Infectious bursal disease virus uptake involves macropinocytosis and trafficking to early endosomes in a Rab5-dependent manner.

Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin- and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.

Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil.

The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analysis by electron microscopy and gel electrophoresis, respectively, revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.

Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus.

Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery.

Lactobacillus plantarum bacteriophages isolated from Kefir grains: phenotypic and molecular characterization.

Two greatly related Lactobacillus plantarum bacteriophages (named FAGK1 and FAGK2) were isolated from Kefir grains of different origins. Both phages belonged to the Siphoviridae family (morphotype B1) and showed similar dimensions for head and tail sizes. The host range of the two phages, using 36 strains as potential host strains, differed only in the phage reactivity against one of them. The phages showed latent periods of 30 min, burst periods of 80+/-10 min and burst size values of 11.0+/-1.0 PFU per infected cell as mean value. Identical DNA restriction patterns were obtained for both phages with PvuI, SalI, HindIII and MluI. The viral DNA apparently did not present extremes cos and the structural protein patterns presented four major bands (32.9, 35.7, 43.0 and 66.2 kDa). This study reports the first isolation of bacteriophages of Lb. plantarum from Kefir grains and adds further knowledge regarding the complex microbial community of this fermented milk.

Identification of a new nucleopolyhedrovirus from naturally-infected Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae) larvae on poplar plantations in South Brazil.

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81+/-2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [(35)S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.

A rapid method to produce high yields of purified rotavirus particles.

A rapid purification method of rotavirus particles to high yield retaining the double shelled structure of infectious virus is described. Group A rotavirus (UK strain) was concentrated through a cushion of colloidal silica (rho=1.10 g/cm(3)) or by precipitating with polyethylene glycol 8000. After concentration, infectious rotavirus was cleared from host cell proteins by density equilibrium centrifugation in gradients of colloidal silica using near vertical rotors. Characterisation of purified virus assessed by electron microscopy and poliacrylamide gel electrophoresis (PAGE) revealed the typical wheel shape structure of rotavirus particles and the presence of the 11 segments of dsRNA arranged in the 4-2-3-2 pattern. Presence of rotavirus structural proteins including VP6, VP4 and VP7 from the outer shell, was demonstrated by SDS-PAGE and Western blot using polyclonal and VP6-specific monoclonal antibodies. This method achieved a approximately 1500 fold purification, which retained approximately 80% infectivity depending on the concentration protocol used, while yielding 160 microg of viral protein per each litre of infected cell culture medium. The time required for the isopycnic centrifugation was only 25 min and the entire completion of the method required 3.5 h. The method is simple technically and applicable to the purification of large as well as minute amounts of virus.

Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539.

A restriction map was constructed of the 37 kb genome of the temperate Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Restriction analysis and Southern hybridization experiments detected variable levels of homologous regions among the genomes of lb539 and the L. delbrueckii reference phages LL-H (virulent) and mv4 (temperate). The principal homology was observed at the regions encoding the structural proteins. These studies allowed us to construct a partial genetic map of phage lb539 for lysin, the main structural tail protein and the packaging region genes. Furthermore, a short 1.5 kb DNA fragment of the prolate-headed JCL1032 phage genome was observed to be highly homologous with the DNA of the isometric-headed lb539, mv4 and LL-H phages. The described distribution of the homologous regions between the genomes of the phages lb539, LL-H, mv4 and JCL1032 presented here supports the modular evolution theory of the bacteriophages.

[A structural protein study of the influenza A (H1N1) virus by polyacrylamide gel electrophoresis]. / Estudio de las proteínas estructurales de influenzavirus A (H1N1) por electroforesis en gel de poliacrilamida.

Influenza is an acute respiratory disease typically appearing as an epidemic. Three immunological types of the influenza virus are known: A, B and C. Continually, antigen changes occur, especially in type A. Therefore, a comparative study was carried out on 4 influenza A(H1N1) virus strains in relation to protein structure (surface antigens), by using polyacrylamide gel electrophoresis by the modified Laemmli method. The objective was to compare the structural proteins of the A/Havana/1292/78 (H1N1) national strain with the proteins of 3 international pattern strains. In all the cases, 6 bands were detected by densitometry. In the 4 strains studied the most abundant protein was M. Great differences between the Cuban strain and the 3 international patterns were not seen.

Further analysis on the structural proteins of infectious pancreatic necrosis virus.

The structural proteins of infectious pancreatic necrosis virus (IPNV) have been analyzed. Two-dimensional gel electrophoresis showed that IPNV proteins are slightly acidic with apparent pIs ranging from 5.8 to 6.6. To identify the IPNV surface-located proteins, purified virus was labelled either with fluorescein isothiocyanate (FITC) or with Na 125I. After analysis by SDS-PAGE, only the major viral protein, VP2, was labelled by either procedure. The accessibility of VP2 to these reagents suggests that this protein is externally located. In addition, using Concanavalin A conjugated with FITC and IPNV labelling with 3H-mannose, evidence is present that VP2 contains carbohydrate residues.