Infectivity of microsporidia spores stored in water at environmental temperatures

To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.     

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.     

Antigenic diversity of Encephalitozoon hellem demonstrated by subspecies-specific monoclonal antibodies

Encephalitozoon hellem is a unicellular, obligate intracellular microsporidian species detected and isolated in HIV-infected patients presenting with keratoconjunctivitis, sinusitis, tracheobronchitis, nephritis, cystitis, and disseminated infection. A total of 24 monoclonal antibodies were produced against E. hellem and characterized. The monoclonal antibodies were of the immunoglobulin (Ig) G and Ig M subclasses, and, when incorporated into indirect immunofluorescence and immunoblotting assays, reacted against 13 isolates of E. hellem originating from three geographic regions. These monoclonal antibodies did not react with one strain each of either Encephalitozoon intestinalis or Encephalitozoon cuniculi, demonstrating their specificity. Two monoclonal antibodies reacted with all karyotype B-E. hellem isolates but did not react with karyotype A-isolates from North America and the Netherlands, thus demonstrating antigenic diversity among E. hellem isolates. These results add to the increasing evidence for diversity among E. hellem, which therefore may be reclassified into subspecies.     

Molecular evolution of the tick-borne encephalitis and Powassan viruses

The problem of emerging viruses, their genetic diversity and viral evolution in nature are attracting more attention. The phylogenetic analysis and evaluationary rate estimation were made for pathogenic flaviviruses such as tick-borne encephalitis virus (TBEV) and Powassan (PV) circulated in natural foci in Russia. 47 nucleotide sequences of encoded protein E of the TBEV and 17 sequences of NS5 genome region of the PV have been used. It was found that the rate of accumulation of nucleotide substitutions for E genome region of TBEV was approximately 1.4 x 10(-4) and 5.4 x 10(-5) substitutions per site per year for NS5 genome region of PV. The ratio of non-synonymous nucleotide substitutions to synonymous substitution (dN/dS) for viral sequences were estimated of 0.049 for TBEV and 0.098 for PV. Maximum value dN/dS was 0.201-0.220 for sub-cluster of Russian and Canadian strains of PV and the minimum - 0.024 for cluster of Russian and Chinese strains of Far Eastern genotype TBEV. Evaluation of time intervals of evolutionary events associated with these viruses showed that European subtype TBEV are diverged from all-TBEV ancestor within approximately 2750 years and the Siberian and Far Eastern subtypes are emerged about 2250 years ago. The PV was introduced into natural foci of the Primorsky Krai of Russia only about 70 years ago and PV is a very close to Canadian strains of PV. Evolutionary picture for PV in North America is similar to evolution of Siberian and Far Eastern subtypes TBEV in Asia. The divergence time for main genetic groups of TBEV and PV are correlated with historical periods of warming and cooling. These allow to propose a hypothesis that climate changes were essential to the evolution of the flaviviruses in the past millenniums.     

First detection and genotyping of human-associated microsporidia in pigeons from urban parks

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.     

Chronic vasodilation produces plasma volume expansion and hemodilution in rats: consequences of decreased effective arterial blood volume

Plasma volume (PV) expansion is required for optimal pregnancy outcomes; however, the mechanisms responsible for sodium and water retention in pregnancy remain undefined. This study was designed to test the "arterial underfill hypothesis" of pregnancy which proposes that an enlarged vascular compartment (due to systemic vasodilation and shunting of blood to the placenta) results in renal sodium and water retention and PV expansion. We produced chronic vasodilation by 14 days administration of nifedipine (NIF; 10 mg·kg(-1)·day(-1)) or sodium nitrite (NaNO2; 70 mg·kg(-1)·day(-1)) to normal, nonpregnant female Sprague-Dawley rats. Mean arterial pressure, monitored by telemetry, was reduced by both NIF and NaNO2 but was unchanged in control rats. At day 14, vasodilator treatment lowered hematocrit and increased PV (determined by Evans blue dye dilution). Plasma osmolarity (Posm), sodium (PNa), and total protein concentrations all fell. These responses resemble the responses to normal pregnancy with hemodilution, marked PV expansion, and decreased Posm and PNa. Our previous work indicates a role of increased inner medullary phosphodiesterase-5 (PDE5) in the sodium retention of pregnancy. Here, we found that inner medullary PDE5A mRNA and protein expression were increased by both NIF and NaNO2 treatment vs. control; however, neither renal cortical nor aortic PDE5 expression was changed by vasodilator treatment. We suggest that a primary, persistent vasodilation drives increased inner medullary PDE5 expression which facilitates continual renal Na retention causing "refilling" of the vasculature and volume expansion.     

Comparative sequences of two type 1 dengue virus strains possessing different growth characteristics in vitro

The complete genome sequences of two dengue-1 virus strains having different growth characteristics (Mochizuki and A88) were compared with other published strains. The sequence analysis indicated several unique amino acid changes throughout the coding region of Mochizuki strain, mostly in envelope (E) protein. A unique amino acid, Ile-69 for Mochizuki strain at E protein resulted in the loss of an Asn-67-linked glycosylation site. A Thr substitution for Ala-114 at C protein and amino acid changes found in E, non-structural NS3, NS4a, and NS5 proteins were unique for A88 strain. These substitutions might be correlated to their different growth characteristics in vitro.     

DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.     

Fine structure of a new human microsporidian, Encephalitozoon hellem, in culture

Encephalitozoon hellem is a new human microsporidian isolated from corneal biopsies and conjunctival scrapings of three AIDS patients and cultured in Madin Darby canine kidney (MDCK) cells. Encephalitozoon hellem and Encephalitozoon cuniculi display different protein profiles with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and unique antibody binding patterns with murine antisera against Western blots of each organism. Developmental stages of E. hellem in culture are similar to E. cuniculi. Meronts are 1.3-2.7 microns in diameter, develop within a parasitophorous vacuole adjacent to the vacuolar membrane, divide by binary fission, and contain one or two discrete nuclei. Sporonts measure 2 x 3 microns, separate from the vacuolar membrane, and have a thickened outer membrane. Sporoblasts display a tri-layered wall and possess the earliest recognized polar filaments. Mature spores measure 1 x 1.5 microns and are more electron-dense than other stages. Each spore contains a single nucleus, a polar tubule with four to nine coils, thin electron-dense exospore and thick, electron-lucent endospore. Although E. hellem and E. cuniculi differ biochemically and immunologically, their fine structure and development are indistinguishable.     

乙型脑炎病毒结构蛋白C＋pre M和E基因的克隆与序列分析

目的对乙型脑炎病毒（Japanese encephalitis virus,JEV）结构蛋白基因进行扩增、克隆和序列测定。方法根据GenBank中发表的乙脑病毒结构蛋白基因C＋pre M基因和E序列,设计合成两对引物,以JEV Nakayama-NIH株RNA为模板,反转录并扩增目的cDNA,然后与pGEM-T easy载体连接并转化大肠杆菌DH5α菌株,提取的重组质粒用电泳、PCR和酶切鉴定后并进行测序。将JEV Nakayama-NIH与GenBank中报道的多株JEV结构蛋白C＋pre M基因和E基因序列进行了比较。结果Nakayama-NIH与其他参考株C＋pre M和E基因的核苷酸同源性为99%,证实扩增克隆的是JEV的结构蛋白C＋pre M和E基因。结论对JEV分子生物学特征进行了分析,为JEV特异性核酸探针的研制奠定了基础。     

Efficient phagocytosis of Klebsiella pneumoniae strains that poorly bind to human polymorphonuclear leukocytes.

The phagocytosis process of unencapsulated MIAT-negative strains that, although binding very poorly to human polymorphonuclear leukocytes (PMN) at 4 degrees C, are efficiently killed by these cells at 37 degrees C, was studied. At 37 degrees C the number of bacteria bound to the PMN external surface was similar to that observed at 4 degrees C (about 100 bacteria/100 PMN after 60 min); on the contrary the number of internalized bacteria was much higher (from 500 bacteria/100 PMN after 60 min). Interactions between phagocytosis-sensitive Klebsiella pneumoniae strains (PSK) and PMN were then compared with those of two isogenic Escherichia coli strains with and without type 1 fimbriae. Whereas PSK strain binding to blocked PMN was very slow and became significant only after 5-6 h, that of phagocytosis-sensitive fimbriated E. coli was rapid and efficient. Phagocytosis-resistant, non fimbriated E. coli strain bound with an efficiency that, within the first 60 min, was not very different from that of the PSK strains. However, longer incubations led to increases in PSK binding, whereas unfimbriated E. coli remained constant. PSK and fimbriated E. coli strains were efficiently internalized and killed, whereas the unfimbriated E. coli strain was not. It is suggested that PMN can phagocytize unopsonized bacteria through two different mechanisms. By one mechanism, observed with the fimbriated E. coli strain, PMN bind many more bacteria than those they can internalize. By the other, observed with PSK strains, PMN bind only the bacteria they can immediately internalize.     

Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1β and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.     

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.     

研究报告Ⅱ型脊灰病毒抗原表位嵌合蛋白的免疫学研究

目的：评价以轮状病毒（RV）重组VP6蛋白为载体插入Ⅱ型脊髓灰质炎病毒（PV2）VP1蛋白上的1个抗原表位构建而成的嵌合蛋白的体外免疫学性质。 方法：采用分子克隆和基因重组技术将PV2抗原表位插入到RV载体蛋白上,在大肠杆菌中表达并用SDS-PAGE确认表达产物,再通过动物免疫、Western blot、免疫荧光和病毒血清抗体中和试验分析嵌合蛋白的免疫学性质。结果：成功构建了以VP6为载体的PV2抗原表位嵌合蛋白6F/PV₂N₁,并且在E.coli系统中高效表达,嵌合蛋白免疫的豚鼠血清抗体对RV和PV2具备较好的中和活性。结论：以RV VP6为载体构建的嵌合蛋白具有较好的免疫原性,免疫豚鼠产生血清抗体可中和RV和PV2在体外细胞上的感染;进一步为研发RV/PV2嵌合疫苗提供了较好的基础。     

Human immunodeficiency virus type 1 intersubtype (B/E) recombination in a superinfected chimpanzee.

Genetic characterization of a large number of human immunodeficiency virus type 1 (HIV-1) isolates indicates that at least 10% of all strains have mosaic genomes generated by recombination between viruses of the same or different subtypes or clades. What is not known, however, is the time between infection with the first and second HIV-1 strains as well as the time between infection with the second strain and the recombinational event. After 32 months of infection with HIV-1(LAI(IIIB)), a chimpanzee was inoculated intravenously and became infected with a subtype E strain, HIV-1(90CR402). With PCR amplification, DNA heteroduplex analysis, and DNA sequencing, both parental strains and two distinct recombinant proviruses were found in genomic DNA from lymph node tissue obtained 24 weeks after exposure to HIV-1(90CR402). These results show (i) that antiviral immune responses established by long-term infection with an HIV-1 subtype B strain did not prevent infection by a subtype E strain and (ii) that both strains actively replicated and produced sufficient quantities of virus to coinfect the same cell(s), resulting in recombinant viruses.     

Genetic and phenotypic intraspecific variation in the microsporidian Encephalitozoon hellem.

Encephalitozoon hellem is a microsporidian species that causes disseminated infections in HIV-positive patients. Identical genotypes of E. hellem, as assessed by the sequence of the rDNA internal transcribed spacer, have been identified in isolates from humans and from a psittacine bird. However, by analysing the rDNA ITS of four E. hellem isolates from Switzerland (three) and Tanzania (one), two new genotypes were identified. Differences among the E. hellem isolates were also detected by Western blot analysis, but there was no absolute match between ITS genotype and antigen profile. Hence, strain variation exists in E. hellem and the ITS sequence seems a valuable marker in obtaining further insight into the epidemiology of this pathogen.     

Fine Structure of a New Human Microsporidian, Encephalitozoon hellem, in Culture

Encephalitozoon hellem is a new human microsporidian isolated from corneal biopsies and conjunctival scrapings of three AIDS patients and cultured in Madin Darby canine kidney (MDCK) cells. Encephalitozoon hellem and Encephalitozoon cuniculi display different protein profiles with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and unique antibody binding patterns with murine antisera against Western blots of each organism. Developmental stages of E. hellem in culture are similar to E. cuniculi. Meronts are 1.3–2.7 μm in diameter, develop within a parasitophorous vacuole adjacent to the vacuolar membrane, divide by binary fission, and contain one or two discrete nuclei. Sporonts measure 2 × 3 μm, separate from the vacuolar membrane, and have a thickened outer membrane. Sporoblasts display a tri-layered wall and possess the earliest recognized polar filaments. Mature spores measure 1 × 1.5 μm and are more electron-dense than other stages. Each spore contains a single nucleus, a polar tubule with four to nine coils, thin electron-dense exospore and thick, electron-lucent endospore. Although E. hellem and E. cuniculi differ biochemically and immunologically, their fine structure and development are indistinguishable.     

N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.     

Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi.

Peritoneal macrophage activation as measured by H2O2 release and histopathology was compared between Swiss mice and Calomys callosus, a wild rodent, reservoir of Trypanosoma cruzi, during the course of infection with four strains of this parasite. In mice F and Y strain infections result in high parasitemia and mortality while with silvatic strains Costalimai and M226 parasitemia is sub-patent, with very low mortality. H2O2 release peaked at 33.6 and 59 nM/2 x 10(6) cells for strains Y and F, respectively, 48 and 50 nM/2 x 10(6) for strains Costalimai and M226, at different days after infection. Histopathological findings of myositis, myocarditis, necrotizing arteritis and absence of macrophage parasitism were found for strains F and Costalimai. Y strain infection presented moderate myocarditis and myositis, with parasites multiplying within macrophages. In C. callosus all four strains resulted in patent parasitemia which was eventually overcome, with scarce mortality. H2O2 release for strains Y and F was comparable to that of mice-peaks of 27 and 53 nM/2 x 10(6) cells, with lower values for strains Costalimai and M226-16.5 and 4.6 nM/2 x 10(6) cells, respectively. Histopathological lesions with Y and F strain injected animals were comparable to those of mice at the onset of infections; they subsided completely at the later stages with Y strain and partially with F strain infected C. callosus. In Costalimai infected C. callosus practically no histopathological alterations were observed.