Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach.

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Identificación de un aislamiento argentino de Spodoptera frugiperda granulovirus / Identification of an Argentinean isolate of Spodoptera frugiperda granulovirus

Abstract The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is an important maize pest. Due to the environmental impact and emergence of resistance caused by chemical pesticides and transgenic events, the use of baculoviruses becomes a safe and useful alternative for its control in integrated pest management strategies. Here we report the identification of a novel isolate of a granulovirus of S. frugiperda native to the central region of Argentina, named SfGV ARG. We observed that larvae infected with SfGV ARG showed a yellowish coloration, swollen body and, in some cases, severe lesions in the last abdominal segments. We confirmed the identity of the isolate by sequencing fragments of the lef-8, lef-9 and granulin genes and by calculating evolutionary distances using the Kimura-2-Parameter model. SfGV ARG DNA restriction pattern allowed to estimate a genome of at least 135 kb.

Resumen La oruga militar tardía, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), es una plaga importante del maíz. Debido al impacto ambiental y a la aparición de resistencia causados por los pesticidas químicos y los eventos transgénicos, el uso de baculovirus resulta una alternativa útil y saludable para su control en estrategias de manejo integrado de plagas. En este trabajo reportamos la identificación de un nuevo aislamiento del granulovirus de la S. frugiperda nativo de la región central de Argentina, SfGV ARG. Se observó que larvas infectadas con SfGV ARG mostraron coloración amarillenta, hinchazón y, en algunos casos, lesiones graves en los últimos segmentos abdominales. Se confirmó la identidad del aislamiento por secuenciación de fragmentos de los genes lef-8, lef-9y granulina, y por cálculo de distancias evolutivas usando el parámetro de Kimura-2. El patrón de restricción generado con el ADN genómico de SfGV ARG permitió estimar un tamaño de genoma de al menos 135 kb.

The complete genome sequence of the first hesperiid-infecting alphabaculovirus isolated from the leguminous pest Urbanus proteus (Lepidoptera: Hesperiidae).

Baculoviruses are insect viruses largely used as expression vectors and biopesticides. These viruses can efficiently infect the larval stage of several agricultural pests worldwide causing a lethal disease. In this work, we found a novel baculovirus isolated from the larval stage of Urbanus proteus (L.), the bean leafroller and characterized its complete genome. This is an important pest of several leguminous plants in Brazil and belongs to the butterfly family Hesperiidae, from where no baculovirus genome sequence has been described. This new virus was shown to have the smallest genome among all alphabaculoviruses sequenced to date, with 105,555 bp and 119 putative ORFs. We found ten unique genes, seven bro, and the 38 baculovirus core genes. UrprNPV was found to be related to the Adoxophyes-infecting baculoviruses AdorNPV and AdhoNPV with high genetic distance and a long branch length. Interestingly, few individual core gene-based phylogenies were found to support the relationship of UrprNPV to both AdorNPV and AdhoNPV. Importantly, the increase in number of completely sequenced baculovirus points to a very exciting way to understand baculovirus and its evolution and could potentially help the use of baculovirus as both biopesticides and expression vectors.

A Novel Betabaculovirus Isolated from the Monocot Pest Mocis latipes (Lepidoptera: Noctuidae) and the Evolution of Multiple-Copy Genes.

In this report, we described the genome of a novel baculovirus isolated from the monocot insect pest Mocis latipes, the striped grass looper. The genome has 134,272 bp in length with a G + C content of 38.3%. Based on the concatenated sequence of the 38 baculovirus core genes, we found that the virus is a betabaculovirus closely related to the noctuid-infecting betabaculoviruses including Pseudaletia unipuncta granulovirus (PsunGV), Trichoplusia ni granulovirus (TnGV), Helicoverpa armigera granulovirus (HearGV), and Xestia c-nigrum granulovirus (XecnGV). The virus may constitute a new Betabaculovirus species tentatively named Mocis latipes granulovirus (MolaGV). After gene content analysis, five open reading frames (ORFs) were found to be unique to MolaGV and several auxiliary genes were found including iap-3, iap-5, bro-a, bro-b, and three enhancins. The virus genome lacked both chitinase and cathepsin. We then looked at the evolutionary history of the enhancin gene and found that betabaculovirus acquired this gene from an alphabaculovirus followed by several duplication events. Gene duplication also happened to an endonuclease-like gene. Genomic and gene content analyses revealed both a strict collinearity and gene expansion into the genome of the MolaGV-related species. We also characterized the granulin gene using a recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and found that occlusion bodies were produced into the nucleus of infected cells and presented a polyhedral shape and no occluded virions within. Overall, betabaculovirus genome sequencing is of importance to the field as few genomes are publicly accessible. Mocislatipes is a secondary pest of maize, rice, and wheat crops in Brazil. Certainly, both the discovery and description of novel baculoviruses may lead to development of greener and safer pesticides in order to counteract and effectively control crop damage-causing insect populations.

Genomic analysis of a Trichoplusia ni Betabaculovirus (TnGV) with three different viral enhancing factors and two unique genes.

The complete genome of a Trichoplusia ni granulovirus (TnGV) is described and analyzed. The genome contains 175,360 bp (KU752557), becoming the third largest genome within the genus Betabaculovirus, smaller only than the Xestia c-nigrum GV (XecnGV) (178,733 pb) and the Pseudaletia unipuncta GV (PsunGV) (176,677 pb) genomes. The TnGV genome has a 39.81% C+G content and a total of 180 ORFs were identified, 96 of them in the granulin gene direction and 84 in the opposite direction. A total of 94.38% of the ORFs showed high identity with those of ClanGV, HaGV, and SlGV. Eight homologous regions (hrs) were identified as well as one apoptosis inhibitor (IAP-3). Interestingly, three viral enhancing factors (VEFs) were located in TnGV genome: VEF-1 (orf153), VEF-3 (orf155), and VEF-4 (orf164), additional to another metalloprotease (orf37). Two ORFs were unique to TnGV (orf100 and orf101) and another one was shared by only TnGV and AgseGV (orf2). Eleven of the deduced proteins showed high identity with proteins from nucleopolyhedroviruses, three with proteins from ascoviruses, and one with an entomopoxvirus protein. The largest deduced protein contains 1,213 amino acids (orf43) and the smallest deduced protein contains only 50 amino acids (orf143). Sequence identity and phylogenetic analyses showed that the closest related genomes to TnGV are, to date, those of PsunGV and XecnGV. This genome analysis may contribute to functional research on TnGV, and may form the bases for the utilization of this betabaculovirus as a pest control agent.

A betabaculovirus encoding a gp64 homolog.

BACKGROUND: A betabaculovirus (DisaGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pests of the sugarcane and other monocot cultures in Brazil. RESULTS: The complete genome sequence of DisaGV was determined using the 454-pyrosequencing method. The genome was 98,392 bp long, which makes it the smallest lepidopteran-infecting baculovirus sequenced to date. It had a G + C content of 29.7% encoding 125 putative open reading frames (ORF). All the 37 baculovirus core genes and a set of 19 betabaculovirus-specific genes were found. A group of 13 putative genes was not found in any other baculovirus genome sequenced so far. A phylogenetic analysis indicated that DisaGV is a member of Betabaculovirus genus and that it is a sister group to a cluster formed by ChocGV, ErelGV, PiraGV isolates, ClanGV, CaLGV, CpGV, CrleGV, AdorGV, PhopGV and EpapGV. Surprisingly, we found in the DisaGV genome a G protein-coupled receptor related to lepidopteran and other insect virus genes and a gp64 homolog, which is likely a product of horizontal gene transfer from Group 1 alphabaculoviruses. CONCLUSION: DisaGV represents a distinct lineage of the genus Betabaculovirus. It is closely related to the CpGV-related group and presents the smallest genome in size so far. Remarkably, we found a homolog of gp64, which was reported solely in group 1 alphabaculovirus genomes so far.

The complete sequence of the first Spodoptera frugiperda Betabaculovirus genome: a natural multiple recombinant virus.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.

Occurrence and phylogenetic characterization of a baculovirus isolated from Culex quinquefasciatus in São Paulo State, Brazil.

The aim of this study was to assess the occurrence of baculovirus infections in mosquitoes and characterize them by using molecular tools. Fortnightly collections were made of mosquito larvae in the city of Caraguatatuba. Six larvae of Culex quinquefasciatus were isolated that had white cysts (nodules) in epithelia cells of the posterior midgut, indicative of infection by a baculovirus. These larvae were subjected to DNA extraction. DNA was amplified, producing a fragment of around 600 nt of the lef-8 gene and 400 nt of Pif-2 gene. The sequences were aligned, using ClustalX 2.0, with partial sequences of lef-8 genes of baculoviruses isolated from members of other insect orders taken from the GenBank database and edited, and phylogenetic analysis was performed. Phylogenetic analysis performed with the lef-8 and pif-2 genes demonstrated that the baculovirus identified in Culex quinquefasciatus in the Caraguatatuba region is most closely related to the deltabaculovirus Culex nigripalpus nucleopolyhedrovirus.

Occurrence and phylogenetic characterization of a baculovirus isolated from Culex quinquefasciatus in São Paulo State, Brazil

The aim of this study was to assess the occurrence of baculovirus infections in mosquitoes and characterize them by using molecular tools. Fortnightly collections were made of mosquito larvae in the city of Caraguatatuba. Six larvae of Culex quinquefasciatus were isolated that had white cysts (nodules) in epithelia cells of the posterior midgut, indicative of infection by a baculovirus. These larvae were subjected to DNA extraction. DNA was amplified, producing a fragment of around 600 nt of the lef-8 gene and 400 nt of Pif-2 gene. The sequences were aligned, using ClustalX 2.0, with partial sequences of lef-8 genes of baculoviruses isolated from members of other insect orders taken from the GenBank database and edited, and phylogenetic analysis was performed. Phylogenetic analysis performed with the lef-8 and pif-2 genes demonstrated that the baculovirus identified in Culex quinquefasciatus in the Caraguatatuba region is most closely related to the deltabaculovirus Culex nigripalpus nucleopolyhedrovirus...

Detection and kinetic analysis of Epinotia aporema granulovirus in its lepidopteran host by real-time PCR.

Epinotia aporema granulovirus (EpapGV) has attracted interest as a potential biocontrol agent of the soybean pest Epinotia aporema in Argentina. Studies on virus/host interactions conducted so far have lacked an accurate method to assess the progress of virus load during the infection process. The present paper reports the development of a real-time PCR for EpapGV and its application to describe viral kinetics following ingestion of two different virus doses by last-instar E. aporema larvae. Real-time PCR was shown to be a reliable method to detect and quantify the presence of EpapGV in the analyzed samples. The increase in virus titer (log) exhibited a sigmoidal pattern, with an exponential growth phase between 24 and 48 h postinfection for both initial doses tested.

Characterization of a granulovirus isolated from Epinotia aporema Wals. (Lepidoptera: Tortricidae) larvae.

A granulovirus (GV) isolated from Epinotia aporema (Lepidoptera: Tortricidae)-a major soybean pest-was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.

Azurexidina, proteína de neutrófilo com funçäo antibiótica: Expressäo, purificaçäo e caracterizaçäo da proteína recombinante / Azurocidin, protein of neutrophilic with function antibiotical: expression, purification and characterization of recombinant protein

Os leucócitos polimorfonucleares têm papel crítico na destruiçäo, digestäo de patógenos, geraçäo e regulaçäo do processo inflamatório. Proteínas que säo estocadas dentro dos grânulos citoplasmáticos têm sido implicadas nos mecanismos microbicidas, independentes de oxigênio, bem como na degradaçäo de proteínas. Os grânulos azurófilos e específicos de polimorfonucleares neutrófilos contêm pelo menos 10 proteínas que podem destruir microorganismos. Dentre estas proteínas, 3 säo serina proteinases: catepsina G, proteinase 3 e elastase. Estas serina proteinases têm atividade antibiótica e também degradam proteínas do tecido conjuntivo. Azurocidina apresenta diversas semelhanças na sequência de aminoácidos e de DNA com as serina proteinases, também possui atividade antibiótica, porém näo apresenta atividade proteolítica, devido a mutaçäoes ocorridas na molécula de DNA, alterando o sítio catalítico típico desta família de proteínas. Esta família de proteínas, incluindo a azurocidina, é denominada de serprocidinas (serina proteinases com atividade microbicida). Azurocidina tem peso molecular de 29 kD e seu DNA codifica 225 aminoácidos da proteína madura e 24 resíduos de aminoácidos do sinal peptídico. O presente trabalho de tese diz respeito à expressäo e produçäo de azurocidina recombinante no sistema de expressäo baculovírus-células de inseto. Azurocidina recombinante expressada e produzida neste sistema foi caracterizada bioquimicamente e sua atividade microbicida conta bactérias Gram negativas e Gram positivas, bem como contra fungo, foi demonstrada. Azurocidina recombinante possui similaridades com a molécula nativa no peso molecular, 29 kD, padräo de glicosilaçäo, ambas possuem manose como resíduo de açucar terminal, sequência N-terminal de aminoácidos e com a atividade microbicida. A LD50 de azurocidina recombinante comtra E. coli, S. faecalis e C. albicans foi 1,2 (mais ou menos) 0,1, 2,9 (mais ou menos) 0,4 e 8,2 (mais ou menos) 0,2 ug/ml, respectivamente e a LD50 de azurocidina nativa foi de 1,3 (mais ou menos) 0,05, 2,3 (mais ou menos) 0,2 e 8 (mais ou menos) 0,8 ug/ml. Também foi demonstrado, com experimentos de deleçäo na molécula de DNA de azurocidina, que as sequências de aminoácidos localizadas entre as posiçöes 114 a 129 e/ou entre 154 a 186, säo necessárias para que azurocidina recombinante seja secretada pelo mio de cultura das células de inseto infectadas com o baculovírus recombinante. Associado a este trabalho de tese, desenvolvemos...