Arrested spread of vesicular stomatitis virus infections in vitro depends on interferon-mediated antiviral activity

A quantitative understanding of the innate immune response will enable its recruitment against emerging, poorly characterized, or weaponized viral pathogens. To gain insights into how the innate responses can limit viral spread, we used quantitative focal infections to study how the spread of recombinant vesicular stomatitis viruses (VSV) on baby hamster kidney (BHK) and delayed brain tumor (DBT) cell monolayers is affected by innate cellular antiviral responses. We observed that rates of infection spread correlated with one-step growth rankings for four ectopic VSV strains: N1, N2, N3, and N4. However, this correlation was lost for M51R, a recombinant VSV mutant that lacks the ability to shut-off host gene expression. In BHK cells, M51R spread at two-thirds the rate of the recombinant control virus, XK3.1, even though their one-step growth was comparable. In DBT cells, M51R infections failed to spread beyond the site of inoculation. Addition of anti-interferon antibody restored M51R spread and one-step growth to wild-type levels. Interestingly, the antibody enhanced the spread of wild-type virus but not its growth. These results suggest that while the rate of viral spread generally correlates with the rate of viral growth, the induction of cellular antiviral activities can be in some cases, the overriding factor in both spread and growth. In summary, focal infections enabled us to visualize and quantify how viral spread was inhibited by cellular antiviral activities. This study demonstrates a mechanism for quantifying how innate cellular responses can mitigate infection spread in vitro.     

Molecular identification of human tuberculosis in recent and historic bone tissue samples: The role of molecular techniques for the study of historic tuberculosis

We describe the molecular identification of the M. tuberculosis complex DNA in bone tissue samples from recent and historic populations. In a first set, archival paraffin material from vertebral bodies of 12 recent cases with clinically/microbiologically proven tuberculosis was compared to 12 further cases without tuberculosis. While eight TB cases revealed a specific mycobacterial amplification product, none of the controls was positive. Interestingly, one case with tuberculous sepsis (Landouzy sepsis), five cases with tuberculous spread beyond the primarily affected organ (i.e., lymph node or miliar involvement), and also two of six cases with restricted pulmonary tuberculosis reacted positively in the vertebral specimens. This indicates that a molecular analysis can detect mycobacteria even in unremarkable bone tissue, proving that organ tuberculosis is present. In addition, the extent of spread is of high significance for the frequency of positive reactions. In addition, we investigated a series of vertebral samples coming from an Egyptian population of the necropolis of Thebes-West dating to approximately 1450-500 BC. In this group of 36 cases, three of five cases with typical macromorphological signs for tuberculous spondylitis, 2 of 12 cases with nonspecific alterations, and 2 of 19 cases without macroscopic pathology revealed a specific amplicon of the M. tuberculosis complex. This suggests a significant frequency of infected people in that ancient population. Finally, a fourth group of 51 long bone samples with pathological alterations coming form a southern German ossuary (between AD 1400-1800) was investigated, and 10 cases were positive for the M. tuberculosis complex. These studies of historic material clearly support the notion that tuberculous infections can be unequivocally identified by molecular techniques. The relatively high frequency of ancient bacterial DNA amplifications in unremarkable bone is well-explained by our analysis of the recent material. Our data form an important basis for the investigation of tuberculosis frequency and spread in historic periods.     

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.     

Mixtures of varying coefficient models for longitudinal data with discrete or continuous nonignorable dropout

The analysis of longitudinal repeated measures data is frequently complicated by missing data due to informative dropout. We describe a mixture model for joint distribution for longitudinal repeated measures, where the dropout distribution may be continuous and the dependence between response and dropout is semiparametric. Specifically, we assume that responses follow a varying coefficient random effects model conditional on dropout time, where the regression coefficients depend on dropout time through unspecified nonparametric functions that are estimated using step functions when dropout time is discrete (e.g., for panel data) and using smoothing splines when dropout time is continuous. Inference under the proposed semiparametric model is hence more robust than the parametric conditional linear model. The unconditional distribution of the repeated measures is a mixture over the dropout distribution. We show that estimation in the semiparametric varying coefficient mixture model can proceed by fitting a parametric mixed effects model and can be carried out on standard software platforms such as SAS. The model is used to analyze data from a recent AIDS clinical trial and its performance is evaluated using simulations.     

Macrophages exposed to lymphotropic and monocytotropic HIV induce similar CTL responses despite differences in productive infection

Macrophages are accessory cells that are vulnerable to infection by HIV-1. HTLV-IIIB, a lymphotropic strain of HIV, infects macrophages poorly resulting in either no or low levels of virus expression compared to high levels of productive infection after exposure of macrophages to the monocytotropic HIV strain Ada-M. Whether this results in an impaired ability of HTLV-IIIB-exposed macrophages to initiate protective cytotoxic T lymphocyte (CTL) immune responses against these strains is not well defined. We investigated the ability of monocyte-derived macrophages (MDM) exposed to lymphotropic and monocytotropic HIV strains to initiate primary CTL responses in vitro. MDM exposed to HTLV-IIIB induced a specific primary CTL response that was comparable to MDM exposed to the monocytotropic strain Ada-M despite marked differences in productive HIV infection in MDM between the two strains. CTL generated in this model were MHC-restricted, strain-specific, and CD8+. These data demonstrate that high levels of productive HIV infection in accessory cells are not a prerequisite for the generation of a primary CTL response, suggesting a novel immunologic interaction between MDM and lymphotropic HIV strains.     

Genomic analysis of Fas and FasL genes and absence of correlation with disease progression in AIDS

Apoptosis has been suggested as a major mechanism for the CD4⁺ T-lymphocyte depletion observed in patients infected with human immunodeficiency virus 1 (HIV-1). To evaluate the impact of genetic variations to apoptosis during progression of acquired immunodeficiency syndrome (AIDS), we have performed an extensive genetic analysis of Fas and Fas ligand (FasL) genes. The coding regions and promoters of these genes were resequenced in a cohort of 212 HIV-1-seropositive patients presenting extreme disease phenotypes and 155 healthy controls of Caucasian origin. Overall, 33 single nucleotide polymorphisms (SNPs) with an allele frequency >1% were identified and evaluated for their association with disease progression. Among them, 14 polymorphisms were newly characterized. We did not find any statistically significant association of Fas and FasL polymorphisms and haplotypes with AIDS progression.     

Effects of education, vaccination and treatment on HIV transmission in homosexuals with genetic heterogeneity

Genetic studies report the existence of a mutant allele Delta32 of CCR5 chemokine receptor gene at high allele frequencies (approximately 10%) in Caucasian populations. The presence of this allele is believed to provide partial or full resistance to HIV. In this study, we look at the impact of education, temporarily effective vaccines and therapies on the dynamics of HIV in homosexually active populations. In our model, it is assumed that some individuals possess one or two mutant alleles (like Delta32 of CCR5) that prevent the successful invasion or replication of HIV. Our model therefore differentiates by genetic and epidemiological status and naturally ignores the reproduction process. Furthermore, HIV infected individuals are classified as rapid, normal or slow progressors. In this complex setting, the basic reproductive number R0 is derived in various situations. The separate or combined effects of therapies, education, vaccines, and genetic resistance are analyzed. Our results support the conclusions of Hsu Schmitz that some integrated intervention strategies are far superior to those based on a single approach. However, treatment programs may have effects which counteract each other, as may genetic resistance.     

Shelter preference in <Emphasis Type="Italic">Macrobrachium</Emphasis> spp. with reference to aquaculture

Macrobrachium malcolmsonii, M. nobilii, and M. lamarrei were used to investigate their preferences among three substrate colors (dark brown, black, and light brown), shelter type (black tile and black PVC pipe), color (black and brown tile), and diameter, and regarding intra- and inter-species habitation. Both M. lamarrei and M. malcolmsonii preferred dark-brown substrate, while M. nobilii preferred black. The choice of shelter type was monitored by time spent by each species in a black PVC tube or in a black tile. Within a 24-h period, M. malcolmsonii spent a greater amount of time (60.5%) within the black tile, while M. nobilii spent more time (71%) inside the black PVC tube. M. lamarrei always occupied an open area and thus was discarded from further experiments. If we consider daytime and nighttime separately, both species spent more time within the black tile during the day than during the night. Choice of shelter color was tested with black and brown shelters of identical size; both species exclusively preferred the black. Between the species, despite the smaller body size both male and female M. nobilii succeeded in evicting M. malcolmsonii, suggesting a stronger shelter affinity. When competing for shelter within the same species, a male was more dominant than a conspecific female and juvenile. Similarly males of both species allowed conspecific juveniles to cohabit for about 16% of the time. Male M. malcolmsonii also allowed cohabitation of a female for 31.2% of the time, unlike the male M. nobilii, which did not allow female cohabitation at all. Both species preferred shelters that were adjusted to their meral spread as opposed to their carapace width. The possibility of taking meral spread as a taxonomic character was also indicated. The study also found a linear relationship between exuvium weight and shelter affinity among the three species. A lower ratio indicated a higher shelter affinity. The exuvium/total body weight ratio confirmed the shelter affinity, which was ranked as M. nobilii (0.275) < M. malcolmsonii (0.312) < M. lamarrei (0.426).     

Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato

A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5′ to the simian immunodeficiency virus (SIV_mac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by G_M1-ganglioside-enzyme-linked immunosorbent assay (G_M1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by G_M1-ELISA showed that CTB-Gag fusion protein made up approx 0.016–0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.     

On the origin and evolution of the human immunodeficiency virus (HIV)

The human AIDS viruses--HIV-1 and HIV-2--impose major burdens on the health and economic status of many developing countries. Surveys of other animal species have revealed that related viruses--the SIVs are widespread in a large number of African simian primates where they do not appear to cause disease. Phylogenetic analyses indicate that these SIVs are the reservoirs for the human viruses, with SIVsm from the sooty mangabey monkey the most likely source of HIV-2, and SIVcpz from the common chimpanzee the progenitor population for HIV-1. Although it is clear that AIDS has a zoonotic origin, it is less certain when HIV-1 and HIV-2 first entered human populations and whether cross-species viral transmission is common among primates. Within infected individuals the process of HIV evolution takes the form of an arms race, with the virus continually fixing mutations by natural selection which allow it to escape from host immune responses. The arms race is less intense in SIV-infected monkeys, where a weaker immune response generates less selective pressure on the virus. Such a difference in virus-host interaction, along with a broadening of co-receptor usage such that HIV strains are able to infect cells with both CCR5 and CXCR4 chemokine receptors, may explain the increased virulence of HIV in humans compared to SIV in other primates.