SummaryThe clinical complications associated with severe and cerebral malaria occur as a result of the intravascular mechanical obstruction of erythrocytes infected with the asexual stages of the parasite, Plasmodiumfakiparum. We now report that a primary P.fakiparum-infected erythrocyte (parasitized red blood cell [PRBC]) isolate from a patient with severe complicated malaria binds to cytokineinduced human vascular endothelial cells, and that this adhesion is in part mediated by endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). PRBC binding to tumor necrosis factor o~ (TNF-c0-activated human vascular endothelial cells is partially inhibited by antibodies to ELAM-1 and ICAM-1 and the inhibitory effects of these antibodies is additive. PRBCs selected in vitro by sequential panning on purified adhesion molecules bind concurrently to recombinant soluble ELAM-1 and VCAM-1, and to two previously identified endothelial cell receptors for PRBCs, ICAM-1, and CD36. Post-mortem brain tissue from patients who died from cerebral malaria expressed multiple cell adhesion molecules including ELAM-1 and VCAM-1 on cerebral microvascular endothelium not expressed in brains of individuals who died from other causes. These results ascribe novel pathological functions for both ELAM-1 and VCAM-1 and may help delineate alternative adhesion pathways PRBCs use to modify malaria pathology.

TOC Summary: Forests harboring these mosquitoes may be a reservoir for transmission of P. knowlesi.

Aichi virus is the type species of a new genus, Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5 end of the genome. Computer-assisted prediction of the secondary structure of the 5 end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5 end of the positive strand, but not at the 3 end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5 end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.Aichi virus was first isolated in 1989 from a stool specimen from a patient with oyster-associated nonbacterial gastroenteritis in Aichi, Japan (42). The complete genome sequence of this virus was determined, and the genome organization revealed that this virus is a member of the family Picornaviridae (45). However, the deduced amino acid sequences of Aichi virus proteins exhibited only 15 to 36% homology to those of other picornaviruses, suggesting that Aichi virus belongs to a distinct genus from the previously identified six genera of Picornaviridae (45). In 1999, this virus was classified into a new genus, Kobuvirus (18), whose name is derived from the characteristic morphology of the virus particles (kobu means bump in Japanese). Sequence analysis of 519-base reverse transcription-PCR (RT-PCR) products corresponding to the 3C-3D junction for 17 isolates of Aichi virus revealed that these isolates could be divided into two groups with an approximately 90% sequence homology (46).Aichi virus has often been detected by enzyme-linked immunosorbent assay of stool specimens collected during oysterassociated gastroenteritis outbreaks in Japan. Between 1989 and 1991, 13 of 47 stool samples from adult patients in five of nine oyster-associated gastroenteritis outbreaks were positive for the Aichi virus antigen (44). Aichi virus has also been isolated from Pakistani children with gastroenteritis and from Japanese travelers with gastroenteritis from Southeast Asia (43). These findings suggest that this virus is widely distributed in Asia and that it is one...

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The feasibility of identifying parasite DNA and specific mRNAs from wild caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria. Keywords: Salivary glands, Plasmodium knowlesi, PCR, RT-PCRMalaria transmission occurs in the forested areas in the southern and central provinces of Vietnam (Erhart et al., 2004) and constitutes a considerable public health problem in these areas. In order to fully understand the complexities of malaria transmission, it is important to identify and characterize malaria parasites in mosquito vectors. Evidence for the generation of genetic diversity during sexual proliferation in mosquito vectors has previously been accumulated using laboratory isolates of genetically diverse parasites, as well as with samples from field settings (Babiker et al., 1994; Menegon et al., 2000 knowlesi, were spotted onto chromatography-grade filter papers (ET31CHR;Whatman, Maidstone, UK). Each blood-spotted filter paper was immediately air-dried and stored in a sealed plastic bag at room temperature until RT-PCR or PCR analysis was performed. To examine the detection limit for mosquito salivary gland-extracted parasite mRNA, salivary glands from laboratory reared Anopheles stephensi were prepared in the same way as were the wild-caughtAn. dirus. Salivary glands were dissected from laboratory colony reared An.stephensi more than 1 week after blood feeding on either mouse or human blood and dried on E31CHR filter paper at room temperature. Gametocyte suspensions were obtained from cultured 3D7-9A by pyrimethamine treatment at 10 -6 M from day 7 after thawing until harvest on day 12. Ten times serially diluted cultured gametocyte suspension was added to the spot on the filter paper where the salivary glands were attached. Extraction of RNA and reverse transcription was carried out as previously described (Maeno et al., 2003). Briefly, dried spotted filter paper was cut into small pieces and total RNA and genomic DNA (gDNA) was extracted with ISOGEN (Nippon gene, Tokyo, Japan) according to the manufacturer's instructions. The extracted total RNA was transcribed to synthesize cDNA which was subsequently subjected to PCR using specific The detection limit of parasite mRNA by RT-PCR was one gametocyte per salivary gland for pfg377 mRNA and 10 gametocytes per gland for pfs16 mRNA (Fig. 1A). We extracted both parasite gDNA and mRNA from the salivary glands of wild caught mosquitoes. Region 3 of pfg377 mRNA was detected in all of the three dried salivary gland samples and both the mRNA and gDNA of pfg377 were detected in two samples (Fig. 1B). The PCR products showed the same molecular size as the RT-PCR products by electrophoresis. No PCR product was observed by ...