Nopporn Sittisombut

Specific identification of Penicillium marneffei by a polymerase chain reaction/hybridization technique

Penicillium marneffei has been described recently as a cause of an emerging mycotic infection in HIV-infected patients. A PCR/hybridization assay was developed to rapidly identify this pathogen. The nucleotide sequence of the 631-bp region of 18S ribosomal DNA of P. marneffei was determined using the standard dideoxy chain termination method. An oligonucleotide probe was designed on the basis of the analysed sequences of P. marneffei and 18S rDNA sequences of other fungi in the GenBank database. A 631-bp PCR product was amplified using primers RRF1 and RRH1 from P. marneffei and seven other fungi, Penicillium spp., Aspergillus fumigatus, A. flavus, Histoplasma capsulatum, Cryptococcus neoformans, Candida albicans and C. krusei. A 15 oligonucleotide segment (Pm3) which was specific for P. marneffei was synthesized and used as a probe. Only the PCR products of P. marneffei isolates hybridized with the Pm3 oligonucleotide probe. The sensitivity of the assay was approximately 0.5 pg/microl and 0.1 pg/microl of DNA by PCR and Southern hybridization, respectively. The usefulness of this method as a diagnostic tool will require further studies.

Western immunoblot analysis of protein antigens of Penicillium marneffei

Protein antigens of Penicillium marneffei prepared during the yeast and mould phases of in vitro growth were analyzed by gel electrophoresis and immunoblot assay. More than 20 yeast phase proteins were detected by Coomassie staining; among these, at least 10 reacted with IgG in the pooled sera of 28 AIDS patients with penicilliosis. Four immunogenic proteins of 200, 88, 54 and 50 kDa were produced in large quantity during the deceleration and early stationary phases of growth. When these proteins were reacted with individual sera derived from 33 AIDS patients with penicilliosis, reactivities to the 200, 88, 54 and 50 kDa protein were detected in 72.7, 93.9, 60.6 and 57.6%, respectively. The bands of 88, 54 and 50 kDa gave strong reactions with about a half of serum samples. In one serum derived from an AIDS patient, reactivities to the 54 and 50 kDa proteins could be strongly detected two months before the definite diagnosis by fungal culture. Protein components from the mould form were of lower yield and gave weaker signal in immunoblot analysis. These results indicate that at least two yeast-phase immunoreactive proteins (54 and 50 kDa) are relatively specific to the P. marneffei infection, thereby suggesting its potential for clinical application to the diagnosis of this emerging disease.

Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement

BACKGROUND Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.

Influence of pr-M Cleavage on the Heterogeneity of Extracellular Dengue Virus Particles

ABSTRACT 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.

Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

ABSTRACT During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.

Studies on Serological Cross-Reaction in Sequential Flavivirus Infections

Acute‐ and convalescent‐phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre‐existing flavivirus antibodies were tested for cross‐reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test. A fourfold or greater rise in N antibody titer in the convalescent‐phase was considered significant. Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital, showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two cross‐reactive JE sera revealed that IgM class antibody was specific for JE, while IgG class antibody was cross‐reactive. Of three JE cases with pre‐existing YF antibody obtained in Okinawa, Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross‐reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test, in sequential flavivirus infection.

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

ABSTRACT 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.

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.

Interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production.

Dengue virus infection is an important mosquito-borne disease and a public health problem worldwide. A better understanding of interactions between human cellular host and dengue virus proteins will provide insight into dengue virus replication and cellular pathogenesis. The glycosylated envelope protein of dengue virus, DENV E, is processed in the endoplasmic reticulum of host cells and therefore reliant on host processing functions. The complement of host ER functions involved and nature of the interactions with DENV E has not been thoroughly investigated. By employing a yeast two-hybrid assay, we found that domain III of DENV E interacts with human immunoglobulin heavy chain binding protein (BiP). The relevance of this interaction was demonstrated by co-immunoprecipitation and co-localization of BiP and DENV E in dengue virus-infected cells. Using the same approach, association of DENV E with two other chaperones, calnexin and calreticulin was also observed. Knocking-down expression of BiP, calnexin, or calreticulin by siRNA significantly decreased the production of infectious dengue virions. These results indicate that the interaction of these three chaperones with DENV E plays an important role in virion production, likely facilitating proper folding and assembly of dengue proteins.

Association of dengue virus NS1 protein with lipid rafts

During the replication of dengue virus, a viral non-structural glycoprotein, NS1, associates with the membrane on the cell surface and in the RNA replication complex. NS1 lacks a transmembrane domain, and the mechanism by which it associates with the membrane remains unclear. This study aimed to investigate whether membrane-bound NS1 is present in lipid rafts in dengue virus-infected cells. Double immunofluorescence staining of infected HEK-293T cells revealed that NS1 localized with raft-associated molecules, ganglioside GM1 and CD55, on the cell surface. In a flotation gradient centrifugation assay, a small proportion of NS1 in Triton X-100 cell lysate consistently co-fractionated with raft markers. Association of NS1 with lipid rafts was detected for all four dengue serotypes, as well as for Japanese encephalitis virus. Analysis of recombinant NS1 forms showed that glycosylated NS1 dimers stably expressed in HEK-293T cells without an additional C-terminal sequence, or with a heterologous transmembrane domain, failed to associate with lipid rafts. In contrast, glycosylphosphatidylinositol-linked recombinant NS1 exhibited a predilection for lipid rafts. These results indicate an association of a minor subpopulation of NS1 with lipid rafts during dengue virus infection and suggest that modification of NS1, possibly lipidation, is required for raft association.