柑桔衰退病毒的提纯

从甜橙病株的嫩梢皮组织提纯柑桔衰退病毒(Citrus Tristeza Virus,CTV),冰冻组织按每克鲜组织加入5ml0.1mol/L Tris缓冲液pH8.4(内含0.15％Triton x—100)进行匀浆。经几次差速离心和两次PEG(分子量6,000)沉淀后,将获得的病毒粗提液铺在不连续蔗糖密度梯度液上,HITACHI RPS_(40)T转头30,000r/m离心3小时,收集位于300mg/ml和400mg/ml梯度层之间的分离带,洗脱、浓缩后获得CTV提纯物。提纯的CTV粒子大小为1.000—1,500x12urn,与美国的CTV抗血清起阳性反应。     

Alteration of Bark Proteins Associated with Citrus Tristeza Virus (CTV) Infection on Susceptible Citrus Species and Scion-Rootstock Combinations

The effect of citrus tristeza virus (CTV) on bark protems of susceptible citrus species and scion-rootstock combinations was studied by polyaerylamide gel electrophoresis (PAGE). Protein pattern of sour orange bark from CTV-infected trees on this rootstock showed reduced intensity in a protein band, about 20,000 daltons molecular weight, as compared with similar CTV-free trees. This protein modification appears specifically associated with decline induced by tristeza since it was observed on trees of different ages and scion-rootstock combinations, grown in various locations and infected with several CTV isolates, but not on trees exhibiting decline from other causes. The observed protein alteration was localized in the ribosomic fraction. No protein alteration, associated with CTV infection could be found on lemon bark, although this citrus species also behaves as a CTV-susceptible rootstock. Electrophoretic profiles obtained from CTV infected Mexican lime and Etrog citron seedlings also showed reduced intensity in a protein band with the same electrophoretic mobility as the tnodified band of sour orange.     

Denial of Risk Behavior Does Not Exclude Asymptomatic Anorectal Sexually Transmitted Infection in HIV-Infected Men

Background

The Centers for Disease Control recommend screening for asymptomatic sexually transmitted infection (STI) among HIV-infected men when there is self-report of unprotected anal-receptive exposure. The study goals were: (1) to estimate the validity and usefulness for screening policies of self-reported unprotected anal-receptive exposure as a risk indicator for asymptomatic anorectal infection with Neisseria gonorrhoeae (GC) and/or Chlamydia trachomatis (CT). (2) to estimate the number of infections that would be missed if anal diagnostic assays were not performed among patients who denied unprotected anorectal exposure in the preceding month.

Methods and Findings

Retrospective analysis in HIV primary care and high resolution anoscopy (HRA) clinics. HIV-infected adult men were screened for self-reported exposure during the previous month at all primary care and HRA appointments. Four sub-cohorts were defined based on microbiology methodology (GC culture and CT direct fluorescent antibody vs. GC/CT nucleic acid amplification test) and clinical setting (primary care vs. HRA). Screening question operating characteristics were estimated using contingency table methods and then pooled across subcohorts. Among 803 patients, the prevalence of anorectal GC/CT varied from 3.5–20.1% in the 4 sub-cohorts. The sensitivity of the screening question for self-reported exposure to predict anorectal STI was higher in the primary care than in the HRA clinic, 86–100% vs. 12–35%, respectively. The negative predictive value of the screening question to predict asymptomatic anorectal STI was ≥90% in all sub-cohorts. In sensitivity analyses, the probability of being an unidentified case among those denying exposure increased from 0.4–8.1% in the primary care setting, and from 0.9–18.8% in the HRA setting as the prevalence varied from 1–20%.

Conclusion

As STI prevalence increases, denial of unprotected anal-receptive exposure leads to an increasingly unacceptable proportion of unidentified asymptomatic anorectal STI if used as a criterion not to obtain microbiologic assays.     

Detection of Australian Field Isolates of Citrus Tristeza Virus by Double-Stranded RNA Analysis

Double-stranded RNA analysis indicated that widespread latent infection by citrus tristeza virus (CTV) occurs naturally in a citrus variety collection at Merbein, Australia. Detection was based on the presence of the putative replicative form (RF) of the virus (ca 19. 5 kilobase pairs) and the presence or absence of three previously known putative subgenomic dsRNAs with molecular sizes of ca 3. 1,1.7 and 0.8 kilobase pairs. With the exception of some citrus relatives normally used as rootstocks, the dsRNAs were readily detectable in all the trees tested. Variability was noticed among the dsRNAs profiles of individual seedling trees of Microcitrus australasica var. sanguinea. By contrast, dsRNA patterns of trees of open-pollinated Ellendale tangor were indistinguishable. A number of dsRNA bands found in West Indian lime infected with CTV were not detected when this isolate was graft-inoculated to healthy sweet orange seedlings. A simple method for enhancing the sensitivity of detection of the CTV dsRNA bands by silver staining is described which involves incubating the gels in RNase A prior to staining; this allowed detection of the putative CTV RF in only 40 mg of green hark material.     

The p23 Protein of Citrus Tristeza Virus Controls Asymmetrical RNA Accumulation

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3' genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3'-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3' genes.     

Virus-Viroid Interactions: Citrus Tristeza Virus Enhances the Accumulation of Citrus Dwarfing Viroid in Mexican Lime via Virus-Encoded Silencing Suppressors

An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd.     

Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10(5) to 10(6) of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.     

Oral Immunization with OspC Does Not Prevent Tick-Borne Borrelia burgdorferi Infection

Oral vaccination strategies are of interest to prevent transmission of Lyme disease as they can be used to deliver vaccines to humans, pets, and to natural wildlife reservoir hosts of Borrelia burgdorferi. We developed a number of oral vaccines based in E. coli expressing recombinant OspC type K, OspB, BBK32 from B. burgdorferi, and Salp25, Salp15 from Ixodes scapularis. Of the five immunogenic candidates only OspC induced significant levels of antigen-specific IgG and IgA when administered to mice via the oral route. Antibodies to OspC did not prevent dissemination of B. burgdorferi as determined by the presence of spirochetes in ear, heart and bladder tissues four weeks after challenge. Next generation sequencing of genomic DNA from ticks identified multiple phyletic types of B. burgdorferi OspC (A, D, E, F, I, J, K, M, Q, T, X) in nymphs that engorged on vaccinated mice. PCR amplification of OspC types A and K from flat and engorged nymphal ticks, and from heart and bladder tissues collected after challenge confirmed sequencing analysis. Quantification of spirochete growth in a borreliacidal assay shows that both types of spirochetes (A and K) survived in the presence of OspC-K specific serum whereas the spirochetes were killed by OspA specific serum. We show that oral vaccination of C3H-HeN mice with OspC-K induced significant levels of antigen-specific IgG. However, these serologic antibodies did not protect mice from infection with B. burgdorferi expressing homologous or heterologous types of OspC after tick challenge.     

ACTG-Toxins Produced by a Strain of Alternaria altemata That Does Not Originate From Citrus Species

AGTG-toxins designated as host-specific toxins were isolated at first from a pathotype of Alternaria alternata attacking Citrus species. A strain of Alternaria alternata isolated from Brassica sinensis in Central Germany and improved to a high tentoxin content, also produces two ACTG-toxins. It is doubtful, therefore, if both substances can be denoted as host-specific toxins.     

Systemic Acquired Resistance Induced in Tobacco Plants by Localized Virus Infection Does Not Operate Against Challenging Viruses That Infect Systemically

Localized infections produced by tobacco necrosis virus (TNV) or tomato mosaic virus (ToMV) in White Burley tobacco induced a systemic acquired resistance in upper, uninoculated leaves. This resistance was effective against challenge infection by TNV or ToMV but not by potato virus Y, necrotic strain (PVYⁿ), tobacco mosaic virus (TMV) or tobacco rattle virus (TRV), viruses giving systemic infections. Systemic acquired resistance against TNV or ToMV was expressed as a reduction in lesion size but not in viral antigen content of the resulting necrotic local lesions. The acquisition of resistance was concurrent with an increased capacity of the resistant leaves to convert 1-aminocyclopropane-1-carboxylic acid into ethylene. Systemic acquired resistance was ineffective to contrast or minimize in whatever way the systemic challenge infection produced by PVY^N, TMV or TRV. Severity of symptoms and virus multiplication did not differ in resistant leaves from controls. This result does not allow any optimistic promise on possible application of the systemic acquired resistance against severe viral diseases of crops.     

A Seminomadic Population in Bangladesh with Extensive Exposure to Macaques Does Not Exhibit High Levels of Zoonotic Simian Foamy Virus Infection

Simian foamy viruses (SVF) are ubiquitous in nonhuman primates (NHP). SFV can be zoonotically transmitted to humans who either work with or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, a PCR assay failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.     

Prior Infection Does Not Improve Survival against the Amphibian Disease Chytridiomycosis

Many amphibians have declined globally due to introduction of the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Hundreds of species, many in well-protected habitats, remain as small populations at risk of extinction. Currently the only proven conservation strategy is to maintain species in captivity to be reintroduced at a later date. However, methods to abate the disease in the wild are urgently needed so that reintroduced and wild animals can survive in the presence of Bd. Vaccination has been widely suggested as a potential strategy to improve survival. We used captive-bred offspring of critically endangered booroolong frogs (Litoria booroolongensis) to test if vaccination in the form of prior infection improves survival following re exposure. We infected frogs with a local Bd isolate, cleared infection after 30 days (d) using itraconazole just prior to the onset of clinical signs, and then re-exposed animals to Bd at 110 d. We found prior exposure had no effect on survival or infection intensities, clearly showing that real infections do not stimulate a protective adaptive immune response in this species. This result supports recent studies suggesting Bd may evade or suppress host immune functions. Our results suggest vaccination is unlikely to be useful in mitigating chytridiomycosis. However, survival of some individuals from all experimental groups indicates existence of protective innate immunity. Understanding and promoting this innate resistance holds potential for enabling species recovery.     

Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

Hepatitis B Virus Infection Does Not Significantly Influence Plasmodium Parasite Density in Asymptomatic Infections in Ghanaian Transfusion Recipients

Background

Areas endemic for malaria and Hepatitis B virus (HBV) infection largely overlap geographically. A recent study has suggested the existence of an interaction between the two pathogens in symptomatic co-infected individuals on the South-American continent. We examined this issue in a hyperendemic area for both pathogens in sub-Saharan Africa.

Methodology and Findings

Pre-transfusion samples from a retrospective cohort of 154 blood transfusion recipients were screened for both serological and molecular markers of HBV and Plasmodium genomes using species-specific nested PCR and quantitative real-time PCR. Thirty-seven individuals met exclusion criteria and were subsequently eliminated from further analysis. Of 117 participants, 90% of recipients exhibited evidence of exposure to HBV, 42% with HBsAg and/or HBV DNA and 48% anti-HBc reactive without detectable HBV DNA. Plasmodium genome prevalence by NAT was 50%. Parasitemic individuals were significantly younger than non-parasitemic individuals (P = 0.04). Parasitemia level was not significantly lower in individuals with HBV DNA positive infections compared to those with HBV DNA negative exposures. HBV DNA load was not significantly different in parasitemic and non-parasitemic individuals.

Conclusion

The data presented suggests that, in sub-Saharan Africa, asymptomatic co-infections with these two ubiquitous pathogens do not appear to significantly affect each other and evolve independently.     

Enhanced PD-1 Expression by T Cells in Cerebrospinal Fluid Does Not Reflect Functional Exhaustion during Chronic Human Immunodeficiency Virus Type 1 Infection

During chronic viral infections, T cells are exhausted due to constant antigen exposure and are associated with enhanced programmed death 1 (PD-1) expression. Deficiencies in the PD-1/programmed death-ligand 1 (PD-L1) pathway are associated with autoimmune diseases, including those of the central nervous system (CNS). To understand the role of PD-1 expression in regulating T-cell immunity in the CNS during chronic infection, we characterized PD-1 expression in cerebrospinal fluid (CSF) and blood of individuals with chronic human immunodeficiency virus type 1 (HIV-1) infection. PD-1 expression was higher on HIV-specific CD8⁺ T cells than on total CD8⁺ T cells in both CSF and blood. PD-1 expression on CSF T cells correlated positively with CSF HIV-1 RNA and inversely with blood CD4⁺ T-cell counts, suggesting that HIV-1 infection drives higher PD-1 expression on CSF T cells. However, in every HIV-positive individual, PD-1 expression was higher on T cells in CSF than on those in blood, despite HIV-1 RNA levels being lower. Among healthy HIV-negative controls, PD-1 expression was higher in CSF than in blood. Furthermore, frequencies of the senescence marker CD57 were lower on CSF T cells than on blood T cells, consistent with our prior observation of enhanced ex vivo functional capacity of CSF T cells. The higher PD-1 expression level on CSF T cells therefore does not reflect cellular exhaustion but may be a mechanism to downregulate immune-mediated tissue damage in the CNS. As inhibition of the PD-1/PD-L1 pathway is pursued as a therapeutic option for viral infections, potential effects of such a blockade on development of autoimmune responses in the CNS should be considered.Programmed death 1 (PD-1; also called CD279) and its ligands, PD-L1 (also called B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD-273), regulate T-cell activation, peripheral tolerance, and autoimmunity (22, 43). PD-1 can be expressed on CD8⁺ and CD4⁺ T cells, B cells, natural killer T cells, and activated monocytes. PD-L1 is expressed on various cells, including T and B cells, dendritic cells, macrophages, mast cells, nonhematopoietic cell types (including vascular endothelial cells, pancreatic islet cells, astrocytes, keratinocytes, and microglial cells), and cells in immune privileged sites, including the placenta and the eye (22). PD-L2 expression is inducible and is restricted to dendritic cells, monocytes, macrophages, and mast cells (22). During chronic infections, the PD-1/PD-L1 pathway inhibits antigen-specific T-cell responses (7, 8, 35, 46). In human immunodeficiency virus type 1 (HIV-1)-infected individuals, PD-1 expression on HIV-specific T cells in peripheral blood is upregulated and correlates positively with plasma viremia and inversely with CD4⁺ T-cell counts (7, 46). PD-1 expression on HIV-specific T cells is also associated with T-cell exhaustion, as defined by a reduced ability to proliferate and produce cytokines (7, 46). Inhibition of the PD-1/PD-L1 pathway augments HIV-specific CD8⁺ and CD4⁺ T-cell function, and antiretroviral therapy is associated with a significant reduction of PD-1 expression on HIV-specific T cells in peripheral blood (8).The PD-1/PD-L1 pathway also limits immune-mediated tissue damage that may be caused by overreactive peripheral T cells, especially in immune privileged sites such as the central nervous system (CNS). In 1999, the importance of PD-1 for peripheral tolerance was first suggested by studies which showed that PD1^−/− mice develop lupus-like autoimmune diseases (32). In humans, polymorphisms in the PDCD1 gene, which encodes PD-1, have been associated with autoimmune diseases, including lupus, diabetes, rheumatoid arthritis, and multiple sclerosis (20, 21, 25). Upregulation of PD-L1 in multiple sclerosis lesions from human brain tissue suggests a role for the PD-1/PD-L1 pathway in regulating T-cell activation and controlling immunopathological damage (33).The CNS is involved by HIV-1 early during primary infection (6, 13), and approximately 40% of patients who develop advanced AIDS without receiving antiretroviral therapy develop cognitive impairment (6, 13, 38). While HIV-1 proteins gp120 (3, 16) and Tat (30) are directly neurotoxic and may contribute to HIV-associated dementia, detrimental neuropathogenic effects have also been postulated for inflammatory and innate immune cells, especially monocytes/macrophages and T cells (11, 19, 49, 50). Immune responses cause neuropathogenesis during other viral infections, and cytotoxic T lymphocytes can worsen the disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (14). However, we recently described higher frequencies of functional HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) than in blood among asymptomatic HIV-positive individuals with little or no HIV-1 RNA in CSF, suggesting that HIV-1-specific CD8⁺ T cells help to control intrathecal viral replication (40).To understand the role of the PD-1/PD-L1 pathway in regulating T-cell responses during viral infection of the CNS, we characterized PD-1 expression on T cells in CSF and peripheral blood among asymptomatic HIV-positive individuals. We hypothesized that T-cell PD1 expression would be lower in CSF than in blood, since HIV-1 RNA concentrations are lower in CSF than in plasma and the magnitude and breadth of IFN-γ-secreting HIV-specific T cells are greater in CSF than in blood (40). We show that, in CSF, HIV-1 RNA correlates directly with PD-1 expression on CD4⁺, CD8⁺, and HIV-specific CD8⁺ T cells. Unexpectedly, PD-1 expression on all T cells is higher in CSF than in blood in HIV-positive patients and healthy HIV-negative controls. In contrast, expression of the senescence marker CD57 is lower in CSF than in blood. These data suggest that higher PD-1 expression on T cells in CSF may be a mechanism to regulate T-cell immunity in the CNS, rather than indicating T-cell exhaustion, and that this regulation is increased by HIV-1 replication.