Codon volatility of hemagglutinin genes of H5N1 avian influenza viruses from different clades

Codon volatility is a method recently developed to estimate selective pressures on proteins on the basis of their synonymous codon usage. Volatility of a codon was defined as the fraction of single nucleotide substitutions that would be nonsynonymous. Higher volatility may indicate that the gene has been under more positive selection in the recent past. We analyzed volatility of hemagglutinin genes of H5N1 viruses in the recent outbreaks and observed differences in the volatility among viruses of different clades. The codon volatility of subclade 2.1 viruses from Indonesia was the lowest among all H5N1 clades and subclades. Time series analyses since the beginning of the epidemic in 2004 showed that codon volatility of subclade 2.1 has gradually decreased, while those of other major clades have been increasing. This may reflect differences in the recent evolution of these viruses.     

Prime-Boost Immunization of Codon Optimized HIV-1 CRF01_AE Gag in BCG with Recombinant Vaccinia Virus Elicits MHC Class I and II Immune Responses in Mice

The HIV-1 CRF01_AE gag gene was modified by codon restriction for Mycobacterium spp. and transformed into BCG; and it was designated as rBCG/codon optimized gagE. This produced 11 fold higher HIV-1 gag protein expression than the recombinant native gene rBCG/HIV-1gagE. In mice, CTL activity could be induced either by a single immunization of the codon optimized construct or by using it as a priming antigen in the prime-boost modality with recombinant Vaccinia virus expressing native HIV-1 gag. Specific secreted cytokine responses were also investigated. Only when rBCG gag was codon optimized did the prime-boost immunization produce significantly enhanced IFN-γ and IL-2 secretion indicating recognition via CD4+ and CD8+ T cells, and these responses seemed to be codon optimized immunogen dose-responsive. On contrary, the prime-boost vaccination using an equal amount of native rBCG/HIV-1gagE instead, or a single rBCG/codon optimized gagE immunization, had no similar effect on the cytokine secretion. These findings suggest that the use of recombinant codon BCG construct with recombinant Vaccinia virus encoding CRF01_AE gag as the prime-boost HIV vaccine candidate, will induce CD4+ Th1 and CD8+ T cell cytokine secretions in addition to enhancing CD8+ CTL response.     

HIV-1 C亚型密码子优化gp120负载人DC疫苗的构建及体外功能

目的构建HIV-1C亚型gp120负载人树突状细胞(dentriti ccell,DC)疫苗,并对其体外功能进行初步检测。方法利用Amaxa细胞核转染技术将pcDNA3.1-gp120质粒转染至人成熟DC,以Western blot检测gp120的表达。通过流式细胞仪检测DC表面共刺激分子的变化、混合淋巴细胞反应、CD8+T细胞表面活化分子CD25的表达及其分泌IFN-γ的变化。结果通过Western blot检测,gp120在DC中得到了正确表达。经流式细胞仪检测,DC表面分子CD80表达率由刺激前的33.34%上升至43.20%,CD86表达率由刺激前的60.08%上升至90.34%;负载gp120DC刺激淋巴细胞增殖率为86.72%;CD8+T细胞表面分子CD25表达率由刺激前的5.27%上升至74.21%,IFN-γ的表达率达37%。结论负载了HIV-1gp120的人树突状细胞能够显著刺激淋巴细胞的增殖、增强CD8+T细胞表面活化分子CD25表达以及促进CD8+T细胞分泌IFN-γ,为下一步DC治疗性疫苗的体内研究奠定基础。     

A reduction in selective immune pressure during the course of chronic hepatitis C correlates with diminished biochemical evidence of hepatic inflammation

It is considered that selection pressure exerted by the host immune response during early HCV infection might influence the outcome of that infection particularly as it relates to persistence or clearance of the agent. However, it is unclear whether positive selection pressure plays a role in determining the severity of hepatitis C during the course of persistent HCV infection. To address the evolutionary mechanism by which HCV escapes from the host immune response and to assess the relationship between viral evolution and hepatic inflammation, we determined 57 sequences (3-5 serial samples per patient) from 5 individuals with persistent HCV infection of genotype 1a who were under long-term follow-up ranging from 15.6 to 21.6 years. We applied a novel method to estimate serial alternations of selective pressure against the HCV enveloped region and compared this to fluctuation in transaminase level over time. Positive selection pressure was reduced over time postinfection, as evidenced by a reduction in nonsynonymous substitutions in the later phase of infection. Furthermore, serum transaminase, as a measure of inflammatory necrosis of hepatocytes, was reduced in parallel with decreased positive selection pressure. These results suggest that during persistent HCV infection, the virus faces diminished immune pressure over time, either from mutation to an immune resistant sequence or from immunologic exhaustion, and that this diminished immune attack is reflected in diminished inflammatory activity. This observation may be applicable to other viruses characterized by a slow rate of disease progression.     

Multicenter quality control of the detection of HIV-1 genome in semen before medically assisted procreation

Couples in whom the man is HIV-1-positive may use medically assisted procreation in order to conceive a child without contaminating the female partner. But, before medically assisted procreation, the semen has to be processed to exclude HIV and tested for HIV nucleic acid before and after processing. The performance was evaluated of the technical protocols used to detect and quantify HIV-1 in 11 centers providing medically assisted procreation for couples with HIV-1 infected men by testing panels of seminal plasma and cells containing HIV-1 RNA and/or DNA. The performance of these tests varied due to the different assays used. False positive results were obtained in 14-19% of cases. The sensitivity for RNA detection in seminal plasma was 500-1,000 RNA copies/ml, over 500 RNA copies/10(6) cells in semen cells, and for DNA detection in semen cells 50-500 DNA copies/10(6) cells. The use of silica-based extraction seemed to increase the assay performance, whereas the use of internal controls to detect PCR inhibitor did not. This first quality control highlights the need for technical improvements of the assays to detect and quantify HIV in semen fractions and for regular evaluation of their performance.     

Thymic selection does not limit the individual MHC diversity

The number of different major histocompatibility (MHC) molecules expressed per individual is widely believed to represent a trade-off between maximizing the detection of foreign antigens, and minimizing the loss of T cell clones due to self-tolerance induction. Using a mathematical model we here show that this argument fails to explain why individuals typically express of the order of 1020 different MHC molecules. Expression of extra MHC types decreases the number of clones surviving negative selection, but increases the number of positively selected clones. Based on experimental parameter estimates, we show that the number of clones in the functional T cell repertoire would in fact increase if the MHC diversity within an individual were to exceed its normal value, until more than one hundred different MHC molecules would be expressed. Since additional MHC types also increase the number of presented pathogen peptides, resistance against pathogens only decreases at unrealistically high MHC diversities exceeding 1,500 different MHC molecules per individual.     

Immunization with HIV-1 SF162-derived Envelope gp140 proteins does not protect macaques from heterologous simian-human immunodeficiency virus SHIV89.6P infection

Immunization by the SF162gp140 or the DeltaV2gp140 HIV-1 envelope proteins results in the generation of strong homologous neutralizing antibodies (NAbs) that offer similar degree of protection from disease-development to macaques challenged with homologous virus. These two immunogens elicit weak cross-reactive NAbs and their effectiveness against heterologous challenge is currently unknown. To examine this issue, we immunized macaques with SIVGag p55 and either the SF162gp140 or the DeltaV2gp140 and challenged them intravenously with SHIV-89.6P. All animals became infected but previous immunization with SF162gp140 accelerated the development of anti-SHIV89.6P neutralizing antibody responses following infection. DeltaV2gp140 is derived from SF162gp140 following the deletion of 30 amino acids and one N-linked glycosylation site from the V2 loop. Our results suggest that even small differences in HIV Envelope immunogen structure can affect the neutralizing antibody responses generated following infection.     

Viral fitness implications of variation within an immunodominant CD8+ T-cell epitope of HIV-1

Cytotoxic T-lymphocyte (CTL) epitopes within the HIV genome are subject to negative and positive selective pressures, the balance of which influences CTL escape at a given epitope. We investigated whether viral fitness requirements dictate conservation of the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Viral clones incorporating changes throughout the SL9 epitope region were compared to consensus SL9 virus in terms of replication kinetics and relative viral fitness. Constructs recapitulating in vivo SL9-CTL escape variants showed markedly little effect on replication and fitness, as did non-natural conservative mutations targeting immunologically relevant positions of the epitope. Although certain residues of the epitope were constrained by viral requirements, our research reveals that there are multiple SL9 variants that are well tolerated virologically but fail to arise in vivo. In light of this data, assumptions regarding the balance of immune and viral selective pressures on this immunodominant epitope sequence need to be reassessed.     

Impact of HIV-1 genetic diversity in China on the measurement of viral load

In this study, 190 HIV-positive samples were collected from different regions of China. The HIV clades of 153 samples were determined successfully based on env sequencing. Specifically, 48, 5, 87, and 13 isolates belonged to clades B', B, BC, and AE, respectively. The viral loads in all samples were measured using three commercial assays, Amplicor HIV-1 monitor v1.5, Nuclisens HIV-1 QT and NucliSens EasyQ HIV-1 assays. The differences and linear correlations for individual assays were compared, with expected 1:1 relationships. Significant differences were found for the following viral loads: clade BC measured by any two assays (P < 0.001); clade AE between Amplicor 1.5 and Easy Q (P = 0.005); clade B' between Amplicor 1.5 and Nuclisens QT (P = 0.002); clade AE between Amplicor 1.5 and Nuclisens QT (P = 0.025); and clade B' between Amplicor 1.5 and EasyQ (P = 0.04). The largest mean difference in the log(10) values was 0.9518, which was found between Amplicor 1.5 and Nuclisens QT. However, the viral loads for clades AE and B' measured by EasyQ and Nuclisens QT, and those for clade B measured by any two assays did not differ significantly. The degrees of correlation for clades B and B' between any two assays (R > 0.8) were higher that those for clades AE and BC between any two assays (R < 0.7), except for clade AE between Amplicor 1.5 and Easy Q. Thus, the clade types, especially clades BC and AE, are most likely to impact on the quantitation of viral load using differentassays.     

ASPCR检测微量HIV-1 K103N耐药突变方法的建立

目的 建立检测微量HIV-1 K103N耐药突变的等位基因特异扩增实时定量PCR(allele-specific real-time PCR,ASPCR)方法,用于微量K103N耐药突变的检测和分析.方法 首先建立检测K103N耐药突变的ASPCR方法,然后对方法进行验证,最后采用ASPCR方法检测对照样本.构建含K103N突变的质粒作为标准品,然后设计特异性引物用以区分野生型和突变型模板,采用SYBR green法进行实时定量PCR,并绘制标准曲线.分别采用特异性和非特异性引物扩增模板,根据二者Ct(cycle threshold)值结合相应的标准曲线判断是否存在K103N突变及突变比例.对ASPCR检测K103N突变位点的特异性、敏感性、准确性、重复性等进行验证.结果 特异性和非特异性引物扩增等量野生型模板的Ct值之差(△Ct)可达13.56;当突变比例小于0.1%时仍具有良好的准确性;批内变异系数小于0.7,批间变异系数小于1.6;检测灵敏度可达0.01%.检测阴性对照样本的△Ct显著高于临界值,阳性对照样本检测结果均为刚性.结论 ASPCR是一种快速、灵敏、准确的检测HIV-1微量耐药突变的方法,可为临床抗病毒治疗提供理论指导.     

Evaluation of supplemental testing with the Multispot HIV-1/HIV-2 Rapid Test and APTIMA HIV-1 RNA Qualitative Assay to resolve specimens with indeterminate or negative HIV-1 Western blots

BackgroundThe use of Western blot (WB) as a supplemental test after reactive sensitive initial assays can lead to inconclusive or misclassified HIV test results, delaying diagnosis.ObjectiveTo determine the proportion of specimens reactive by immunoassay (IA) but indeterminate or negative by WB that could be resolved by alternative supplemental tests recommended under a new HIV diagnostic testing algorithm.Study designRemnant HIV diagnostic specimens that were reactive on 3rd generation HIV-1/2 IA and either negative or indeterminate by HIV-1 WB from 11 health departments were tested with the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test (Multispot) and the Gen-Probe APTIMA HIV-1 RNA Qualitative Assay (APTIMA).ResultsAccording to the new testing algorithm, 512 (89.8%) specimens were HIV-negative, 55 (9.6%) were HIV-1 positive (including 19 [3.3%] that were acute HIV-1 and 9 [1.6%] that were positive for HIV-1 by Multispot but APTIMA-negative), 2 (0.4%) were HIV-2 positive, and 1 (0.2%) was HIV-positive, type undifferentiated. 47 (21.4%) of the 220 WB-indeterminate and 8 (2.3%) of the 350 WB-negative specimens were HIV-1 positive.ConclusionApplying the new HIV diagnostic algorithm retrospectively to WB-negative and indeterminate specimens, the HIV infection status could be established for nearly all of the specimens. IA-reactive HIV-infected persons with WB-negative results had been previously misclassified as uninfected, and HIV diagnosis was delayed for those with WB-indeterminate specimens. These findings underscore the limitations of the WB to confirm HIV infection after reactive results from contemporary 3rd or 4th generation IAs that can detect HIV antibodies several weeks sooner than the WB.     

Ramp rate of blood pressure changes does not affect aortic afferent sensitivity in anesthetized rats

To investigate whether the rate of change in blood pressure affects the sensitivity of the aortic baroreceptor afferent response, the change in aortic nerve activity (ANA) to two different rates of ramp increase in mean blood pressure (MBP), elicited by phenylephrine administration, was determined in the rat under urethane (1.5 g kg⁻¹) anesthesia. The sensitivity of the increase in ANA following a rapid (average ramp rate, 9.14 ± 0.60 mmHg s⁻¹, n = 11) or gradual (1.78 ± 0.24 mmHg s⁻¹, n = 11) increase in MBP was 2.03 ± 0.14% and 1.81 ± 0.20% of baseline mmHg⁻¹, respectively. These values were not significantly different from each other (P = 0.16). Furthermore, we found no correlation between the rate of ramp increase in MBP and the sensitivity of the increase in ANA (r = 0.24, P = 0.29, n = 22). These results suggest that, at least within the normal physiological range of MBP, the rate of the ramp change in blood pressure does not affect aortic baroreceptor afferent sensitivity in the anesthetized rat.     

Structural alterations of the chloroplast genome found in grasses are not common in monocots

The distribution of structural rearrangements of the chloroplast genome found in grass cpDNA in comparison to that of tobacco was systematically checked in the cpDNAs of representative monocots. The physical map of lily cpDNA, which shares a key position in the diversity of monocotyledonous plants, was constructed to assess whether three inversions found in grass cpDNA are common in monocots. Specific probes for the detection of (1) intron loss in the rpoC1 gene, (2) insertional sequence gain in rpoC2, (3) deletion of ORF2280 in the inverted repeats, (4) non-reciprocal translocation of rpl23, and (5) rearrangements of ORF512, were hybridized to cpDNAs of lily, onion, spiderwort, two turf grasses, and wheat. The existence of intervening sequences in the rpoC1 and rpoC2 genes was also confirmed by PCR analysis. All markers used in the study revealed that structural rearrangements of the chloroplast genome were restricted to grasses, indicating that drastic structural alterations of the chloroplast genome had occurred in the ancestor(s) of grasses. These results also suggest that structural analysis of the chloroplast genome is applicable to the phylogenetic reconstruction of related plants.     

Primary CCR5 only using HIV-1 isolates does not accurately represent the in vivo replicating quasi-species

Most HIV-1 isolates depend on CCR5 or CXCR4 to infect target cells, and efficient use of other coreceptors is rare. We cloned HIV-1 envelopes from virus at acute infection and found that most use CCR3 efficiently. This result contradicts prevailing data, suggesting that CCR3 usage is rare. We hypothesized that direct isolation into PBMC biases selection of viruses that use CCR5 and not CCR3. We therefore compared coreceptor use of isolates obtained by PBMC coculture with envelopes cloned directly from patient blood samples, which should represent actively replicating species. Viruses derived by cloning generally used CCR3 and CCR5 with equally efficiently. In contrast, we found that viruses isolated by PBMC coculture largely, or exclusively, used CCR5. Regardless of whether CCR3 use contributes to HIV-1 transmission or pathogenesis, our results demonstrate that "primary isolates" generated by PBMC culture are unlikely to accurately represent the in vivo replicating quasi-species.     

Circulation of HIV-1 subtype A within the subtype C HIV-1 epidemic in Tamil Nadu, India

The HIV‐1 epidemic in India is caused mainly by subtype C viruses that are transmitted sexually and by injecting drug use. The state of Tamil Nadu in Southern India has an HIV‐1 median prevalence of 16.8% among injecting drug users, 6.6% in men who have sex with men, and 4.6% in female sex workers. In the rural district of Namakkal, a prevalence >3% was detected among antenatal women. The goal of this study was to determine the HIV‐1 molecular epidemiology in Tamil Nadu. Blood samples were collected from 40 high‐risk HIV‐seropositive individuals from Chennai and Namakkal. HIV‐1 subtype was determined by envelope nucleotide sequencing. Among the samples studied, 85% were subtype C, however, a cluster of subtype A samples (12.5%) and one subtype E recombinant form CRF01_AE (close to the Thailand strains) were detected. The average genetic distance of subtype C samples from Chennai and Namakkal were 9.44 ± 0.77% and 11.8 ± 0.7%, respectively indicating an evolved epidemic. This pilot study confirmed that subtype C was predominant in these regions but an outbreak of subtype A was detected in Namakkal. These results stress the importance of periodic monitoring of circulating HIV‐1 subtypes in South India. J. Med. Virol. 84:1507–1513, 2012. © 2012 Wiley Periodicals, Inc.     

HIV-1 down-regulates the expression of CD1d via Nef

HIV-1 has evolved several strategies to subvert host immune responses to the infected cells. One is to inhibit CTL recognition by HIV-1 Nef-mediated down-regulation of MHC-I expression on the surface of infected cells. Here we report that Nef also reduces the expression of the non-classical MHC-I like CD1d molecule, a third lineage of antigen-presenting molecule, which presents lipid antigens. Nef achieves this by increasing internalization of CD1d molecules from the cell surface and retaining CD1d in the trans-Golgi-network (TGN). This effect depends on a tyrosine-based motif present in CD1 cytoplasmic tail as well as the actions of four Nef motifs, which are known to be involved in the down-regulation of MHC-I or CD4. These results suggest that Nef regulates intracellular trafficking of CD1d via a distinct but shared pathway with MHC-I and CD4. Thus, HIV-1 reduces the visibility of its infected cells not only to MHC-I-restricted T cells but also to CD1d-restricted NKT cells. Given that CD1d-restricted T cells have unique effector and regulatory functions in innate and adapted immune responses as compared with their counterpart MHC-restricted T cells, our data provide additional new insights into molecular basis of HIV-1-mediated damage to the immune system.     

Proliferative activation up-regulates expression of CD4 and HIV-1 co-receptors on NK cells and induces their infection with HIV-1

NK cells play important roles in immune surveillance against malignancy and virus-infected cells. NK cell functions are affected in patients infected with HIV-1; however, whether there is direct interaction between NK cells and HIV-1 remains controversial. In this study the expression of CD4, an important receptor for HIV-1, was up-regulated on NK cells co-cultured with an NK cell-selective stimulating cell line, HFWT, and rIL-2. Although the level of CD4 was lower on NK cells than on CD4+ T cells, expression of the HIV-1 co-receptor CCR5 was clearly up-regulated on CD4+ NK cells. CD4+ NK cells expressed higher levels of HLA-DR and CD25 than CD4- NK cells, suggesting that they were highly activated. Cell-free HIV-1 could not infect the NK cells, but NK cells were infected when co-cultured with HIV-1-infected T cells. Using this co-culture system, we can better understand how HIV-1 infects NK cells and how NK cell functions are affected in AIDS.