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.     

柑橘衰退病毒柚类分离株的分子鉴定

应用限制性片段长度多态性(RFLP)、双向逆转录-聚合酶链式反应(BD-PCR)、序列分析等技术对我国部分地区柑橘衰退病毒(Citrus tristeza virus,CTV)柚类分离株进行了鉴定分析。结果表明:柚类以受相对单一的CTV株系侵染为主;柚类上的优势流行株系属于p25/HinfⅠRFLP 6组群(占79.4%)和p23/BD-PCRⅢ组群(占84.1%);在对柚类CTV分离株S051~S058及国外CTV分离株PB61、T30、T36、T385、SY568、VT、NUAGA的p23、p25基因序列分析中,S051与弱毒株PB61的同源性最高;S052与弱毒株T30、T385的同源性最高;S053、S054、S055、S056、S057、S058与其它CTV分离株的同源关系均较远,并在系统发生树上形成了独立的分枝。     

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.     

Preactivation of B Lymphocytes Does Not Enhance Mouse Mammary Tumor Virus Infection

We investigated whether mouse mammary tumor virus (MMTV) favors preactivated or naive B cells as targets for efficient infection. We have demonstrated previously that MMTV activates B cells upon infection. Here, we show that polyclonal activation of B cells leads instead to lower infection levels and attenuated superantigen-specific T-cell responses in vivo. This indicates that naive small resting B cells are the major targets of MMTV infection and that the activation induced by MMTV is sufficient to allow efficient infection.     

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.     

柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析

通过改进提纯方法获得了柑橘衰退病毒(Citrustristezavirus,CTV)的提纯液,其产量为1mg/100g植物组织。用CTV免疫大耳白兔,获得多克隆抗体,间接ELISA效价为1∶25600。用CTV免疫小鼠,经细胞融合、ELISA筛选和克隆化培养,获得18株能稳定分泌抗CTV单克隆抗体的杂交瘤单细胞株。对其中4株单克隆腹水抗体进行分析的结果表明,这些抗体的ELISA效价为1∶51200~1∶204800,其中2G和3H的抗体类型及亚类为IgG2a,1E和4H为IgG2b。用所制备抗体对不同来源柑橘样品的CTV检测结果显示,单克隆和多克隆抗体结合使用,采用三抗体夹心ELISA(TAS-ELISA)可以获得理想的检测效果,其特异性强、灵敏度高。同时发现所分析4株单克隆抗体对不同的CTV分离物鉴别能力存在差异,但有关这些CTV分离物的特性及其血清学关系还需进一步研究。     

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.     

Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus (Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism (SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18, p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting that they may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed that the p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequence homology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination events in isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.     

HIV超感染防御研究进展

机体在原发感染病毒后,体内能产生某种可能的干扰以阻挡、防止被同种（或同类别）株病毒再次感染现象,HIV感染中也存在这种超感染防御现象。显然,该的研究对于改进HIV疫苗研制策略及其他抗病毒策略十分重要。目前研究认为感染细胞在分子水平上产生的超感染抵抗（superinfection resistance,SIR）和机体免疫反应是感染个体能防御超感染的主要原因。但以上各种假说都没有得到充分验证,HIV超感染防御依然尚未明确。本文将这些研究进行总结,以期找出新的突破口,推进该项研究。     

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.     

Expanded Strain‐Specific RT‐PCR Assay for Differential Detection of Currently Known Citrus Tristeza Virus Strains: a Useful Screening Tool

Genotypic characterization of Citrus tristeza virus (CTV) strains has progressed significantly, but their phenotypic expression is poorly established as CTV naturally occurs as mixed‐strain populations. A screening system for the analysis of mixed‐strain populations is required for population studies and the correlation with symptom expression. In this study, a published CTV strain‐specific detection assay was expanded and improved to facilitate detection of currently known CTV strains. Supplementary RT‐PCR assays were developed for two variant groups of the RB strain and the HA16‐5 strain, and assays for the T36 strain and generic CTV detection were improved. The value of the strain‐specific assays was shown by the ability to identify the strain components of two CTV cross‐protecting sources, GFMS35 and LMS6, used in the South African budwood certification scheme and to demonstrate the segregation of strains in budwood source trees.     

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.     

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.     

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.