Distribution by Scientific Domains
Distribution within Medical Sciences

Kinds of Post-infection

  • day post-infection
  • h post-infection
  • week post-infection

  • Selected Abstracts

    Correction: IL-10 is crucial for the transition from acute to chronic disease state during infection of mice with Schistosoma mansoni

    C. H. Sadler
    Vol. 33(4) 2003, pp 880-888 Pages 882 (Fig. 2) and 883 (Fig. 5) The x-axis label in Fig. 2 should have the same sampling times post-infection as Fig. 1. The legend to Fig. 5 should be amended to read: blue nuclei, red collagen and yellow connective tissue or hepatic parenchyma. [source]

    Transient viral-mediated overexpression of ,-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of ,-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence

    B. F. Singer
    Abstract Calcium/calmodulin-dependent protein kinase II (CaMKII) activity is necessary for the long-lasting expression of locomotor sensitization and enhanced drug-taking observed in rats previously exposed to psychostimulants. Exposure to these drugs also transiently increases ,CaMKII levels in the nucleus accumbens (NAcc), an effect that, when mimicked by transient viral-mediated overexpression of ,CaMKII in NAcc shell neurons, leads to long-lasting enhancement in locomotor responding to amphetamine and NAcc ,-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA). The present experiments characterized the dopamine (DA) dependence of the functional AMPA receptor upregulation observed long after transient overexpression of ,CaMKII. Rats infected with herpes simplex virus-,CaMKII in the NAcc shell showed a transient increase in ,CaMKII levels that peaked at 4 days post-infection and returned to baseline 8 days later. When challenged with AMPA (0.8 nmol/side) in the NAcc shell at 20 days post-infection, these rats showed enhanced locomotion compared with controls. This sensitized locomotor response was blocked when AMPA was coinfused with either the DA type-1 receptor antagonist SCH23390 (0.8 nmol/side) or the protein kinase A inhibitor Rp-cAMPS (80 nmol/side). Neither SCH23390 nor Rp-cAMPS produced locomotor effects when infused by itself into the NAcc shell. Furthermore, these antagonists did not block the acute non-sensitized locomotor response to AMPA observed in control rats. These findings show that transient viral-mediated overexpression of ,CaMKII in neurons of the NAcc shell leads to long-lasting functional upregulation of AMPA receptors that is DA type-1 receptor and protein kinase A dependent. Thus, transient increases in levels of ,CaMKII in the NAcc shell produce long-lasting changes in the way that DA and glutamate interact in this site to generate locomotor behavior. [source]

    Analysing scots pine defence-related transcripts and fungal DNA levels in seedlings single- or dual-inoculated with endophytic and pathogenic Rhizoctonia species

    FOREST PATHOLOGY, Issue 6 2009
    H. Grönberg
    Summary Fungal DNA and induction of host defence-related transcripts were monitored by real-time PCR in young Scots pine seedlings inoculated with pathogenic uninucleate (UNR) and endophytic binucleate (BNR) Rhizoctonia species. The UNR (teleomorph Ceratobasidium bicorne) causes root dieback in conifer seedlings following invasion of the vascular cylinder via root apex and destroying apical meristems whilst the BNR, representing anastomosis group AG-I of genus Ceratobasidium, is primarily restricted to the cortex in basal root regions. In the experiment 1 the fungi were simultaneously inoculated on roots, while in experiment 2, BNR was pre-inoculated 168 h before inoculation with UNR. Nucleic acids were extracted from infected roots at intervals up to 192 h post-infection (hpi), and the genomic DNA levels of the host and fungi and the transcript levels of a house-keeping gene (glyceraldehyde-3-phosphate dehydrogenase) and nine putative defence genes were quantified. In simultaneous inoculation UNR was more competitive than BNR whereas pre-inoculation of BNR suppressed but did not completely prevent root colonization by UNR. Stilbene synthase (STS) transcription was significantly up-regulated in single-inoculations with both fungi and in dual inoculation in both experiments. Maximum STS transcript levels were observed in roots single-inoculated with UNR; the peak level at 48 hpi in experiment 2 was significantly higher than in seedlings single-inoculated with BNR or co-inoculated with both fungi, the latter two treatments showing relatively similar STS transcript levels. Similarly, transcript levels of phenylalanine ammonia lyase at 48 hpi in experiment 2 were significantly higher in roots single-inoculated with UNR compared with BNR or in UNR+BNR co-inoculations. The other seven putative defence genes monitored did not show any clear-cut up-regulation following fungal inoculation. We conclude that BNR suppresses UNR in Scots pine roots via direct competition for infection sites, since the studied transcripts showed no evidence of BNR induced resistance against UNR. [source]

    Problem of Distinguishing False-Positive Tests from Acute or Transient Helicobacter pylori Infections

    HELICOBACTER, Issue 2 2006
    Zhannat Z. Nugalieva
    Abstract Background:, Reliable detection of acute Helicobacter pylori infections remains problematic. The high prevalence of false-positive non-invasive tests in low H. pylori prevalence populations makes identification of acute and transient infections difficult. Methods:, We explored the use of serum pepsinogens (PG) for diagnosis of acute infection in patients following H. pylori challenge such that the onset of the infection was known. We then compared those findings to a group of children with presumed acute infections defined as a positive urea breath test (UBT) and negative IgG serology. Results:, We examined the pattern and calculated cut-off values of PG levels in 18 adult volunteers with known acute H. pylori infection. We then compared the results with sera from nine symptomatic children with presumed acute H. pylori infection and a matched control group of nine children who did not meet criteria for acute H. pylori infection. In acute infection, both PGI and II levels increased following H. pylori infection reaching a peak by 2 weeks post-infection. The frequency of a positive test defined as a value > mean +2 SD was 17, 71, and 94% at week 1, 2, and 4 post-infection, respectively. Only one child with presumed acute H. pylori infection had an elevated serum PGI and one had an elevated PGII. Five of the children had follow-up UBTs and four were negative consistent with the diagnosis of false-positive UBT. H. pylori infection was confirmed in the child with an elevated PGI level. Conclusions:, These data suggest that a single positive noninvasive test in populations of low prevalence is most likely a false-positive result. This suggests that a single positive test requires confirmation preferably using a test that measures a different parameter (e.g., UBT confirmed by stool antigen test). It appears that most "transient"H. pylori infections are diagnosed on the basis of false-positive tests. PG levels are possible candidates as the confirmatory test. [source]

    Murid herpesvirus-4 induces chronic inflammation of intrahepatic bile ducts in mice deficient in gamma-interferon signalling

    Babunilayam Gangadharan
    Aim:, Infection of gamma interferon receptor defective mice with murid herpesvirus-4 also known as murine gammaherpesvirus-68 results in multi-organ fibrosis. In this paper we characterise the pathological changes occurring in the liver in this model. Methods:, Standard immunohistochemistry and in situ hybridisation techniques were used to identify the cellular changes and the presence of virus at different times post infection. Results:, In liver sections from infected gamma interferon receptor defective mice sampled on day 16 to at least day 120, 79% showed proliferating intrahepatic bile ducts associated with a chronic mononuclear cell inflammation. Only 8% of wild type mice showed similar lesions. Coincident with the inflammatory response bile duct epithelial cells were positive for arginase 1. Around day 50 post infection onwards focal fibrotic lesions appeared in approximately 30% of gamma interferon receptor defective mice resulting in destruction of intrahepatic bile ducts. In contrast to the chronic persisting inflammatory response the presence of virus infected cells were only observed between day 12,20 post-infection. Conclusion:, Infection of gamma interferon receptor defective mice with a murine gammaherpesvirus initiates a chronic persisting inflammatory response with a pathological profile similar to the human fibrotic liver disorder Primary Sclerosing Cholangitis. [source]

    Antibody response to influenza infection of mice: different patterns for glycoprotein and nucleocapsid antigens

    IMMUNOLOGY, Issue 4 2003
    Robert Sealy
    Summary Our previous studies of C57BL/6 mice intranasally infected with influenza virus (A/PR8) revealed a spike of virus-specific immunoglobulin A (IgA)-secreting antibody-forming cells (AFC) in the mediastinal lymph node (MLN) 7 days post-infection. Here we show that these AFC are directed only against viral glycoprotein, and not nucleocapsid antigens. The early IgA spike associates with a decline in glycoprotein-specific AFC during week 2 post-infection. In contrast to the glycoprotein-specific AFC, nucleocapsid-specific, IgA-secreting AFC develop gradually in the MLN and persist for more than 3 weeks post-infection. As peripheral lymph node reactions wane, the nucleocapsid-specific AFC appear as long-sustained populations in the bone marrow. Microanatomical examination of the respiratory tract in infected mice shows foci of infection established in the lung 2 days post-infection, from which virus spreads to infect the entire lining of the trachea by day 3. At this time, viral haemagglutinin can be seen within the MLN, probably on projections from infected dendritic cells. This feature disappears within a day, though viral antigen expression continues to spread throughout the respiratory tract. Total IgA- and IgG-secreting AFC appear histologically in large numbers during the first week post-infection, significantly preceding the appearance of germinal centres (revealed by peanut agglutinin staining in week 2). To explain these results, we suggest that the initial immunogenic encounter of B cells with viral antigens occurs about 3 days post-infection in the MLN, with antigens transported by dendritic cells from airway mucosa, the only site of viral replication. Viral glycoproteins expressed as integral membrane components on the surface of infected dendritic cells [probably in the absence of cognate T helper (Th) cells] promote members of expanding relevant B-cell clones to undergo an IgA switch and terminal local plasmacytoid differentiation. Anti-glycoprotein specificities are thus selectively depleted from progeny of activated B-cell clones which are channelled to participate in germinal centre formation (perhaps by cognate T helper cells when they become sufficiently frequent). One product of the germinal centre reaction is the long-sustained, bone marrow-resident population, which is accordingly rich in anti-nucleoprotein, but not anti-glycoprotein specificities. Of note, we find that AFC responses toward influenza virus and Sendai virus differ, even though viral replication is limited to the airway mucosa in each case. The response towards Sendai virus exhibits neither the early appearance of anti-glycoprotein AFC expressing IgA in draining lymph nodes, nor the subsequent relative deficit of this specificity from bone marrow AFC populations. [source]

    Expression and immunocytochemical analysis of Autographa californica nucleopolyhedrovirus (AcMNPV) orf74 gene

    INSECT SCIENCE, Issue 5 2006
    Abstract Autographa californica nucleopolyhedrovirus orf74 (Ac74) is located between 62 311 and 63 108bp in the AcMNPV genome, which encodes 265 amino acid residues with a predicted 31 kDa molecular weight. The homologues of Ac74 were searched using BLASTP in protein databases, GenBank/EMBL and SWISS-PROT. The result revealed that deduced Ac74 protein was homologous to the predicted products from 10 lepidoptera NPV ORFs. The multiple sequence alignments of Ac74 and its 10 homologues manifested only one amino acid residue was completely conserved. The transcript analysis revealed that the transcript of Ac74 was detected from 24,72 hours post-infection (hpi). The product of Ac74 was detected at 24 hpi and lasted until 72 hpi by Western blot using anti-Ac74 antiserum, consistent with reverse transcriptase polymerase chain reaction results. These results suggested Ac74 was expressed during the later stages of infection. The product of Ac74 was 31 kDa in size, consistent with predicted molecular weight. The subcellular localization of Ac74 proteins manifested Ac74 protein in the cytoplasm, and was hardly present in the nucleus at 24 hpi. The fluorescence was also observed in polyhedra, except cytoplasm at 72 hpi. Together, Ac74 is a functional protein with 3 1kDa molecular weight and is located in the cytoplasm and the polyhedra. [source]

    Experimental acute respiratory Burkholderia pseudomallei infection in BALB/c mice

    Mark S. Lever
    Summary Burkholderia pseudomallei is the causative agent of melioidosis, which is considered a potential deliberate release agent. The objective of this study was to establish and characterise a relevant, acute respiratory Burkholderia pseudomallei infection in BALB/c mice. Mice were infected with 100 B. pseudomallei strain BRI bacteria by the aerosol route (approximately 20 median lethal doses). Bacterial counts within lung, liver, spleen, brain, kidney and blood over 5 days were determined and histopathological and immunocytochemical profiles were assessed. Bacterial numbers in the lungs reached approximately 108 cfu/ml at day 5 post-infection. Bacterial numbers in other tissues were lower, reaching between 103 and 105 cfu/ml at day 4. Blood counts remained relatively constant at approximately 1.0 × 102 cfu/ml. Foci of acute inflammation and necrosis were seen within lungs, liver and spleen. These results suggest that the BALB/c mouse is highly susceptible to B. pseudomallei by the aerosol route and represents a relevant model system of acute human melioidosis. [source]

    Effect of undernourishment on Herpes Simplex Virus Type 1 ocular infection in the Wistar rat model

    Abstract. ,We have studied the susceptibility to Herpes Simplex Virus Type 1 (HSV-1) infection in malnourished rats. Groups of 10 rats were undernourished during suckling by offspring duplication. The animals were put on commercial diet and at 1, 2, 3, 5 and 8 weeks after weaning, infected in the eye by scarification with HSV-1, strain F. Significant differences in morbidity and mortality were observed between malnourished and control groups infected three weeks after weaning. Viral titres were higher in ocular washings and brains obtained from the malnourished group. This group showed a diminution in antigen dependent lymphocyte proliferation compared to control, and significantly lower delayed type hypersensitivity reaction against inactivated virus (malnourished = 0.16 ± 0.02 mm, control = 0.26 ± 0.03 mm, p < 0.05). Neutralizing antibodies in serum were lower in the malnourished group and lower levels of interferon were obtained in the malnourished group 24 h post-infection. We conclude that malnutrition during suckling induces a delay in the capability to overcome HSV infection. [source]

    Morphological features of Murray Valley encephalitis virus infection in the central nervous system of swiss mice

    Vance Matthews
    We have examined the histological and ultrastructural features of CNS infection with Murray Valley encephalitis (MVE) virus in mice inoculated with a virulent parental strain (BH3479). Light microscopic examination revealed neuronal necrosis in the olfactory bulb and hippocampus of MVE-infected brains by 5 days post-infection (pi). Electron microscopy of these regions showed endoplasmic reticulum membrane proliferation, and tubular and spherical structures in the cisternae of the endoplasmic reticulum, Golgi complex and nuclear envelope. At seven to eight days pi, infected neurones exhibited chromatin condensation and extrusion, nuclear fragmentation, loss of segments of the nuclear envelope, reduced surface contact with adjacent cells and loss of cytoplasmic organelles. This cell injury was particularly noticeable in the proximal CA3 and distal CA1 regions of the hippocampus. The inflammatory cell profile consisted of macrophages, lymphocytes and especially neutrophils, and many of these inflammatory cells were apoptotic. High mortality rates in the BH3479-infected population of mice correlated with the intense polymorphonuclear and mononuclear leucocyte inflammatory infiltrate in the CNS. [source]

    Strain-dependent activation of the mouse immune response is correlated with Porphyromonas gingivalis -induced experimental periodontitis

    Asaf Wilensky
    Abstract Aims: To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response. Materials and Methods: Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti- P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model. Results: The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups (p<0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 (p0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1, levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2±260.0, p<0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8±131.6, p<0.05). Conclusions: The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response. [source]

    Alternative measures of response to Pseudomonas aeruginosa infection in Drosophila melanogaster

    Abstract Studies of invertebrate immune defence often measure genetic variation either for the fitness cost of infection or for the ability of the host to clear the parasite. These studies assume that variation in measures of resistance is related to variation in fitness costs of infection. To test this assumption, we infected strains of the fruit fly, Drosophila melanogaster, with a pathogenic bacterium. We then measured the correlation between host bacterial load and the ability to survive infection. Despite the presence of genotypic variation for both traits, bacterial load and survival post-infection were not correlated. Our results support previous arguments that individual measures of immune function and the host's ability to survive infection may be decoupled. In light of these results, we suggest that the difference between tolerance and resistance to infection, a distinction commonly found in the plant literature, may also be of value in studies of invertebrate immunity. [source]

    Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus

    P P Chiou
    Abstract We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer, muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process. [source]

    Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

    J Melena
    Abstract Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0,1 × 103 copies ,g,1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0,2 × 103 copies ,g,1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response. [source]

    Experimental transmission of sleeping disease in one-year-old rainbow trout, Oncorhynchus mykiss (Walbaum), induced by sleeping disease virus

    S Kerbart Boscher
    Abstract Sleeping disease (SD) is a serious disease of rainbow trout, Oncorhynchus mykiss, reared in fresh water caused by sleeping disease virus (SDV). In this study a detailed clinical, histological, virological and serological description of the experimental reproduction of SD in 1-year-old rainbow trout exposed to SDV was carried out. Two hundred disease-free fish were intraperitoneally inoculated with a SDV isolate and 100 fish were inoculated with an uninfected cell culture lysate as a negative control. Infected and control fish were randomly removed at days 4, 7, 14, 21, 42 and 70 post-infection. Blood and tissues were collected for virus isolation, histopathological examination and serum neutralization. SDV was detected in serum, kidney and brain of infected fish from 4 to 21 days post-infection (dpi). Characteristic pathological lesions were observed in infected fish as early as 7 dpi. Lesions were first detected in exocrine pancreas and subsequently observed in heart and skeletal muscle. Neutralizing antibodies to SDV were detected in infected fish from 14 to 70 dpi. Infected fish displayed typical signs of SD 1-month pi and the mortality reached 18.7% within 44 days. This study experimentally reproduced all the pathognomonic features of natural outbreaks of SD in 1-year-old rainbow trout. [source]

    Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

    J-R Hong
    Abstract In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 °C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 °C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 °C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 °C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 °C) compared with the non-permissive temperature of 28 °C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis. [source]

    Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique

    JOURNAL OF FISH DISEASES, Issue 11-12 2003
    A S Holzer
    Abstract Sphaerospora truttae is an important pathogen of Atlantic salmon parr in Scottish aquaculture. To trace the early development of S. truttae and to overcome the common problem of detecting low numbers of cryptic, early myxosporean stages, a DNA-based approach was applied in this study. Specific primers were designed for S. truttae from Atlantic salmon, based on 18S rDNA sequences, obtained from isolated myxosporean spores. These were 5, biotin-labelled and used in an optimized and rapid in situ hybridization (ISH) protocol, which provided a strong and specific signal of the parasite within host tissue sections and, at the same time, minimized structural damage to tissues due to processing. This methodology provided a reliable tool enabling the detection of S. truttae stages down to single cell level. Using ISH the epithelium of the gills was identified as the predominant entry locus of the parasite. By 3 days after infection S. truttae had penetrated the vascular epithelia and thereafter proliferated in the blood for at least 10 days before exiting the vascular system through capillary walls. From day 12 post-infection onwards, the kidney, as well as the spleen and the liver, were invaded. Numbers of S. truttae invading the kidney (37.3%) differed little from numbers found in the spleen (35.3%) and the liver (27.4%). The latter organs represented a dead end in the development of S. truttae as all stages in these organs degenerated and sporogony was found to take place exclusively inside the renal tubules. Early sporogonic stages were found from day 25 post-infection but mature spores only developed after at least 15 days of proliferation within the tubules. [source]

    Non-specific immune response of turbot, Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagius

    L Villamil
    Abstract The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 × 103 and 5 × 104 bacteria mL,1) and higher doses of the heat-killed bacteria (5 × 104,5 × 106 bacteria mL,1). In both cases, the NO inhibitorN- , -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mm). [source]

    Detection of nodavirus in barramundi, Lates calcarifer (Bloch), using recombinant coat protein-based ELISA and RT,PCR

    The coat protein encoded by the nodavirus RNA2 gene originally isolated from greasy grouper, Epinephelus tauvina, was cloned, expressed as a recombinant polyhistidine-tailed fusion protein and characterized by immunoblot analysis. The purified recombinant protein was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) to detect body exudate and plasma antibodies against the coat protein in both experimentally infected and commercial barramundi. In addition, the nucleotide sequence was employed to develop a RT,PCR detection assay based on the T4 region. The results showed that the virus could be detected as early as 3 days post-infection by RT,PCR while antibodies against the recombinant coat protein were detectable on day 6 post-infection. Among 112 commercial barramundi samples collected from October 1999 to April 2000, 9% showed positive ELISA results which were further verified by Western blot. [source]

    Characterization of grouper nervous necrosis virus (GNNV)

    S C Chi
    Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10,12 with a 4 log10 drop in infectivity. Purified GNNV was analysed by SDS,PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 106 and 0.50 × 106 Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart. [source]

    The effect of temperature and salinity on the settlement and survival of copepodids of Lepeophtheirus salmonis (Krřyer, 1837) on Atlantic salmon, Salmo salar L.

    C S Tucker
    The effects of temperature and salinity on the settlement, subsequent survival and development of the copepodids of Lepeophtheirus salmonis on Atlantic salmon were investigated experimentally. There was a significantly greater settlement and survival of copepodids at 10 days post-infection (dpi) at 12 °C compared with at 7 °C at a constant salinity of 34,. Development of L. salmonis was also more rapid at 12 °C. Settlement was significantly greater at a salinity of 34, than at 24,. In one experiment, survival at 10 dpi was significantly greater at 34,; however, a second experiment found that there was no significant difference between the two saline levels. This may have been because of a rise in water temperature for 2 dpi, which appears to have overridden the effect of low salinity. Development of L. salmonis was more rapid at 34,. Copepodids settled on all of the external surfaces of the salmon, although the proportion on different surfaces varied between experiments. The gills, particularly at low temperatures, the body surface, and the pectoral and dorsal fins were especially favoured. [source]

    Effects of infection with the ectoparasite Argulus japonicus (Thiele) and administration of cortisol on cellular proliferation and apoptosis in the epidermis of common carp, Cyprinus carpio L., skin

    A L Van Der Salm
    The host-parasite interaction between juvenile carp, Cyprinus carpio, and the ectoparasitic branchiuran, Argulus japonicus, together with the role of cortisol in this interaction, was examined at the level of the host skin epidermis. Epidermal mucous cell numbers, and proliferation and apoptosis of the epithelial cells were studied over 32 days. Apoptotic cell numbers in the uppermost epidermis were reduced at 26 days post-infection with A. japonicus, while the other parameters were unaffected. Administration of cortisol-containing food resulted in reduced apoptosis in the cells in the upper skin epidermis at 24 h and at 28 days post-feeding. Cortisol feeding combined with A. japonicus infection reduced numbers of apoptotic cells in the upper epidermis more than either individual treatment. Further, combining the treatments also significantly increased apoptosis in the lower epidermis in cells morphologically identified as leucocytes apparently migrating macrophages and lymphocytes. Using immunohistochemistry, we demonstrated cortisol receptor presence and cellular localization in the teleost epidermis. Receptors only occurred in pavement cells in the upper epidermis and in leucocytes in the lower parts of the epidermis. The ectoparasites, or administered cortisol, induced effects which may be functionally adaptive in the upper pavement cells, while combining the two treatments also induced changes indicative of immunosuppression. [source]

    Longitudinal follow up of SIVmac pathogenesis in rhesus macaques of Chinese origin: emergence of B cell lymphoma

    B. Ling
    Abstract: Two subspecies of rhesus (Rh) macaques, the Chinese (Ch) and Indian (Ind) subspecies were infected intravenously with 100TCID50 SIVmac239. CD4+, CD8+ T cells, plasma viral loads, depletion of intestinal lymphocytes with memory phenotype, humoral immune responses and clinical courses were monitored for 600 days. The pathogenesis of SIVmac was also compared with primary human immunodeficiency virus (HIV) infection of humans. Plasma viral loads in Ch Rh were lower in the acute and chronic phases compared with Ind Rh. SIVmac pathogenesis in Ch Rh was closer to virus loads in untreated HIV infected humans. Ch Rh had higher CD4/CD8 ratios, stronger antibody responses and interestingly, less depletion of intestinal memory CCR5+ CD4+ T lymphocytes compared with Ind Rh. One Ch Rh developed B cell origin lymphoma at 570 days post-infection, the first such report in this subspecies. Three of four Ind Rh developed AIDS within 6 months. The findings indicate that Ch Rh are more resistant to SIVmac pathogenesis compared with Ind Rh and that Ch Rh paralleled HIV-1 infections in untreated adult humans. The SIVmac infected Ch Rh subspecies are an acceptable model for HIV/AIDS. [source]

    A new panel of NS1 antibodies for easy detection and titration of influenza A virus,

    Zhihao Tan
    Abstract The non-structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42,53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross-reactivities. On the other hand, mAb 1G1 binds to residues 206,215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post-infection while the conventional method using cytopathic effect yields results at 3 days post-infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467,475, 2010. © 2010 Wiley-Liss, Inc. [source]

    Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of HIV-1,,

    Kelly A. Curtis
    Abstract HIV diagnosis at the point-of-care or in resource-limited settings poses considerable challenges due to time and cost limitations. Currently, nucleic acid-based tests are the only reliable method for diagnosing recent infections during the window period post-infection and pre-seroconversion, but these tests are only suitable for well-equipped laboratory settings. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology exhibits characteristics that are ideal for the development of a rapid, cost-effective nucleic acid-based test for detection of HIV DNA and RNA. In this study, a sequence-specific detection method was developed for immediate, naked-eye visualization of RT-LAMP products with high sensitivity and specificity. The rapid detection method was incorporated into the HIV-1-specific RT-LAMP assay and validated using minute volumes of whole blood from HIV-1-infected individuals. Together with the minimal sample preparation time and one-step, isothermal amplification reaction, the sequence-specific detection method adds to the overall versatility of the RT-LAMP assay and enhances the applicability for use at point-of-care or resource-limited sites. J. Med. Virol. 81:966,972, 2009. Published 2009 Wiley-Liss, Inc. [source]

    Dendritic cell susceptibility to hepatitis C virus genotype 1 infection

    Maria-Cristina Navas
    Abstract In vitro infection of human monocyte-derived dendritic cells was carried out to study their susceptibility to hepatitis C virus (HCV) infection. Immature dendritic cells and mature dendritic cells were incubated overnight at 37°C with HCV-positive (genotype 1) serum samples; the presence of the viral genome associated with the production of its replicative intermediate was used as evidence of infection. In immature dendritic cells, HCV RNA was detectable from days 1,10 post-infection (p.i.), and de novo synthesis of negative-strand HCV RNA could be demonstrated by a strand-specific rTth reverse transcription-polymerase chain reaction at day 2. In mature dendritic cells, the positive-strand form was detectable from days 1,5 p.i., while the negative-strand HCV RNA appeared at days 1 and 2 p.i. Quasispecies present in the inoculum and 6 days p.i. were analyzed by sequencing hypervariable region 1 of the E2 protein. Only two of seven HVR variants present in the inoculum were found in HCV-infected immature dendritic cells. Another two HVR variants not found in the inoculum were recovered from infected immature dendritic cells, suggesting serum minor variants selection or virus evolution during in vitro replication. Analysis by single-strand conformation polymorphism assay of 5, untranslated region of HCV sequences showed that the patterns obtained from the inoculum and infected immature dendritic cells and mature dendritic cells differed slightly. These findings indicate that both immature dendritic cells and mature dendritic cells are susceptible to HCV genotype 1 infection, supporting at least HCV RNA replication. This model should be a valuable tool for the study of modulation of dendritic cell functions in HCV infection. J. Med. Virol. 67:152,161, 2002. © 2002 Wiley-Liss, Inc. [source]

    Basal replication of hepatitis C virus in nude mice harboring human tumor

    Patrick Labonté
    Abstract Hepatitis C virus (HCV) can infect and propagate in humans and chimpanzees. Whereas the chimpanzee has been used as an animal model for infection, ethical considerations, conservation, and the prohibitively high cost preclude progress for experimental research on the biology of the virus. The development of a small animal model for HCV infection is thus desirable to facilitate studies on the infectious cycle of the virus and for the evaluation of drugs for the treatment of HCV infections in humans. As an alternative to the chimpanzee model, we have established a model based on ex vivo infection of orthotopically-implanted human hepatocellular carcinoma cells (HCC) in athymic nude mice. The results show that up to 42 days post-infection, HCV RNA was present in the tumor cells as well as in the liver and serum of infected mice. Furthermore, a direct correlation between size of the tumor and the presence of HCV RNA in the liver was observed, which is concordant with the finding that HCV RNA was detectable only in mice harboring human tumor. Immunohistochemistry analysis of infected liver specimens showed cells expressing the HCV encoded NS5B protein. A few mice developed a humoral response against the nonstructural viral proteins, providing further evidence for expression of these proteins during viral infection. In summary, these results suggest that mice harboring orthotopic tumors support a basal level of HCV replication in vivo. J. Med. Virol. 66:312-319, 2002. © 2002 Wiley-Liss, Inc. [source]

    Mosquito cells bind and replicate hepatitis C virus

    Raphaële Germi
    Abstract Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells. J. Med. Virol. 64:6,12, 2001. © 2001 Wiley-Liss, Inc. [source]

    Induction of early murine cytomegalovirus infection by different reporter gene-associated recombinant viruses

    U. Drebber
    Summary., Murine cytomegalovirus (MCMV) has provided useful models for acute, chronic and latent CMV infection because of its similarities in structure and biology with human CMV. We report the induction of acute MCMV hepatitis with different bacterial artificial chromosome (BAC)-cloned virus constructs [MCMV-SEAP which includes the gene for secreted alkaline phosphatase (SEAP) under Rous sarcoma virus (RSV)-promoter control, MCMV-GFP which includes the gene for enhanced green fluorescent protein (eGFP) under HCMV-ie promoter control, MCMV-HBs includes the gene for hepatitis B surface antigen (HBsAg) under simian virus (SV)40-promoter control and the DeltaMC95.21 virus in which the m152 gene was deleted and substituted by the reporter gene lacZ] in order to elucidate the histopathological changes together with different reporter-gene products in the liver tissue and the effect of the deletion of a certain gene. All the virus constructs induced a similar mild acute hepatitis which had its climax from days 3 to 5 post-infection in immunocompetent mice. In situ, the reporter-gene products beta-galactosidase and secreted alkaline phosphatase could be visualized in relation to the inflammatory changes. The composition of the invading cell populations did not change even in the absence of the m152 gene. Additionally discrete inflammatory changes were seen in kidney and serosa while the other organs were not involved. This model helps us understand the immunological and histopathological mechanisms of the CMV-induced hepatitis, which plays an important role especially in the immunocompromised patient. The morphological changes can be analysed while the respective reporter gene product is expressed by the virus construct. [source]

    Expression of ,re,y luciferase gene in Erwinia amylovora

    Giovanna Gentilomi
    Abstract In this study we describe an ef,cient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the ,re,y luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our ,ndings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments. [source]