Extinction and rapid emergence of strains of dengue 3 virus during an interepidemic period.

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.

Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies.

Three of 41 IgM monoclonal antibodies derived from dengue 1 virus immunized mice neutralized dengue 1 infection in vitro. All three neutralizing monoclonal antibodies reacted with spatially related epitopes on the E protein of dengue 1 which were also recognized by antibodies in sera from dengue patients. Two neutralization-resistant populations of dengue 1 virus, D1-M10 and D1-M17, were selected by sequential passage of virus in C6/36 cells in the presence of neutralizing IgM monoclonal antibodies M10 and M17, respectively. Single nucleotide changes occurred in the E protein gene of each of these virus populations resulting in single amino acid substitutions at E279 (Phe-Ser) in D1-M10 and at E293 (Thr-Ile) in D1-M17. Both neutralization-resistant populations of virus were more sensitive to elevated temperature than was the wild-type dengue 1 virus and the infectivity and haemagglutinating ability of the neutralization-resistant populations decreased more slowly than that of wild-type virus when exposed to pH in the range 5.8 to 7.0. These are the first epitopes involved in neutralization to have been identified in dengue 1 virus and the first outside domain III of the E protein on any dengue virus.

Identification of epitopes on the envelope (E) protein of dengue 2 and dengue 3 viruses using monoclonal antibodies.

Of a panel of forty-six anti-dengue 3 monoclonal antibodies (MAbs) only three neutralised infection of BHK cells by dengue 3 virus. Attempts to select neutralisation escape mutants (n.e.m.) with two of these antibodies failed. The n.e.m. population selected in the presence of the third neutralising antibody, 1H9, had a nucleotide change at position 1157 of the E protein gene resulting in a non-conservative amino acid change at E386 for a Lys to an Asn. A dengue 2 n.e.m. was selected with the flavivirus crossreactive IgG monoclonal antibody 4G2, had deduced amino acid changes at E169 (Ser to Pro) and E275 (Gly to Arg). This dengue 2 n.e.m. population produced smaller plaques in BHK cells than the parental virus, decreased fusion activity (FFWI) and had lost the ability to agglutinate gander erythrocyes at pH 6.0 to 6.7.

Functional expression of dipeptidyl peptidase I (Cathepsin C) of the oriental blood fluke Schistosoma japonicum in Trichoplusia ni insect cells.

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel-nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS-PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH(2)-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues -180 to -175, NH(2)-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH(2)-terminal sequence, LDXNQLY, corresponding to residues -73 to -67 of the proregion peptide and thus were generated by removal of 126 residues from the NH(2)-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37 degrees C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k(cat)/K(m)) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM(-)1 s(-1), respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) High Five cells.

Surveillance for Ross River virus infection using blood donors.

The number of clinical Ross River virus (RRV) infections (epidemic polyarthritis) each year in Australia continues to grow despite extensive vector control programs. There is a need, therefore, for a surveillance program that can give sufficient warning of outbreaks of the disease so that highly focused preventative measures may be undertaken. The ability of a surveillance program, based on voluntary Red Cross blood donations, to predict outbreaks of epidemic polyarthritis was evaluated. Anti-RRV IgM antibody was detected in significant numbers of blood donors from throughout the state of Queensland 6-9 weeks prior to an increase in the number of notified cases of epidemic polyarthritis. At a local level, significant numbers of anti-RRV IgM blood donors were detected in Brisbane in 1996 four weeks prior to an increase in the number of notified cases of epidemic polyarthritis. This system of surveillance is technically simple, rapid (results are obtained in 2-3 days), it samples the human population from throughout the state, and it gives timely warning of outbreaks of epidemic polyarthritis.

Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system.

In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.

Antibody-dependent enhancement and persistence in macrophages of an arbovirus associated with arthritis.

Ross River virus (RRV) is the aetiological agent of epidemic polyarthritis (EPA) a predominantly rheumatic disease afflicting up to 5000 Australians annually. We show here for the first time that macrophages can be productively infected by RRV. Subneutralizing titres of anti-RRV IgG (but not IgM) also showed classical antibody-dependent enhancement (ADE) of RRV infection in macrophage and monocyte cell lines. No correlation between development of EPA and the pre-existence of ADE titres was apparent, nor could sera raised against a related arbovirus, Barmah Forest, enhance RRV infection. Tumour necrosis factor-alpha, implicated in the immunopathogenesis of rheumatoid arthritis, was not secreted by RRV-infected monocytes or macrophages. Macrophage cell lines infected with RRV were, however, capable of producing virus for over 50 days. RRV-induced arthritis may therefore be due to the persistent productive infection of macrophages, perhaps established by a brief period of ADE early in infection.

Analysis of functional epitopes on the dengue 2 envelope (E) protein using monoclonal IgM antibodies.

Forty-two hybridomas secreting IgM antibody against dengue virus were derived from spleen cells of dengue 2 infected mice. Antibody from 27 of these recognised the E protein of this virus. Of the 22 antibodies which neutralised dengue 2, only two cross-reacted with other flaviviruses. These 22 antibodies recognised three discrete domains on dengue virions. Competitive binding studies with IgG monoclonal antibodies suggested that two of the three domains were recognised by both IgG and IgM antibodies and that there were two additional domains which may be recognised exclusively by either IgG or IgM antibodies. IgM antibodies reacting with domains recognised by IgG antibodies that enhanced infection of susceptible cells by dengue 2, had no enhancing properties. None of the IgM antibodies activated the serum complement system after reacting with dengue 2 virions.

Development of a candidate vaccine against Ross River virus infection.

Ross River virus is a mosquito-borne alphavirus which causes several thousand cases of arthritis (epidemic polyarthritis) each year. In this study, binary ethylenimine (BEI) was used to destroy the infectivity of this virus without abolishing the antigenicity or immunogenicity of the virion. Mice immunized intramuscularly with BEI-inactivated virus, with or without Alhydrogel adjuvant, produced antibody which neutralized Ross River virus in vitro, and the mice also failed to develop viraemia when challenged intravenously with live virus. Serum neutralization and in vivo protection were greatest when BEI-inactivated virus was administered without adjuvant.

Processing of the dengue virus type 2 proteins prM and C-prM.

A glycoprotein C-prM of 35,000 M(r) was immuno-precipitated from lysates of Aedes albopictus cells infected with dengue virus type 2 (DEN-2) using antisera directed against the C protein or an amino-terminal fragment of the prM glycoprotein. C-prM was not detected in infected Vero cells. The prM glycoprotein synthesized in infected A. albopictus and Vero cells was cleaved to produce the membrane-associated virion protein (M) and the non-M fragment (pr) immediately preceding or occurring simultaneously with the release of viral particles from cells. The cleavage was less efficient in mosquito cells. The pr fragment was found only in the medium and was not rapidly degraded. To obtain pr-specific and M-specific antisera for these studies, proteins containing fragments of DEN-2 prM fused with staphylococcal Protein A were synthesized in Escherichia coli using the expression vector pRIT2T. The fusion proteins were stable and were used to raise antisera in rabbits for immunoprecipitation of radiolabelled cell extracts and culture medium. This is the first report of the detection of a C-prM protein in flavivirus-infected cells and the identification of the pr component of prM.

Nuclear localization of dengue 2 virus core protein detected with monoclonal antibodies.

Anti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-shocked cells, but were first detected in infected cells approximately 32 h post-infection. PEPSCAN epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (9RNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (6KKAR9) and spans a possible second site for the initiation of synthesis of core protein (12PFN decreases MLKR18).

Isolation of Kunjin virus from a patient with a naturally acquired infection.

OBJECTIVE: To describe the first isolation of Kunjin virus from a patient with a natural infection. CLINICAL FEATURES: A 48-year-old female egg collector presented with muscle weakness, fatigue and extreme lethargy three weeks after developing rigors, headache, photophobia and nausea. Kunjin virus was isolated from an acute phase serum sample. INTERVENTION AND OUTCOME: The patient made a partial recovery after treatment for 10 days with Catovit (Boehringer Ingelheim), one tablet twice a day, and then declined further medical contact. CONCLUSION: The isolation of Kunjin virus from this patient confirms previous serological observations which suggested that this mosquito-borne virus caused febrile episodes in humans accompanied, on occasion, by polyarthralgia or mild central nervous system signs and symptoms.

Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.

A comparison of the sequence of the dengue-2 16681 virus with that of the candidate vaccine strain (16681-PDK53) derived from it identified 53 of the 10,723 nucleotides which differed between the strains. Nucleotide changes occurred in genes coding for all virion and nonvirion proteins, and in the 5' and 3' untranslated regions. Twenty-seven of the nucleotide changes resulted in amino acid alterations. The greatest amino acid sequence differences in the virion proteins occurred in prM (2.20%; 2/91 amino acids) followed by the M protein (1.33%; 1/75 amino acids), the C protein (0.88%; 1/114 amino acid), and the E protein (0.61%; 3/495 amino acids). Differences in the amino acid sequence of nonvirion proteins ranged from 1.51% (6/398 amino acids) in NS4 to 0.33% (3/900 amino acids) in NS5. The encoded protein sequences of 16681-PDK53 were also compared with the published sequences of other flaviviruses to obtain a detailed classification of 17 flaviviruses using the neighbor-joining tree method. The analyses of the sequence data produced dendrograms which supported the traditional groupings based on serological evidence, and they suggested that the flaviviruses have evolved by divergent mutational change and there was no evidence of genetic recombination between members of the group. Comparisons of the sequences of the flavivirus polymerase and helicase-like proteins (NS5 and NS3, respectively) with those from other viruses yielded a classification of the flaviviruses indicating that the primary division of the flaviviruses was between those transmitted by mosquitoes and those transmitted by ticks.

Viral-like characteristics of particle aggregates in a lymphoblastic cell line.

A lymphoblastic cell line (K45) established from a child with acute lymphoblastic leukemia (ALL) is characterized by a profusion of intracytoplasmic particle aggregates (PA) similar in morphology to those occurring in fresh childhood ALL cells. The PA in K45 cells were examined for morphology and capacity for nucleic acid synthesis to test the hypothesis that they are identical to those in fresh ALL cells, and also to identify characteristics which might distinguish PA from cell organelles and which might determine if they are viral-like. In contrast to cell organelles, the PA in both K45 and ALL cells were found to be characterized by a localized thickening of the particle wall. Furthermore, autoradiography of K45 cells showed uptake by PA of 3H uridine rather than 3H thymidine indicating an RNA composition. The presence of PA in profusion was associated with, but preceded necrotic death of K45 cells. These combined features suggest that the PA in K45 and in ALL cells are identical, that they are distinct from cell organelles, are not formed as a consequence of the initiation of cell death and that, while their exact nature remains unknown, a viral origin cannot be excluded.

Routine phenotyping of native and desialyzed alpha 2HS-glycoprotein from blood stains.

The application of a polyacrylamide gel isoelectric focusing (PAGIEF) and immunoblotting procedure for the identification of native alpha 2HS-glycoprotein (AHSG) in routine casework blood stains has produced reportable results on 57.2% of samples. This reporting rate is lower than that for group specific component (GC) (83.8%) and phosphoglucomutase (PGM 1) (72.8%) phenotyping of the same samples. Blood stain samples were desialyzed with 1 U/ml neuraminidase, overnight at room temperature prior to PAGIEF in gels containing pharmalyte pH 5-6 and 2.5 M urea. Simple AHSG patterns were developed by immunoblotting. This procedure was five times as sensitive as the native AHSG method and desialyzation was reproducible over a range of incubation times and neuraminidase concentrations. The application of the desialyzed AHSG analysis to routine casework samples has resulted in a significant increase in the number of reportable results (762 reported out of 1027 samples). This reporting rate (74.2%) compares favourably with that for GC (79.1%) and PGH 1 (69.6%) phenotyping of the same samples. The three AHSG alleles (AHSG*1, 2 and 3) are clearly resolved after sample desialyzation and separation in gels containing pharmalyte pH 5-6 and 2.5 M urea. The sensitivity of desialyzed AHSG phenotyping approaches that of GC and this technique is worthy of inclusion in blood stain screening protocols of forensic laboratories in regions where the population has a limited range of rare AHSG alleles.

Clinical and subclinical Barmah Forest virus infection in Queensland.

Barmah Forest virus is a mosquito-borne agent (alphavirus) reported to cause both clinical and subclinical infections in New South Wales. This report describes 29 cases of clinical Barmah Forest virus infection diagnosed between July 1988 and March 1989 (21 from Queensland, six from New South Wales and two from Victoria) and provides evidence of extensive subclinical infection with this virus (0.23% of the population per annum) throughout Queensland. It also includes a description of the first isolation of Barmah Forest virus from a patient. Data obtained in the course of the study suggest that Barmah Forest virus infections may not be diagnosed correctly in many instances because of the similarity of the symptoms of this disease to those of epidemic polyarthritis and the small number of laboratories providing the necessary serological services.

The application of immunoblotting to the phenotyping of group-specific component.

A sensitive immunoblotting method for the routine detection of group-specific component (GC) from fresh serum, and from control and casework bloodstains has been developed. GC phenotypes were separated in a thin layer polyacrylamide gel by isoelectric focusing, transferred to nitrocellulose by a rapid capillary blotting procedure, and detected using a double antibody enzyme immunoassay. This method is capable of phenotyping 8 ng of GC extracted from bloodstains, a four-fold increase in sensitivity when compared to immunofixation and silverstaining. A total of 2424 casework bloodstains have been analysed and GC phenotypes identified in 78% of samples. The method is suitable for use in routine laboratories and is more sensitive than other methods for GC phenotyping of casework bloodstains.