Yellow Fever Virus Maintained by Sabethes Mosquitoes during the Dry Season in Cerrado, a Semiarid Region of Brazil, in 2021.

In recent decades, waves of yellow fever virus (YFV) from the Amazon Rainforest have spread and caused outbreaks in other regions of Brazil, including the Cerrado, a savannah-like biome through which YFV usually moves before arriving at the Atlantic Forest. To identify the vectors involved in the maintenance of the virus in semiarid environments, an entomological survey was conducted after confirmation of yellow fever (YF) epizootics at the peak of the dry season in the Cerrado areas of the state of Minas Gerais. In total, 917 mosquitoes from 13 taxa were collected and tested for the presence of YFV. Interestingly, mosquitoes of the Sabethes genus represented 95% of the diurnal captured specimens, displaying a peak of biting activity never previously recorded, between 4:30 and 5:30 p.m. Molecular analysis identified three YFV-positive pools, two from Sabethes chloropterus-from which near-complete genomes were generated-and one from Sa. albiprivus, whose low viral load prevented sequencing. Sa. chloropterus was considered the primary vector due to the high number of copies of YFV RNA and the high relative abundance detected. Its bionomic characteristics allow its survival in dry places and dry time periods. For the first time in Brazil, Sa. albiprivus was found to be naturally infected with YFV and may have played a role as a secondary vector. Despite its high relative abundance, fewer copies of viral RNA were found, as well as a lower Minimum Infection Rate (MIR). Genomic and phylogeographic analysis showed that the virus clustered in the sub-lineage YFVPA-MG, which circulated in Pará in 2017 and then spread into other regions of the country. The results reported here contribute to the understanding of the epidemiology and mechanisms of YFV dispersion and maintenance, especially in adverse weather conditions. The intense viral circulation, even outside the seasonal period, increases the importance of surveillance and YFV vaccination to protect human populations in affected areas.

Potential Action Mechanism and Inhibition Efficacy of Morinda citrifolia Essential Oil and Octanoic Acid against Stagonosporopsis cucurbitacearum Infestations.

The use of plant-based products has been shown to efficiently inhibit fungi-mediated diseases in agricultural crops. Here, we extracted and evaluated the composition of noni, Morinda citrifolia L., essential oil and assessed its activities against Stagonosporopsis cucurbitacearum in Cucumis melo L. Using in silico molecular approaches, potential interactions between the essential oil major components and S. cucurbitacearum tyrosine-tRNA ligase were predicted. Finally, we also measured the potential interference of plant physiology (the stomatal conductance and net photosynthesis) mediated by the application of the M. citrifolia essential oil. Chromatographic analysis revealed that octanoic acid (75.8%), hexanoic acid (12.8%), and isobutyl pent-4-enyl carbonate (3.1%) were the major essential oil compounds. Octanoic acid and noni essential oil, when used as preventive measures, reduce fungal mycelial growth at a concentration of 5 mg/mL without causing significant damage to the treated leaves, which reinforces their efficacies as preventive tools against S. cucurbitacearum. Molecular docking analyses predicted very stable interactions between the major essential oil constituents and S. cucurbitacearum tyrosine-tRNA ligase, suggesting the interference of these plant-based molecules upon enzyme activation. Octanoic acid and M. citrifolia essential oil at concentrations of 20 mg/mL decreased the stomatal conductance and net photosynthesis rate of melon plants, resulting in robust phytotoxicity. Collectively, our findings indicated that despite the phytotoxicity risks at higher concentrations, M. citrifolia essential oil and octanoic acid, have potential as alternative tools for the integrative management of S. cucurbitacearum.

Complete Genome Sequences of Two Bovine Alphaherpesvirus 5 Subtype C Strains from Southeast Brazil.

Bovine alphaherpesvirus 5 causes meningoencephalitis in cattle, belongs to the Herpesviridae family, and can be divided into subtypes a, b, and c. Limited information is available about subtype c. Here, we report the complete genome sequences of two strains, P160/96, and ISO97/45, isolated from cattle in southeast Brazil.

Identification of potential new mosquito-associated viruses of adult Aedes aegypti mosquitoes from Tocantins state, Brazil.

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.

Real-Time Genomic Surveillance during the 2021 Re-Emergence of the Yellow Fever Virus in Rio Grande do Sul State, Brazil.

The 2021 re-emergence of yellow fever in non-human primates in the state of Rio Grande do Sul (RS), southernmost Brazil, resulted in the death of many howler monkeys (genus Alouatta) and led the state to declare a Public Health Emergency of State Importance, despite no human cases reported. In this study, near-complete genomes of yellow fever virus (YFV) recovered from the outbreak were sequenced and examined aiming at a better understanding of the phylogenetic relationships and the spatio-temporal dynamics of the virus distribution. Our results suggest that the most likely sequence of events involved the reintroduction of YFV from the state of São Paulo to RS through the states of Paraná and Santa Catarina, by the end of 2020. These findings reinforce the role of genomic surveillance in determining the pathways of distribution of the virus and in providing references for the implementation of preventive measures for populations in high risk areas.

Comparative genomic analysis and mosquito larvicidal activity of four Bacillus thuringiensis serovar israelensis strains.

Bacillus thuringiensis serovar israelensis (Bti) is used to control insect vectors of human and animal diseases. In the present study, the toxicity of four strains of Bti, named T0124, T0131, T0137, and T0139, toward Aedes aegypti and Culex quinquefasciatus larvae was analyzed. The T0131 strain showed the highest larvicidal activity against A. aegypti (LC50 = 0.015 µg/ml) and C. quinquefasciatus larvae (LC50 = 0.035 µg/ml) when compared to the other strains. Furthermore, the genomic sequences of the four strains were obtained and compared. These Bti strains had chromosomes sizes of approximately 5.4 Mb with GC contents of ~35% and 5472-5477 putative coding regions. Three small plasmids (5.4, 6.8, and 7.6 kb) and three large plasmids (127, 235, and 359 kb) were found in the extrachromosomal content of all four strains. The SNP-based phylogeny revealed close relationship among isolates from this study and other Bti isolates, and SNPs analysis of the plasmids 127 kb did not reveal any mutations in δ-endotoxins genes. This newly acquired sequence data for these Bti strains may be useful in the search for novel insecticidal toxins to improve existing ones or develop new strategies for the biological control of important insect vectors of human and animal diseases.

Evaluation of the anti-apoptotic activity of bovine alphaherpesvirus type 5 US3 protein kinase in insect cells using a recombinant baculovirus.

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.

Complete Sequences of Two Plasmids Found in a Brazilian Bacillus thuringiensis Serovar israelensis Strain.

Plasmids play a crucial role in the evolution of bacterial genomes by mediating horizontal gene transfer. In this work, we sequenced two plasmids found in a Brazilian Bacillus thuringiensis serovar israelensis strain which showed 100% nucleotide identities with Bacillus thuringiensis serovar kurstaki plasmids.

First detection of porcine circovirus type 3 in Colombia and the complete genome sequence demonstrates the circulation of PCV3a1 and PCV3a2.

Porcine circovirus 3 (PCV3) was recently discovered and is a new species of the genus circovirus. Clinically, it is associated with absence of symptoms or with different clinical syndromes. It has been reported in different countries of America, Europe and Asia. Last year, in Colombia, some farms have reported symptoms similar to those caused by PCV2. Samples were taken from two farms located in the centre of the country, and the presence of PCV3 was determined by PCR in two samples, one from a pool of sera and another from mesenteric lymph node. The strains were fully sequenced (GenBank accession numbers MH327784 and MH327785) and classified into subgroups a1 and a2. According to this classification and its analysis, strain a2 is located within the group called "Linker" that may be evolving towards group "b". In addition to the above, the two Colombian strains were compared with 104 strains reported in the GenBank database. The phylogenetic tree obtained grouped according to the classification of subgroups a1, a2, b1 and b2. It was found that subgroups a1 and a2 were well grouped when comparing whole genomes, but the same was not observed with the strains of group "b". In the latter, no subgroups were evidenced when comparing complete genomes. It is suggested that a new classification of PCV3 subgroups should be proposed, based on whole genome sequences. This is the first report of PCV3 in Colombia and its complete genome sequence.

Genome Sequence of a New Siphoviridae Phage Found in a Brazilian Bacillus thuringiensis Serovar israelensis Strain.

During the fermentation process, Bacillus thuringiensis (Bt) phages can result in bacterial death and decreased yield. In this work, we describe the genome of a new phage related to the Siphoviridae viral family from a Brazilian strain of Bt which showed high nucleotide sequence identity to the genomes of phages phi4l1 and BtCS33.

Genome Characterization of the First Mimiviruses of Lineage C Isolated in Brazil.

The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of ∼1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.

Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.

Genome Sequence of KP-Rio/2015, a Novel Klebsiella pneumoniae (Podoviridae) Phage.

Klebsiella pneumoniae is a pathogen frequently associated with antibiotic-resistant nosocomial infections. Here, we describe the genome of KP-Rio/2015, a novel phage of K. pneumoniae belonging to the family Podoviridae.

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5.

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.

Soroprevalência de herpesvírus bovinos tipos 1 e/ou 5 no Estado do Rio Grande do Sul / Seroprevalence of bovine herpesvirus types 1 and/or 5 in the state of Rio Grande do Sul

Este estudo objetivou estimar a prevalência de anticorpos contra os herpesvírus bovinos tipos 1 e 5 (BoHV-1 e BoHV-5) no Estado do Rio Grande do Sul (RS), Brasil, frente a diferentes cepas de BoHV-1 e BoHV-5. As amostras de soro utilizadas foram extraídas de uma amostragem mais ampla, desenhada para estimar a prevalência de brucelose bovina no Estado. Todos os soros foram coletados de vacas com idade igual ou superior a 24 meses de idade, não vacinadas contra herpesvírus bovinos, de rebanhos de corte e leite. O cálculo amostral foi baseado em uma expectativa de prevalência média de infecção de 33 por cento, considerando-se um erro padrão não superior a 1 por cento e um intervalo de confiança de 95 por cento. Com base nesse cálculo foram examinados 2.200 soros, provenientes de 390 propriedades e 158 municípios. Os soros foram analisados na busca de anticorpos contra BoHV-1 e BoHV-5 pela técnica de soroneutralização (SN), executada frente a quatro cepas de vírus distintas: EVI123/98 e Los Angeles (BoHV-1.1); EVI88/95 (BoHV-5a) e A663 (BoHV-5b). A prevalência média de anticorpos contra o BoHV-1 e BoHV-5 nos animais amostrados foi de 29,2 por cento (642/2200); animais soropositivos foram identificados em 57,7 por cento (225/390) dos rebanhos. As estimativas de prevalência variaram de acordo com a cepa e/ou vírus utilizado para o desafio nos testes de SN. A prevalência e a sensibilidade mais altas foram obtidas quando os resultados positivos à SN frente aos quatro vírus distintos foram somados. O uso de somente um vírus de desafio na SN levaria a redução de sensibilidade de 20,4 por cento a 34,6 por cento quando comparada com os resultados positivos combinados. Estes achados evidenciam que anticorpos contra BoHV-1 e BoHV-5 estão amplamente difundidos nos rebanhos do RS, embora a prevalência em distintas regiões geográficas seja bastante variada. Os resultados obtidos nas estimativas de prevalência foram fortemente afetados pelas diferentes ...

This study was carried out to estimate the prevalence of antibodies to bovine herpesviruses types 1(BoHV-1) and 5 (BoHV-5) in the state of Rio Grande do Sul (RS), Brazil, by testing serum samples against different BoHV-1 and BoHV-5 strains. The sera examined were obtained from a larger sample designed to estimate the prevalence of bovine brucellosis within the state. All sera were collected from cows 24 months or older, not vaccinated to bovine herpesviruses, from both dairy and beef herds. The number of samples to be tested was calculated based on an estimated prevalence of infection of 33 percent, with an average standard deviation of £1 percent and a 95 percent limit of agreement. Sera from 2.200 cattle from 390 farms distributed in 158 counties were tested by serum neutralization (SN) tests in search for antibodies to the following strains: BoHV-1.1 (strains EVI123/98 and Los Angeles), BoHV-5a (strain EVI88/95) and BoHV-5b (strain A663). The overall seroprevalence to BoHV-1 and BoHV-5 in the sampled herds was 29.2 percent (642/2.200); seropositive animals were detected in 225 (57.7 percent) of the sampled farms. Prevalence estimates varied according to the virus used for challenge in SN tests. The highest prevalence and sensitivity were attained when positive SN results against the four different strains were added together. The use of only one virus for challenge in SN tests would lead to a loss in sensitivity from 20.4 percent to 34.6 percent when compared to the combined SN-positive results. These findings provide evidence that antibodies to BoHV-1 and BoHV-5 are largely spread in dairy and beef herds in RS, although prevalence in distinct geographic regions is quite variable. The results were strongly affected by the virus strains used for challenge in SN testing. This must be taken into account when performing serologic tests to detect BoHV-1 and BoHV-5 antibodies. As SN test is not capable of discriminating between antibody ...

Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine / Eficácia de uma vacina inativada, gE-deletada, contra o herpesvírus bovino tipo 1 (BoHV-1)

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 10(7.0) fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

O Herpesvírus bovino tipo 1 (BoHV-1) é reconhecido como um importante agente de perdas econômicas em bovinos. Vacinação tem sido amplamente empregada para minimizar as perdas conseqüentes a infecções com o BoHV-1. Reportamos previamente o desenvolvimento de uma vacina diferencial para BoHV-1, baseada em um recombinante do qual a glicoproteína gE (gE) foi deletada (265gE-). No presente trabalho foi realizada a avaliação da eficácia de tal recombinante como vacina inativada. Cinco bovinos soronegativos para BoHV-1 foram vacinadas por via intramuscular no dia 0 e revacinadas 30 dias após com uma vacina inativada com adjuvante oleoso, contendo massa antigênica equivalente a 10(7.0) doses infectantes para 50 por cento dos cultivos celulares (DICC50) de 265gE-. Três animais foram mantidos como controles não vacinados. No dia 60 pós-vacinação, os animais vacinados e controles foram desafiados com a amostra virulenta parental. Nenhum sinal clínico ou efeito adverso foi observado após ou durante a vacinação. Após o desafio, 2 dos 5 animais vacinados apresentaram sinais leves de infecção, enquanto que todos os animais não vacinados apresentaram intensa rinotraqueíte e disseminaram vírus por mais tempo e em títulos mais elevados do que os animais vacinados. Respostas sorológicas foram detectadas em todos os animais vacinados depois da segunda dose de vacina, mas não nos animais do grupo controle. Após a administração de corticosteróide visando a reativação de infecções latentes, não foram observados sinais clínicos em nenhum dos 5 animais vacinados, enquanto os animais não vacinados apresentaram sinais leves de doença respiratória. Em vista de sua imunogenicidade e efeito protetor frente ao desafio com BoHV-1 virulento, a preparação oleosa com o recombinante 265gE- inativado foi demonstrada ser adequada para uso como vacina.