Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells.

UNLABELLED: During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. IMPORTANCE: Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.

Maturation of flaviviruses starts from one or more icosahedrally independent nucleation centres.

Flaviviruses assemble as fusion-incompetent immature particles and subsequently undergo conformational change leading to release of infectious virions. Flavivirus infections also produce combined 'mosaic' particles. Here, using cryo-electron tomography, we report that mosaic particles of dengue virus type 2 had glycoproteins organized into two regions of mature and immature structure. Furthermore, particles of a maturation-deficient mutant had their glycoproteins organized into two regions of immature structure with mismatching icosahedral symmetries. It is therefore apparent that the maturation-related reorganization of the flavivirus glycoproteins is not synchronized across the whole virion, but is initiated from one or more nucleation centres. Similar deviation from icosahedral symmetry might be relevant to the asymmetrical mode of genome packaging and cell entry of other viruses.

A replication competent luciferase-secreting DENV2 reporter for sero-epidemiological surveillance of neutralizing and enhancing antibodies.

Dengue virus (DENV) specific neutralizing and enhancing antibodies play crucial roles in dengue disease prevention and pathogenesis. DENV reporters are gaining popularity in the evaluation of these antibodies; their accessibility and acceptance may improve with more efficient production systems and indications of their antigenic equivalence to the wild-type virus. This study aimed to generate a replication competent luciferase-secreting DENV reporter (LucDENV2) and evaluate its feasibility in neutralizing and infection-enhancing antibody assays in comparison with wild-type DENV2, strain 16681, and a luciferase-secreting, single-round infectious DENV2 reporter (LucSIP). LucDENV2 replicated to similarly high levels as that of the parent 16681 virus in a commonly used mosquito cell line. LucDENV2 was neutralized in an antibody concentration-dependent manner by a monoclonal antibody specific to the flavivirus fusion loop and two antibodies specific to the E domain III, which closely resembled the neutralization patterns employing the LucSIP and wild-type DENV2. Parallel analysis of LucDENV2 and wild-type DENV2 revealed good agreement between the luciferase-based and focus-based neutralization and enhancement assays in a 96-well microplate format when employed against a set of clinical sera, suggesting comparable antigenic properties of LucDENV2 with those of the parent virus. The high-titer, replication competent, luciferase-secreting DENV reporter presented here should be a useful tool for fast and reliable quantitation of neutralizing and infection-enhancing antibodies in populations living in DENV-endemic areas.

Influence of pr-M cleavage on the heterogeneity of extracellular dengue virus particles.

During dengue virus replication, an incomplete cleavage of the envelope glycoprotein prM, generates a mixture of mature (prM-less) and prM-containing, immature extracellular particles. In this study, sequential immunoprecipitation and cryoelectron microscopy revealed a third type of extracellular particles, the partially mature particles, as the major prM-containing particles in a dengue serotype 2 virus. Changes in the proportion of viral particles in the pr-M junction mutants exhibiting altered levels of prM cleavage suggest that the partially mature particles may represent an intermediate subpopulation in the virus maturation pathway. These findings are consistent with a model suggesting the progressive mode of prM cleavage.

Prevalence and Molecular Characterization of Rickettsia spp. from Wild Small Mammals in Public Parks and Urban Areas of Bangkok Metropolitan, Thailand.

Rural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia.

Generation and characterization of luciferase-secreting, single-round infectious DENV-2 reporter for functional antibody assays.

Flavivirus reporters provide a robust tool for viral pathogenesis studies, anti-viral drug screening, disease diagnosis and functional antibody assays. In this study, we generated a luciferase-secreting, single-round reporter virus by replacing the capsid coding region in a DENV-2 genome with the secretory form of Lucia luciferase gene to produce infectious viral particles in a stable capsid-expressing mosquito cell line. Replication of the reporter virus in trans-complementing mosquito cells was sustained for up to two weeks. There were strong correlations between the extracellular luciferase activity and infectious reporter virus inocula upon infection of mosquito and mammalian cell lines with graded quantities of the reporter virus. A set of anti-E and anti-prM monoclonal antibodies affected the infectivity of reporter virus with similar dose-effect relationships as the parent virus. This simplified version of DENV-2 reporter provides a rapid and reliable method for the detection of neutralizing and infection-enhancing antibodies against dengue virus.

Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction.

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.

Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies.