Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.     

Neuropathogenesis of influenza virus infection in mice

The neurovirulent WSN strain of influenza A virus, introduced into the olfactory bulb of C57BL/6 mice, selectively attacks several brain nuclei which are highly implicated in the pathogenesis of neuropsychiatric disturbances. The virus-infected neurons are eradicated through apoptotic neurodegeneration. On the other hand, activated microglia serve the neuroprotection against virus infection.     

Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection.

The emergence of influenza A viruses which had acquired resistance to rimantadine during a clinical trial (C. B. Hall, R. Dolin, C. L. Gala, D. M. Markovitz, Y. Q. Zhang, P. H. Madore, F. A. Disney, W. B. Talpey, J. L. Green, A. B. Francis, and M. E. Pichichero, Pediatrics 80:275-282, 1987) provided the opportunity to determine the genetic basis of this phenomenon. Analysis of reassortant viruses generated with a resistant clinical isolate (H3N2) and the susceptible influenza A/Singapore/57 (H2N2) virus indicated that RNA segment 7 coding for matrix and M2 proteins conferred the resistant phenotype. Resistant viruses isolated from seven patients each contained a single change in the nucleotide sequence coding for the M2 protein which resulted in substitutions in amino acid 30 (two viruses) or 31 (five viruses) in the transmembrane domain of the molecule. These changes occurred in locations identified in influenza viruses selected for resistance to amantadine in tissue culture and indicate a common mechanism of action of the two compounds in cell culture and during chemotherapeutic use.     

Mechanisms of cell entry by influenza virus

A wide range of viruses, including many human and animal pathogens representing various taxonomic groups, contain genomes that are enclosed in lipid envelopes. These envelopes are generally acquired in the final stages of assembly, as viruses bud from regions of the membrane of the infected cell at which virally encoded membrane proteins have accumulated. The viruses procure their membranes during this process and mature particles 'pinch off' from the cellular membranes. Under most circumstances, initiation of another round of infection is dependent on two critical functions supplied by the envelope proteins. The virus must bind to cell-surface receptors of a new host cell, and fusion of the viral and cellular membranes must occur to transfer the viral genome into the cell. Enveloped viruses have evolved a variety of mechanisms to execute these two basic functions. Owing to their relative simplicity, studies of binding and fusion using enveloped viruses and their components have contributed significantly to the overall understanding of receptor-ligand interactions and membrane fusion processes - fundamental activities involved in a plethora of biological functions.     

Protection of inactivated influenza virus vaccine against lethal influenza virus infection in diabetic mice

Influenza virus infection frequently causes complications and some excess mortality in the patients with diabetes. Vaccination is an effective measure to prevent influenza virus infection. In this paper, antibody response and protection against influenza virus infection induced by vaccination were studied in mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized once or twice with inactivated influenza virus vaccine at various dosages. Four weeks after the first immunization or 1 week after the second immunization, the mice were challenged with influenza virus at a lethal dose. The result showed that the antibody responses in diabetic mice were inhibited. Immunization once with high dose or twice with low dose of vaccine provided full protection against lethal influenza virus challenge in diabetic mice, however, in healthy mice, immunization only once with low dose provided a full protection.     

Cord factor (trehalose-6-6'-dimycolate) of in vivo-derived Mycobacterium lepraemurium.

Harvests of Mycobacterium lepraemurium obtained from livers of moribund infected mice yielded M. lepraemurium cell walls that were extracted with solvent to provide crude M. lepraemurium cell wall lipids. By solvent fractionation and chromatography on DEAE cellulose and cellulose, a cord factor-like glycolipids contaminated with mycoside C was obtained. Additional solvent treatment provided the purified glycolipid, which was identified as 6,6'-trehalose dimycolate, by infrared and chromatographic comparison with authentic samples from M. tuberculosis, by identification of trehalose and specific mycolates of M. lepraemurium, and by permethylation analysis. This constitutes the first unequivocal identification of cord factor as a product of in vivo-derived mycobacteria.     

Mechanisms of innate resistance to Toxoplasma gondii infection.

The interaction of protozoan parasites with innate host defences is critical in determining the character of the subsequent infection. The initial steps in the encounter of Toxoplasma gondii with the vertebrate immune system provide a striking example of this important aspect of the host-parasite relationship. In immuno-competent individuals this intracellular protozoan produces an asymptomatic chronic infection as part of its strategy for transmission. Nevertheless, T. gondii is inherently a highly virulent pathogen. The rapid induction by the parasite of a potent cell-mediated immune response that both limits its growth and drives conversion to a dormant cyst stage explains this apparent paradox. Studies with gene-deficient mice have demonstrated the interleukin-12 (IL-12)-dependent production of interferon gamma (IFN-gamma) to be of paramount importance in controlling early parasite growth. However, this seems to be independent of nitric oxide production as mice deficient in inducible nitric oxide synthase (iNOS) and tumour necrosis factor receptor were able to control early growth of T. gondii, although, they later succumbed to infection. Nitric oxide does, however, seem to be important in controlling persistent infection; treating chronic infection with iNOS metabolic inhibitors results in disease reactivation. Preliminary evidence implicates neutrophils in effector pathways against this parasite distinct from that described for macrophages. Once initiated, IL-12-dependent IFN-gamma production in synergy with other proinflammatory cytokines can positively feed back on itself to induce ''cytokine shock''. Regulatory cytokines, particularly IL-10, are essential to down-regulate inflammation and limit host pathology.     

Mechanisms underlying resistance to nematode infection

Lambs show considerable genetic variation in faecal egg count following natural, predominantly Ostertagia circumcinta infection. This genetic variation is acquired and not innate. Worm length is positively associated with worm fecundity. The genetic variation in faecal egg count is a consequence of genetic variation in worm length and hence worm fecundity, and not of genetic variation in worm burdens. In contrast to lambs, mature sheep may be able to regulate both fecundity and worm numbers. In lambs, three factors account for the majority of the variation in worm length: the strength of the local IgA response against fourth-stage larvae, the specificity of this response against four molecules in particular, and the density-dependent influence of worm number.     

Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium

An ELISA with cord factor (trehalose-6,6'-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6'-tetraacyl trehalose 2'-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria.     

RNAseq expression analysis of resistant and susceptible mice after influenza A virus infection identifies novel genes associated with virus replication and important for host resistance to infection

Background

The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suited to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilized a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual mouse after H1N1 infection.

Results

We performed a detailed expression analysis to identify (i) correlations between changes in expression of host and virus genes, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between two mouse strains that strongly differ in resistance to influenza infections. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection.

Conclusions

Using RNAseq analysis we identified novel genes important for viral replication or the host defense. This study adds further important knowledge to host-pathogen-interactions and suggests additional candidates that are crucial for host susceptibility or survival during influenza A infections.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1867-8) contains supplementary material, which is available to authorized users.     

Trichomonas vaginalis resistance to Mengo virus infection.

We have investigated the susceptibility of Trichomonas vaginalis to Mengo virus infection by comparing the outcome of Mengo virus or purified Mengo virus RNA infection in T. vaginalis and in CCL-1 mouse fibroblasts. While the adsorption and entry of Mengo virus into T. vaginalis occurred in the same manner as in fibroblasts, the uncoating was much slower. In addition, Mengo virus infection of T. vaginalis displayed no eclipse nor any subsequent production of infectious virus. Purified RNA failed to initiate productive infection in T. vaginalis, whereas it provoked viral replication in the fibroblast controls. It was shown by assessment of protein synthesis in T. vaginalis and mouse fibroblasts cell-free systems that the protozoan ribosomes were able to translate endogenous mRNA and poly-U, but not viral RNA.     

Enhanced susceptibility of Ago1/3 double-null mice to influenza A virus infection

RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation.