HIV-1准种的变化与疾病进展关系的研究

目的　研究HIV 1准种的变化对疾病进展的影响。方法　PCR方法扩增HIV 1V3～V5区片段 ,采用HMA(异源双链泳动分析 )的方法分析 4 8例HIV/AIDS准种的变化 ,并结合HIV/AIDSCD4 T淋巴细胞计数及病毒载量的结果分析HIV 1准种的变化对疾病进程的影响。结果　在不同的疾病状态下 ,体内病毒变异是不相同的 ,在体内CD4 T淋巴细胞计数较高 ,血浆病毒载量低的个体内 ,病毒毒株有很强的变异特征 ,体内具有很高的准种复杂度 ;而在CD4 细胞数很低 ,病毒载量较高的个体 ,病毒基因变异较少 ,病毒准种较为单一。结论　HIV 1准种的变化与疾病进展相关。     

Invariant aphthovirus consensus nucleotide sequence in the transition to error catastrophe

RNA viruses replicate as complex distributions of non-identical but closely related variant genomes termed viral quasispecies. When the error rate during genome replication exceeds a threshold value, the genetic information cannot be maintained and the system enters error catastrophe. This violation of the error threshold results in virus extinction and it is currently being investigated as a new antiviral strategy, based on antiviral activity of some mutagenic agents. Previous studies with the important animal pathogen foot-and-mouth disease virus (FMDV) have shown that FMDV entry into error catastrophe is associated with an increase of complexity (mutation frequency and Shannon entropy) of the mutant spectrum of the quasispecies and that mutated, pre-extinction RNA interferes with the infectivity of standard RNA. Here, we report that despite the increase of complexity, the genomic consensus nucleotide sequence of pre-extinction FMDV RNA remains invariant, and that the fitness of pre-extinction FMDV is at least six-fold lower than the fitness of the parental viral clone, prior to mutagenic treatments. Thus, a low fitness genome ensemble can suppress replication of high fitness virus. Furthermore, the results show that profound genetic modifications associated with fitness decrease of a virus population can take place without any manifestation in the consensus genomic sequence. Thus, increase in mutant spectrum complexity and invariance of the consensus sequence characterizes FMDV extinction through error catastrophe.     

Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C

AIM： To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C.
METHODS： Forty seven patients with hepatitis C [32 with chronic active hepatitis （CAH）, 9 with cirrhosis, and 6 with hepatocellular carcinoma （HCC）] were screened for the presence of quasispecies by single stranded conformational polymorphism （SSCP） analysis in the hypervariable region （HVR） and non-structural 5B （NS5B） viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients （11 non-responders and 12 showing a sustained virological response） was sequenced for the assessment of mutations.
RESULTS： The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association （P 〈 0.05） with the non-responders, and leads to persistence of infection. Significant differences （P 〈 0.05） in viral load （log10 IU/mL） were observed among patients infected with complex quasispecies （CQS）, those infected with simple quasispecies （SQS） and those with no quasispecies （NQS）, after 12 wk （CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4） and 24 wk （CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, NQS-2.1 ± 2.3） in the HVR region. However, a statistically significant difference （P 〈 0.05） was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk （CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3） and 48 wk （CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3） of therapy. Disease severity was significantly associated with viral load during th     

Population variation of West Nile virus confers a host-specific fitness benefit in mosquitoes

West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens.     

Expansion of quasispecies diversity but no evidence for adaptive evolution of SHIV during rapid serial transfers among seronegative macaques

Four successive, rapid serial passages of the nonpathogenic, CCR5-tropic simian-human immunodeficiency virus SHIV(SF162) in rhesus macaques resulted in an increase in acute plasma viremia with each passage and the emergence of a pathogenic isolate SHIV(SF162P3) in one of the passage three transfer animals (macaque T353). To explore the mechanism(s) underlying increased virulence of SHIV(SF162) upon in vivo passage, the evolution of the HIV-1 envelope gene was characterized in plasma and PBMC samples obtained from animals before (week 1) and after (week 3) the time of virus transfer. We found no evidence in support of adaptive evolution of the HIV gp120 during rapid serial passage; however, the animals which later received passage virus had more diverse quasispecies. SHIV(SF162P3)-like gp120 sequences were first detected in macaque T353 at week 6, after seroconversion. These sequence changes increased in frequency and number at later time points. The first sequence change conferred neutralization escape but not an increase in viral infectivity that could account for the apparent increase in replicative capacity of the later passage viruses. Collectively, our data argue against any host-specific adaptation of the HIV-1 envelope gp120 as the basis for the generation of more aggressive SHIV variants during rapid serial transfers in seronegative macaques, and support the model of quasispecies diversity as a predictor of pathogenesis. Envelope sequence changes accumulate principally in response to immune pressure exerted by the host, generating viral variants that can persist in the presence of a strong host immune response.     

丙型肝炎病毒感染自然过程中准种的构成变化

目的 观察慢性丙型肝炎患者与自然阴转者外周血HCV准种构成的变化规律。方法 应用基因扩增、分子克隆和测序的方法,对未接受过治疗的4例慢性丙型肝炎患者与4例自然阴转者前后10年血清中丙型肝炎病毒(hepatitis C virus,HCV)核心(C)区与包膜2(E2)区基因片段进行了序列分析及遗传进化关系比较。结果 4例慢性丙型肝炎患者10年前后血清HCV C区和其中2名患者E2区准种群体组内与组间遗传距离没有明显差异。与自然阴转者相比,慢性丙型肝炎患者外周血HCV C区和E2区准种群体组内平均遗传距离较大。在同一患者体内．HCV E2区准种群体组内遗传距离比C区组内遗传距离大,且二者遗传距离大小之间存在相关关系(rs=0．8095)。结论 在丙型肝炎疾病的自然病程中HCV准种构成可长期保持稳定;HCV准种遗传变异度大小可能与丙型肝炎疾病的转归相关。     

急性乙型肝炎患者血清中乙型肝炎病毒序列的研究

目的:研究急性乙型肝炎患者血清中乙型肝炎病毒序列。方法:提取急性乙型肝炎患者血清中HBV DNA,扩增其表面抗原序列,测序并相互比较。结果:测定5例急性乙型肝炎患者血清中乙型肝炎病毒均为adw亚型,其中3例同源性>99%。结论:adw亚型HBV感染后较多为急性病变,由于病毒变异少,机体有可能清除HBV。     

The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice

To define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype.     

Minimization of genetic distances by the consensus, ancestral, and center-of-tree (COT) sequences for HIV-1 variants within an infected individual and the design of reagents to test immune reactivity

Eliciting maximal immune responses to highly divergent viruses is a challenge and a focus in AIDS vaccine development. Another challenge is to identify the immune correlates of protective immunity. Recent AIDS vaccine design approaches attempt to use reconstructed centralized viral sequences that minimize genetic differences to circulating viruses. Using these approaches, we derive and analyze consensus (CON), ancestral (ANC), and center-of-tree (COT) sequences to represent intra-individual HIV-1 env variants encoding a range of diversities and phylogenetic structures. Each reconstructed sequence significantly minimized genetic distances to extant sequences throughout the first 5 years of infection of an individual. Interestingly, ANC sequences diverged and were not significantly better than extant sequences in minimizing genetic distances at later stages of infection and disease, likely due to the development of a substantially asymmetric phylogeny. COT or CON sequences derived from autologous virus samplings may be useful for increasing the sensitivity of assessments of immune reactivity against HIV.