Draft genome of neotropical Bacillus thuringiensis UFT038 and its potential against lepidopteran soybean pests.

Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner. Bt UFT038 and HD-1 were equally toxic against S. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.

Novel plant-associated genomoviruses from the Brazilian Cerrado biome.

The discovery rate of new plant viruses has increased due to studies involving high-throughput sequencing (HTS), particularly for single-stranded DNA viruses of the family Genomoviridae. We carried out an HTS-based survey of genomoviruses in a wide range of native and exotic trees grown in the Brazilian Cerrado biome, and the complete genome sequences of two novel members of the family Genomoviridae from two distinct genera were determined. Specific primers were designed to detect these genomoviruses in individual samples. A new gemykolovirus (Tecoma stans associated gemykolovirus) was detected in Tecoma stans, and a new gemykibivirus (Ouratea duparquetiana associated gemykibivirus) was detected in Ouratea duparquetiana. A gemykrogvirus related to Gila monster associated gemykrogvirus (80% pairwise identity) was also detected in foliar samples of Trembleya parviflora. Our pilot study paves the way for a better characterization of this diverse collection of genomoviruses as well as their interactions with the associated tree species.

Characterization of Cucurbit Aphid-Borne Yellows Virus (CABYV) from Passion Fruit in Brazil: Evidence of a Complex of Species within CABYV Isolates.

High-throughput sequencing (HTS) has been an important tool for the discovery of plant viruses and their surveillance. In 2015, several virus-like symptoms were observed in passion fruit (PF) plants in Bahia state, Brazil. Using HTS technology, bioinformatics tools, RT-PCR, and Sanger sequencing, we identified the cucurbit aphid-borne yellows virus (CABYV, Polerovirus, Solemoviridae) in co-infection with cowpea aphid-borne mosaic virus (CABMV, Potyvirus, Potyviridae) in PF, in green manure, and spontaneous plants in several localities in Bahia. Complete genomes of CABYV-PF isolates were determined and analyzed with other CABYV isolates available in GenBank that have been identified in various countries. Phylogenetic analysis and pairwise identity comparison with CABYV isolates showed that CABYV-PFs are more closely related to French and Spanish isolates. Overall, analyses of all the CABYV genomes revealed that these could represent ten distinct species, and we thus proposed reclassifying these CABYV as isolates into ten species, tentatively named "Polerovirus curcubitaeprimum" to "Polerovirus curcubitaenonum", and "Polerovirus melo". CABYV-PF is a member of "Polerovirus curcubitaeprimum".

Impact of Early Pandemic SARS-CoV-2 Lineages Replacement with the Variant of Concern P.1 (Gamma) in Western Bahia, Brazil.

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.

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.

Heterogeneity in the Response of Different Subtypes of Drosophila melanogaster Midgut Cells to Viral Infections.

Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Here, public scRNA-seq datasets from Drosophila melanogaster midgut cells were used to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication.

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.

Nanopore sequencing of tomato mottle leaf distortion virus, a new bipartite begomovirus infecting tomato in Brazil.

During a survey in a tomato field in Luziânia (Goiás State, Brazil), a single plant with mottling, chlorotic spots, and leaf distortion was found. A new bipartite begomovirus sequence was identified using nanopore sequence technology and confirmed by Sanger sequencing. The highest nucleotide sequence identity match of the DNA-A component (2596 bases) was 81.64% with tomato golden leaf deformation virus (HM357456). Due to the current species demarcation criterion of 91% nucleotide sequence identity for DNA-A, we propose this virus to be a new member of the genus Begomovirus, named "tomato mottle leaf distortion virus".

Tobamoviruses of two new species trigger resistance in pepper plants harbouring functional L alleles.

Tobamoviruses are often referred to as the most notorious viral pathogens of pepper crops. These viruses are not transmitted by invertebrate vectors, but rather by physical contact and seeds. In this study, pepper plants displaying mild mottle and mosaic symptoms were sampled in four different regions of Peru. Upon double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, seven samples cross-reacted weakly with antibodies against pepper mild mottle virus (PMMoV), suggesting the presence of tobamoviruses. When employing RT-PCR, conserved primers amplified cDNA fragments of viruses from two putative new tobamovirus species in the samples. The complete genome of two representative isolates were, therefore, sequenced and analysed in silico. These viruses, which were tentatively named yellow pepper mild mottle virus (YPMMoV) and chilli pepper mild mottle virus (CPMMoV), shared highest nucleotide genome sequence identities of 83 and 85 % with bell pepper mottle virus (BpeMV), respectively. Mechanical inoculation of indicator plants with YPMMoV and CPMMoV isolates did not show any obvious differences in host ranges. These viruses were also inoculated mechanically on pepper plants harbouring different resistance L alleles to determine their pathotypes. Pepper plants carrying unfunctional L alleles (L0) to tobamoviruses were infected by all isolates and presented differential symptomatology for YPMMoV and CPMMoV. On the other hand, pepper plants carrying L1, L2, L3 and L4 alleles were resistant to all isolates, indicating that these viruses belong to pathotype P0.

Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales.

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5' ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

Transmission of the Bean-Associated Cytorhabdovirus by the Whitefly Bemisia tabaci MEAM1.

The knowledge of genomic data of new plant viruses is increasing exponentially; however, some aspects of their biology, such as vectors and host range, remain mostly unknown. This information is crucial for the understanding of virus-plant interactions, control strategies, and mechanisms to prevent outbreaks. Typically, rhabdoviruses infect monocot and dicot plants and are vectored in nature by hemipteran sap-sucking insects, including aphids, leafhoppers, and planthoppers. However, several strains of a potentially whitefly-transmitted virus, papaya cytorhabdovirus, were recently described: (i) bean-associated cytorhabdovirus (BaCV) in Brazil, (ii) papaya virus E (PpVE) in Ecuador, and (iii) citrus-associated rhabdovirus (CiaRV) in China. Here, we examine the potential of the Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) to transmit BaCV, its morphological and cytopathological characteristics, and assess the incidence of BaCV across bean producing areas in Brazil. Our results show that BaCV is efficiently transmitted, in experimental conditions, by B. tabaci MEAM1 to bean cultivars, and with lower efficiency to cowpea and soybean. Moreover, we detected BaCV RNA in viruliferous whiteflies but we were unable to visualize viral particles or viroplasm in the whitefly tissues. BaCV could not be singly isolated for pathogenicity tests, identification of the induced symptoms, and the transmission assay. BaCV was detected in five out of the seven states in Brazil included in our study, suggesting that it is widely distributed throughout bean producing areas in the country. This is the first report of a whitefly-transmitted rhabdovirus.

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.

Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE-088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE-088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE-088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

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.

Double-Stranded RNA High-Throughput Sequencing Reveals a New Cytorhabdovirus in a Bean Golden Mosaic Virus-Resistant Common Bean Transgenic Line.

Using double-strand RNA (dsRNA) high-throughput sequencing, we identified five RNA viruses in a bean golden mosaic virus (BGMV)-resistant common bean transgenic line with symptoms of viral infection. Four of the identified viruses had already been described as infecting common bean (cowpea mild mottle virus, bean rugose mosaic virus, Phaseolus vulgaris alphaendornavirus 1, and Phaseolus vulgaris alphaendornavirus 2) and one is a putative new plant rhabdovirus (genus Cytorhabdovirus), tentatively named bean-associated cytorhabdovirus (BaCV). The BaCV genome presented all five open reading frames (ORFs) found in most rhabdoviruses: nucleoprotein (N) (ORF1) (451 amino acids, aa), phosphoprotein (P) (ORF2) (445 aa), matrix (M) (ORF4) (287 aa), glycoprotein (G) (ORF5) (520 aa), and an RNA-dependent RNA polymerase (L) (ORF6) (114 aa), as well as a putative movement protein (P3) (ORF3) (189 aa) and the hypothetical small protein P4. The predicted BaCV proteins were compared to homologous proteins from the closest cytorhabdoviruses, and a low level of sequence identity (15⁻39%) was observed. The phylogenetic analysis shows that BaCV clustered with yerba mate chlorosis-associated virus (YmCaV) and rice stripe mosaic virus (RSMV). Overall, our results provide strong evidence that BaCV is indeed a new virus species in the genus Cytorhabdovirus (family Rhabdoviridae), the first rhabdovirus to be identified infecting common bean.

A new virus found in garlic virus complex is a member of possible novel genus of the family Betaflexiviridae (order Tymovirales).

Plant vegetative propagation strategies for agricultural crops cause the accumulation of viruses, resulting in the formation of virus complexes or communities. The cultivation of garlic is based on vegetative propagation and more than 13 virus species from the genera Potyvirus, Allexivirus and Carlavirus have been reported. Aiming for an unbiased identification of viruses from a garlic germplasm collection in Brazil, total RNA from eight garlic cultivars was sequenced by high-throughput sequencing (HTS) technology. Although most viruses found in this study were previously reported, one of them did not belong to any known genera. This putative new virus was found in seven out of eight garlic cultivars and phylogenetic data positioned it as representative of an independent evolutionary lineage within family Betaflexiviridae. This virus has been tentatively named garlic yellow mosaic-associated virus (GYMaV), sharing highest nucleotide identities with African oil palm ringspot virus (genus Robigovirus) and potato virus T (genus Tepovirus) for the replicase gene, and with viruses classified within genus Foveavirus for the coat protein gene. Due to its high frequency in garlic cultivars, GYMaV should be considered in upcoming surveys of pathogens in this crop and in the development of virus-free garlic plants.

Viola phlebovirus is a novel Phlebotomus fever serogroup member identified in Lutzomyia (Lutzomyia) longipalpis from Brazilian Pantanal.

BACKGROUND: High throughput sequencing (HTS) boosted the discovery of novel viruses and new variants of known viruses. Here we investigated the presence of viruses in 12 pools of sand flies captured in three climatic periods in RAPELD grids at Rio Claro, Chapada dos Guimarães and at Pirizal, North Pantanal, Mato Grosso State, Midwestern Brazil by HTS, viral isolation of a putative Phlebovirus positive pool in Vero cells, RT-PCR and transmission electron microscopy (TEM). RESULTS: One pool containing three Lutzomyia (Lutzomyia) longipalpis sand flies captured in the transitional climatic period in North Pantanal showed a tripartite genomic sequence of a putative novel Phlebovirus belonging to the phlebotomus fever serogroup. Phylogenetic analysis revealed this virus is closely related and share a common ancestor with phleboviruses included in the same clade: Chagres, Urucuri and Uriurana virus. RNA-dependent RNA polymerase (RdRP) presented 60%, 59% and 58% of amino-acid (aa) similarity with these phleboviruses, respectively. Similarity of Nucleoprotein and NSs protein codified by ambissense strategy of segment S was of 49% and 37%, respectively, with the proteins of the closest phlebovirus, Uriurana virus. Glycoproteins (G1, G2) and NSm protein presented 49% and 48% aa similarity with Chagres and Uriurana virus, respectively. Uriurana virus was isolated from sand flies in Brazilian Amazon and Urucuri from rodents in Utinga forest, Pará State. Chagres virus is an arbovirus responsible for outbreaks of febrile illness in Panama. This phlebovirus was isolated in Vero cells, confirmed by TEM and RT-PCR for the L segment of the virus, and named Viola phlebovirus. CONCLUSIONS: HTS, viral isolation, RT-PCR and TEM showed the presence of one virus in sand flies from North Pantanal with identity to a putative novel Phlebovirus from phlebotomus fever serogroup, named Viola phlebovirus.

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil.

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae: passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus. Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12-30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891-921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7-3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina, Arabidopsis thaliana, and Passiflora edulis.