Culex flavivirus isolates from mosquitoes in Guatemala.

A new strain of Culex flavivirus (family Flaviviridae, genus Flavivirus, CxFV), an insect virus first described in Japan, was isolated from adult Culex quinquefasciatus Say (Diptera: Culicidae) collected in 2006 from Izabal Department on the Caribbean coast of Guatemala. Mosquito pools were assayed for flavivirus RNA by using flavivirus group-specific primers that amplified a 720-bp region of the nonstructural (NS) 5 gene by standard reverse transcriptase-polymerase chain reaction. From 210 pools (1,699 mosquitoes), eight tested positive, and six of these mosquito pools produced virus isolates in Aedes albopictus Skuse C6/36 cells. Nucleotide sequence comparison of the eight flavivirus RNA-positive pools showed that there was 100% identity among them, and phylogenetic analysis of the NS5 and envelope gene regions indicated that they represent a strain of the recently described CxFV from Japan. This is the first report of an insect flavivirus from Central America.

Use of the squirrel monkey Saimiri sciureus to optimize serological tests for diagnosis of Bunyavirus infection in humans.

Several arthropod-borne viruses of the large Bunyaviridae virus family have been isolated in South America. There are few data about the incidence of these viruses in man, except for the Oropuche virus. Since haemagglutination inhibition tests are difficult to perform, only enzyme-linked immunosorbent assays (ELISAs) are used. Nevertheless, positive controls are necessary for ELISA, and infected humans are rare. Squirrel monkeys (Saimiri sciureus) were therefore infected experimentally to assess their value as positive controls in such assays. The kinetics of viraemia and of antibody responses after infection with eight Bunyaviruses present in the Amazonian forest were studied. No viraemia was seen in most cases, but, with every virus studied, immunoglobulin (Ig)M and IgG antibody responses were observed, beginning between days 5 and 14 after infection for IgM and days 14--18 after infection for IgG. This model thus provides reliable positive controls for ELISAs in humans. Their availability will allow determination of the seroprevalence of Bunyaviruses in the human population of French Guiana.

Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton.

Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites.

Plasmodium falciparum: immune pressure in Saimiri sciureus monkeys can select for a parasite population inducing a protective immunity that is not controlled by antibody.

Protective immunity against a Plasmodium falciparum blood infection can be passively transferred by antibodies in humans and in the primate experimental malaria model Saimiri sciureus. We report here the emergence of a novel virulent parasite population after such passive transfer of hyperimmune serum in splenectomized monkeys. These FUP-2 parasites have been partially genotyped and phenotyped. Although no genotypic variation was detected for four polymorphic loci compared to the original FUP-1 parasite population, FUP-2-infected erythrocytes exhibit little or no detectable surface determinants, including those reacting with antibodies raised against FUP-1 surface antigens. In addition, FUP-2-infected erythrocytes exhibit no rosetting or autoagglutination. Interestingly, although Saimiri monkeys control efficiently FUP-2 parasites after repetitive infections, this protection cannot be passively transferred to naive recipients. Our results suggest that antibody-mediated and antibody-independent T-cell-mediated protective responses may cooperate in controlling P. falciparum infection in splenectomized Saimiri monkeys.

Non-detergent sulphobetaines enhance the recovery of membrane and/or cytoskeleton-associated proteins and active proteases from erythrocytes infected by Plasmodium falciparum.

A better understanding of the causative agent's biology and the definition of new targets for the development of drugs and/or specific immune responses is necessary to face the spred of drug-resistant malaria in developing countries and the absence of an efficient vaccine against this most important infectious disease. Non-detergent sulphobetaines enhance the recovery and isoelectric focussing of active Plasmodium falciparum proteases, cytoskeleton-associated proteins and Maurer's cleft-associated proteins. This is a significant advantage for the purification of such proteins and might help pinpoint their role for red blood cell rupture and merozoite release.

Plasmodium falciparum: altered expressions of erythrocyte membrane-associated antigens during antigenic variation.

The O and R antigenic variants of the Plasmodium falciparum Palo Alto strain present differences in the morphology of the infected red blood cell membrane, in their adhesion properties, surface immunofluorescence, and agglutination specificities and importantly, induce a variant-specific protection after a primary infection in Saimiri sciureus monkeys. To identify potential targets of variant-specific immunity, we have compared the antigenic makeup of both variants by immunoblot. O-specific monkey sera generated similar profiles on both parasite types, while R-specific sera showed a consistent difference on a high-molecular-mass undefined antigen. Distinct antibody specificities were eluted from the surface of O- or R-infected erythrocytes, generating variant-specific agglutination, surface immunofluorescence, and immunoblot profiles. An antiserum raised to Pf60.1, predicted to cross-react with the cytoplasmic domain of PfEMP1, reacted with specific, SDS-soluble antigens in both variants. Antigens associated with the membrane of the infected red blood cells were further investigated using several specific antisera. The 85-kDa HRP1 gene product was more abundant in O than in R parasites, while the reverse was observed for the PfEMP3 protein. These data indicate that O and R parasites differ in the expression of several antigens associated with the membrane of the infected red blood cell.

Malaria parasites: enzymes involved in red blood cell invasion.

Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a preformed serine-protease active site but no detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erythrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented.

Malaria parasites: enzymes involved in red blood cell invasion

Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a performed serine-protease active site but not detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erytrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented