The drug sensitivity and transmission dynamics of human malaria on Nias Island, North Sumatra, Indonesia.

Nias Island, off the north-western coast of Sumatra, Indonesia, was one of the first locations in which chloroquine-resistant Plasmodium vivax malaria was reported. This resistance is of particular concern because its ancient megalithic culture and the outstanding surfing conditions make the island a popular tourist destination. International travel to and from the island could rapidly spread chloroquine-resistant strains of P. vivax across the planet. The threat posed by such strains, locally and internationally, has led to the routine and periodic re-assessment of the efficacy of antimalarial drugs and transmission potential on the island. Active case detection identified malaria in 124 (17%) of 710 local residents whereas passive case detection, at the central health clinic, confirmed malaria in 77 (44%) of 173 cases of presumed 'clinical malaria'. Informed consenting volunteers who had malarial parasitaemias were treated, according to the Indonesian Ministry of Health's recommendations, with sulfadoxine-pyrimethamine (SP) on day 0 (for P. falciparum) or with chloroquine (CQ) on days 0, 1 and 2 (for P. vivax). Each volunteer was then monitored for clinical and parasite response until day 28. Recurrent parasitaemia by day 28 treatment was seen in 29 (83%) of the 35 P. falciparum cases given SP (14, 11 and four cases showing RI, RII and RIII resistance, respectively). Recurrent parasitaemia was also observed, between day 11 and day 21, in six (21%) of the 28 P. vivax cases given CQ. Although the results of quantitative analysis confirmed only low prevalences of CQ-resistant P. vivax malaria, the prevalence of SP resistance among the P. falciparum cases was among the highest seen in Indonesia. When the parasites present in the volunteers with P. falciparum infections were genotyped, mutations associated with pyrimethamine resistance were found at high frequency in the dhfr gene but there was no evidence of selection for sulfadoxine resistance in the dhps gene. Night-biting mosquitoes were surveyed by human landing collections and tested for sporozoite infection. Among the five species of human-biting anophelines collected, Anopheles sundaicus was dominant (68%) and the only species found to be infective--two (1.2%) of 167 females being found carrying P. vivax sporozoites. The risk of malarial infection for humans on Nias was considered high because of the abundance of asymptomatic carriers, the reduced effectiveness of the available antimalarial drugs, and the biting and infection 'rates' of the local An. sundaicus.

Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in-vivo drug resistance in West Papua, Indonesia.

This study (conducted in 1996-99) examines the association of mutations in pfmdr1, dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum with in-vivo drug resistance in West Papua, Indonesia. Initially, 85 patients infected with P. falciparum were treated with chloroquine, of whom 21 were cleared of parasites, 49 had parasitaemias classified as RI, RII or RIII resistance and 1 patient had recrudescent parasitaemia. Fansidar (pyrimethamine-sulfadoxine) was the second-line treatment and 18 patients were cleared of parasites and 31 had continuing infections classified as RI, RII or RIII resistance and 1 patient had recrudescent parasitaemia. The pfmdr1, dhfr and dhps genes were examined for mutations previously shown to be associated with resistance to these drugs. In this study, mutations in pfmdr1 were associated with chloroquine resistance and mutations in both dhfr and dhps were associated with Fansidar resistance in vivo. Interestingly, Gly-437 in dhps along with Arg-59/Asn-108 in dhfr were associated with RI, RII and RIII resistance whereas Glu-540 was highly associated with only RII and RIII Fansidar resistance. This finding supports the hypothesis that the molecular basis of RI, RII and RIII Fansidar resistance involves an accumulation of mutations in both dhfr and dhps. These results suggest that mutations in both dhfr and dhps genes are a good predictor of potential Fansidar treatment failure.

A single chain Fv antibody displayed on phage surface recognises conformational group-specific epitope of bluetongue virus.

A single chain fragment variable (scFv) antibody gene was isolated from hybridoma cell line secreting monoclonal antibody (MAb) 20E9 that recognises bluetongue virus (BTV) VP7. DNA fragments encoding variable regions of heavy and light chains were amplified by RT-PCR and library of scFv was constructed in phage vector. Two scFv clones that were selected showed specific reactivity with conformational epitope VP7. The N-terminal 22 amino acid residues of 20E9 light chain were identical to that deduced from scFv DNA sequence. An in-frame TAG stop codon was found in the coding sequence and its potential role in regulating the expression and stability of scFv in phage is discussed.

The presence of cross-reactive antibodies to rabbit haemorrhagic disease virus in Australian wild rabbits prior to the escape of virus from quarantine.

Sera collected from Australian wild rabbits prior to the escape of rabbit haemorrhagic disease virus (RHDV) from Wardang Island were examined for RHDV antibodies using purified recombinant capsid protein VP60 expressed from baculovirus. A VP60-based indirect ELISA showed that 196 of 392 wild rabbit sera reacted (OD(450) >0.15) with VP60. Twenty sera (OD(450) ranging from 0.15-2.47), randomly chosen from the 196 positive sera, recognized the 64 kDa VP60 in Western blot analysis, indicating that the reactivity detected in ELISA is indeed specific to the VP60 antigen. In a separate study, sera of 23 rabbits from an RHD-free area after the escape of RHDV were tested by ELISA and 21 of the 23 rabbits were found to be positive. When these rabbits were challenged with a lethal dose of RHDV, 11 out of the 23 rabbits survived. The presence of RHDV-protective antibodies in some of these rabbits suggested that they had been exposed to a pre-existing non-virulent rabbit calicivirus closely related to RHDV. These results highlight the need to study the prevalence of, and to characterize, this viral agent in order to effectively control rabbit populations in Australia and New Zealand.

Bacterial expression of the major antigenic regions of porcine rotavirus VP7 induces a neutralizing immune response in mice.

The outer capsid protein of rotavirus, VP7, is a major neutralization antigen. A chimeric protein comprising Escherichia coli (E. coli) outer membrane protein A (OmpA) and part of porcine rotavirus VP7 containing all three antigenic regions (217 amino acids) was expressed in Salmonella and E. coli as an outer-membrane associated protein. Mice immunized intraperitoneally or orally, respectively, with live E. coli or Salmonella cells expressing this chimeric protein produced antibodies against native VP7 as determined by enzyme-linked immunosorbent assays and neutralization tests. This indicates that the VP7 fragment from a porcine rotavirus which is antigenically similar to human rotavirus serotype 3, when expressed in bacteria as a chimeric protein, can form a structure resembling its native form at least in some of the major neutralization domains. These results indicate that the use of a live bacterial vector expressing rotavirus VP7 may represent a strategy for the development of vaccines against rotavirus-induced diarrhoea in infants.

Virus-like particles of calicivirus as epitope carriers.

The VP60 of rabbit haemorrhagic disease virus (RHDV), when expressed in baculovirus, self-assembles into virus-like particles (VLP) which are antigenically and immunogenically indistinguishable from native virions. When the N-terminal 30 amino acid residues of VP60 were deleted and substituted by a well characterized six residue epitope from bluetongue virus capsid protein VP7 (Btag), the fusion protein retained its ability to self-assemble into VLPs. However, the size of these particles was only 27 nm, compared to 40 nm of VLPs derived from native VP60. The antigenicity of both VP60 and the Btag was retained as evident from ELISA and Western blot analyses. When Btag was fused at the C-terminus of VP60 without deletion, the fusion proteins formed VLPs of 40 nm in size and also retained their antigenicity, but the Btag antigenicity appeared weak at this fusion site.

Expression of the major inner capsid protein of the epizootic haemorrhagic disease virus in baculovirus and potential diagnostic use.

The RNA 7 encoding the major capsid protein (VP7) of epizootic haemorrhagic disease virus (EHDV), Australian serotype 2 (strain CS439), was cloned and the complete nucleotide sequence was determined. The coding region contained 1047 nucleotides (nt) capable of encoding a predicted 349 amino acid (aa) polypeptide with a calculated molecular size of 38.087 kDa. When the VP7 gene was expressed in bacterial or yeast expression systems, the expression product showed weak or no reactivity with polyclonal antibodies to EHDV. Therefore, the expression of the VP7 gene in baculovirus was pursued. The expressed EHDV VP7 was similar in antigenicity to that of the native virus as revealed by its reactivity in ELISA with monoclonal antibody (MAb) specific to EHDV. Preliminary ELISA results indicated that the recombinant protein binds to EHDV antibodies in serum and that these antibodies block the binding of EHDV-specific MAb. The availability of a reliable EHDV recombinant VP7 could enhance our existing assay for detection of EHDV-specific antibodies.

Application of linker-ligation-PCR for construction of phage display epitope libraries.

An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.

Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus.

Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

Comparative sequence analysis of VP4s from five Australian porcine rotaviruses: implication of an apparent new P type.

VP4s from five Australian porcine rotaviruses (CRW-8, BEN-307, BMI-1, MDR-13, and TFR-41) were sequenced and analyzed. Strains with distinct VP7s were demonstrated to have closely related VP4s similar to OSU. However, MDR-13 which manifested both G3 and G5 specificity was found to contained a unique VP4 which contained 2368 nucleotides, six bases longer than the longest VP4 ever reported.

Analysis of the nucleotide sequence of five genes at the left end of the unique short region of the equine herpesvirus 4 genome.

Eco RI fragment G of equine herpesvirus 4 strain 405/76 (EHV 4.405/76) is located at the left end of the unique short region close to or extending into the internal repeat region of the prototypic arrangement of the genome. The nucleotide sequence of two subclones designated HS and G 19, contiguous within Eco RI fragment G, was determined for each strand by obtaining a nested set of deletion clones of these double-stranded DNA plasmids. Analysis of the nucleotide sequence revealed that the two subclones contain 5449 base pairs with four complete open reading frames (ORFs) and part of a fifth ORF. Comparison of the predicted amino acid sequences of these reading frames showed that they correspond to ORFs 67, 68, 69, 70, and 71 of equine herpesvirus type 1 (EHV 1) [41], of which ORFs 68, 69, and 70 are homologous to human herpes simplex virus (HSV) genes in the unique short (US) region, i.e., US 2, US 3, and US 4. ORF 67' of EHV 4 and ORF 67 of EHV 1 are homologous (65.7%) but these genes have no homologue in HSV 1.

Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure.

A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained and used to construct restriction maps for four restriction endonucleases. Hybridization experiments indicated that the EHV5.2-141 genome does not contain large terminal or internal repeats, although some evidence for very short repeated sequences in the genomic termini was obtained. Such a genome structure makes EHV5 unique among the equine herpesviruses but similar to the mouse, rat, and guinea pig cytomegaloviruses and the tupaiid herpesvirus. Sequence analysis of one of the genomic termini of EHV5.2-141 revealed the presence of a 30-bp sequence (pac-1; Deiss et al. (1986) J. Virol. 59, 605-618) which is highly conserved among herpesviruses.

A variant serotype G3 rotavirus isolated from an unusually severe outbreak of diarrhoea in piglets.

About 80% of faecal samples from severe outbreak of porcine diarrhoea (scours) were positive for rotavirus. Rotavirus positive samples were analyzed for their antigenic properties and amino acid sequences of the glycoprotein genes. These viruses could not be assigned to any serotypes using serotyping monoclonal antibodies (MAbs) developed for porcine rotaviruses [Nagesha and Holmes: Journal of Medical Virology 35:206-211, 1991b]. When two such viruses were isolated in cell culture and analyzed by neutralization tests using hyperimmune sera they showed only one way antigenic relation with both human and porcine viruses belonging to serotype G3. In addition none of the serotyping MAbs neutralized these two virus isolates. There was no base variation between VP7 genes of faecal and cell culture isolates. Predicted amino acid sequences of the VP7 gene showed marked epitope variation from other porcine type G3 isolates with amino acid substitutions and an additional glycosylation site at residue 238. This antigenic variation seen in rotaviruses appears similar to that of influenza viruses undergoing antigenic drift.

Identification of equine herpesvirus 4 glycoprotein G: a type-specific, secreted glycoprotein.

Equine herpesvirus 4 (EHV4) glycoproteins of M(r) 63K and 250K were identified in the supernatant of infected cell cultures. The 63K glycoprotein was type-specific; that is, it reacted with monospecific sera from horses that had been immunized or infected with EHV4, but not with monospecific sera from horses immunized or infected with EHV1, a closely related alphaherpesvirus. It was postulated that the secreted protein may be the homologue of similarly secreted glycoproteins of herpes simplex virus 2 glycoprotein G (HSV2 gG) and pseudorabies virus (PRV) gX, which is the homologue of HSV2 gG. The US region of the EHV4 genome, toward the internal repeat structure, was sequenced. Four open reading frames (ORFs) were identified of which ORF4 showed 52% similarity to the gene-encoding PRV gX in a 650-nucleotide region. ORF4 coded for a primary translational product of 405 amino acids which has a predicted size of 44K. The amino acid sequence of ORF4 showed 28% identity with PRV gX and 16% identity with HSV2 gG, although significantly greater identity was observed in the N-terminal region including the conservation of 4 cysteine residues. Accordingly, we designate ORF4 as EHV4 gG. The predicted amino acid sequence of the EHV4 gG showed characteristics of an envelope glycoprotein. Expression of the entire EHV4 gG gene in the bacterial expression vector pGEX-3X produced a type-specific fusion protein of M(r) 70K of which the gG portion composes 43K. Antibody that was affinity purified from selected portions of Western blots containing the 70K gG fusion protein reacted with the 63K secreted glycoprotein. Conversely, antibody affinity purified to the 63K secreted product reacted with the 70K gG fusion protein. These results showed that the EHV4 63K secreted glycoprotein was EHV4 gG, the third alphaherpesvirus gG homologue known to be, at least in part, secreted. The type-specificity of this glycoprotein provides, for the first time, the opportunity to differentiate between antibodies present in polyclonal sera from EHV4, EHV1, and dual-infected horses and this has important implications for understanding the epidemiology of these viruses.

Molecular and serological analyses of two bovine rotaviruses (B-11 and B-60) causing calf scours in Australia.

Fecal specimens from 78 calves involved in outbreaks of calf diarrhea which occurred in three farms in Victoria, Australia, in 1988 were analyzed for rotaviruses. Thirty-eight samples were positive for group A virus antigen by enzyme-linked immunosorbent assay, and 20 of these contained viral double-stranded RNAs that could be detected by polyacrylamide gel electrophoresis. Two major electropherotypes could be observed, and a representative isolate of each electropherotype (isolates B-11 and B-60) was successfully adapted to grow in MA104 cells. Sequencing of the VP7 genes directly from RNA transcripts of fecal and cell culture-adapted viruses demonstrated that no base changes occurred in this gene upon adaptation to growth in MA104 cells. Sequencing also revealed that the VP7 protein of B-60 was closely related to G serotype 6 (G6) strains, whereas the B-11 sequence was significantly different from all previously published sequences except the recently reported VP7 sequences of bovine isolates 61A and B223, particularly across the antigenic regions A, B, and C. The other strains most closely related to B-11 by VP7 amino acid sequence analysis were G4 porcine strains BMI-1 and BEN-144 and G8 human strain 69M. Serotyping of B-11 and B-60 gave results that were in good agreement with the sequencing data. Hyperimmune typing sera clearly identified B-60 as a member of G6, whereas the B-11 strain reacted to moderate titers only with antisera to some G10 strains. Antiserum raised against B-11 neutralized some strains of G10 cross-reacted with porcine G4 type isolates BMI-1 and BEN-144 but not with other G4 strains or with rotaviruses of other mammalian G serotypes. Northern blot hybridization showed that B-11 was closely related to the recently reported bovine G10 strain B223, and they both possessed a similar segment 4 that was different from that of either UK bovine or NCDV rotavirus.

Cloning and restriction endonuclease mapping of the genome of an equine herpesvirus 4 (equine rhinopneumonitis virus), strain 405/76.

Purified virion DNA of an Australian isolate of equine herpesvirus 4(EHV 4.405/76) was digested with restriction enzymes and the DNA fragments were cloned into pUC19. The resulting recombinant plasmid library, representing 92% of the virus genome, was used in hybridization analyses to construct restriction maps for BamHI, EcoRI, and SalI for the EHV4 genome. The results show that the genome of EHV 4.405/76 was approximately 145 kb and comprised a unique long (UL) region of 112 kb and a unique short (US) region of 12.4 kb. US is flanked by an internal and terminal repetitive sequence (IRS and TRS) of about 10.3 kb. The BamHI and EcoRI restriction maps are similar to those previously published for an English isolate EHV 4.1942 strain although some differences such as location of an additional fragment and changes in positions of two other small fragments were found.

Direct serotyping of porcine rotaviruses using VP7-specific monoclonal antibodies by an enzyme immunoassay.

Employing a serotyping EIA test using MAbs both cell culture adapted and faecal porcine rotaviruses were classified into serotypes G3, G3/5, G4, and G5. The MAbs have confirmed and extended the serotyping results obtained using polyclonal antisera. These MAbs are therefore potential reagents for serotyping of porcine rotaviruses. Using subgroup specific MAbs serotypes G3, G3/5, and G5 were found to contain subgroup I antigens while G4 rotaviruses contained either subgroup II or subgroup I antigens.

VP4 relationships between porcine and other rotavirus serotypes.

VP4 relationship of Australian porcine rotaviruses were identified using genetic reassortants and MAbs. All porcine virus isolates except BEN-144 appeared to share VP4 antigenicity with OSU virus. VP4 and BEN-144 virus (Gottfried-like virus) showed some antigenic relationships with the human neonatal viruses ST-3 and RV-3. In addition, VP4 of porcine CRW-8 showed antigenic relationships with simian SA-11. RRV and also canine K9 viruses, while that of porcine TFR-41 showed at least one way VP4 antigenic relatedness with UK bovine rotavirus. Furthermore, BMI-1 virus which is antigenically similar to an American virus SB1-A (a naturally occurring reassortant) may have arisen similarly by gene reassortment in nature in Australia.

A porcine rotavirus strain with dual VP7 serotype specificity.

Porcine rotavirus MDR-13, which on original isolation showed a two-way antigenic relationship with human rotavirus RV-3, shows VP7 relationships with serotype G5 as well as G3 viruses upon gene reassortment. Analysis of porcine MDR-13 and the MD-UK reassortant revealed marked nucleotide and amino acid similarity of VP7 genes of these viruses with those of both serotype G3 and G5 viruses. Evolution of such a strain, possibly by sequential mutations in the VP7 gene, is discussed.

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.