed for the rapid detection and identification of 14 Brazilian alphaviruses. Using Alphavirus genus-specific primers in a RT-PCR, we obtained amplified products of 434 bp. Species-specific primers were selected and simultaneously tested in a multiplex nested PCR. The nested PCR increased the test sensitivity 1000-fold and was capable of identifying Brazilian Alphavirus showing the expected bands with diagnostic sizes for Venezuelan (400 bp), Eastern (124 bp), and Western (208 bp) equine encephalitis, Aura (86 bp), and Mayaro (270 bp) viruses. This strategy for diagnosis is fast, sensitive, specific and it can be used as a reliable alternative for routine Brazilian Alphavirus diagnosis.

Diagnosis of Oropouche virus infection by RT-nested-PCR.

Using the RT-PCR with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the S segment of Simbu serogroup viruses in a nested PCR it was possible to amplify the Oropouche virus (ORO) genome from the sera of three patients. These results show that this RT-nested-PCR is a useful tool for rapid diagnosis of Oropouche fever infections.