YVDD mutation of hepatitis B Virus, a dominant Lamivudine-resistant type in Guangzhou, South China

The epidemiological effects of native and mutated YMDD motif in the HBV genome under the selective pressure of lamivudine were investigated. YMDD wild and mutation motif in HBV genome were detected by flow through reverse dot blots (FT-RDB) with KaiPuTM DNA HybriMax Rapid Hybridization Machine based on the principle of "Flow-through hybridization" and by the traditional Reverse Dot Blot assay. Sera from 1021 suspected lamivudine-resistant chronic HBV carders after more than 8 months of lamivudine therapy and the corresponding archived sera were collected and assayed. We found 35.94% were single type infections with 8.03% YMDD, 7.93% YIDD and 19.98% YVDD. It was also found that 64.06% were mixed infections including 1.96% YMDD and YIDD, 51.62% YMDD and YVDD, 1.96% YIDD and YVDD, 8.52% YMDD, YIDD and YVDD. The levels of infections containing YVDD motif reached 82.08%. The pretreatment infectious status were: YMDD single infection was 36.93%; YIDD single infection was 6.07%; YVDD single infection was 17.04%; YMDD and YIDD mixed infection was 0.97%; YMDD and YVDD mixed infection was33.99%; YIDD and YVDD mixed infection was 0.98%; YMDD, YIDD and YVDD mixed infection was 4.02%. Infections containing YVDD motif were only 56.03%. The 34.32% mutation rate of YMDD motif to YVDD was significantly higher than the 10.97% of YMDD to YIDD (U=10.98, P<0.05), as estimated by Mann-Whitney U-test for non-parametric data. HBV containing YVDD motif might have an evolutionary ascendancy and become the dominant type under the selective pressure of lamivudine.     

采用熔点曲线法研究乙肝病毒准种与拉米夫定临床耐药相关性

为了研究乙型肝炎病毒(HBV)准种与拉米夫定耐药的关系以指导临床用药,随机选取拉米夫定治疗后发生YMDD变异的慢性乙型肝炎(CHB)患者30例,以及停用拉米夫定后发生病毒反弹的CHB患者30例作为研究对象,同时以未经拉米夫定治疗的CHB患者30例为对照,采用聚合酶链反应(PCR)扩增这些病人体内HBV的P区,再用熔点曲线法分析3组患者体内的HBV准种情况。另外,采用相同方法对HBV C区、S区准种也进行了对比。结果显示,病毒变异组患者体内HBV P区准种数量为2.50±0.86个,病毒反弹组为5.30±0.95个,未治疗组为8.37±0.93个,3组间准种数量两两比较均有显著性差异(P<0.05)。HBV C区病毒变异组准种数量为6.10±1.86个,病毒反弹组为6.37±1.81个,未治疗组为6.33±1.64个,3组准种数量无显著性差异(P>0.05)。HBV S区病毒变异组准种数量为5.23±1.85个,病毒反弹组为6.17±1.93个,未治疗组为5.77±2.11个,3组准种数量无显著性差异(P>0.05)。HBV P区熔点曲线图显示,病毒变异组优势病毒群的熔点与病毒反弹组和未治疗组相比,已发生明显的偏移,而在HBV C区和S区熔点曲线图中,3组的波峰数和优势病毒群的熔点均没有明显变化。可见,在拉米夫定的作用下HBV P区准种数量减少,准种的性质也发生变化,发生变异后劣势耐药病毒株变为优势病毒株易被检测到。拉米夫定对HBV C区、S区作用不明显。     

Dynamic changes of hepatitis B virus polymerase gene including YMDD motif in lamivudine-treated patients with chronic hepatitis B

The mutation of YMDD motif of hepatitis B virus (HBV) polymerase gene is the most frequent cause in HBV resistant to lamivudine. The aim of the study was to investigate variation features of HBV polymerase gene in chronic hepatitis B (CHB) patients before and after lamivudine treatment. From the serum samples of five CHB patients before and after 12 months of lamivudine treatment, HBV polymerase gene was amplificated and positive DNA fragments were cloned into JM105 competent cell. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Among five patients after 12 months of lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except two patients with negative HBV DNA consecutively. In summary, D553G mutation is probably one of the reasons that caused non-responders during lamivudine treatment. The mutations of YMDD motif occurred after lamivudine treatment are caused by the induction of lamivudine.     

Evaluation of intra-host variants of the entire hepatitis B virus genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.     

Tracking hepatitis C virus quasispecies major and minor variants in symptomatic and asymptomatic liver transplant recipients.

To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.     

Subpopulations of equine infectious anemia virus Rev coexist in vivo and differ in phenotype

Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.     

乙型肝炎病毒逆转录酶区基因序列准种与变异特点

乙型肝炎病毒(Hepatitis B Virus,HBV)P基因编码产物从功能上分为末端蛋白(1～178aa)、间隔区(179～336aa)、逆转录酶区(337～682aa)和RNA酶H区(683～816aa),各区有相应的生物学功能;逆转录酶区包含S基因主蛋白编码区.近年来的研究提出HBV感染者体内存在有准种[1,2]的假说.我们以逆转录酶区序列为研究靶区域,应用聚合酶链反应(PCR)技术扩增慢性乙型肝炎患者血清中的靶基因序列,随机选择克隆测序,比较其结果,证明了HBV准种特点的存在,并发现多种基因突变形式.     

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).     

VSD负压引流技术结合游离植皮治疗大面积皮肤缺损的 临床疗效研究

目的：探讨核苷类似物（NAs）拉米夫定（LAM）用药时间与乙型肝炎（cHB）患者体内HBV病毒血清HBV-DNA载量的关系。方法：选取214例HBeAg阳性患者,在LAM（100mg/d）的36个月用药治疗前使用实时荧光定量PcR进行血清HBV-DNA载量进行测定,并使用特异性引物鉴定HBV病毒DNA保守位点YMDD的抗药性突变以及病毒株的变化情况。结果：（1）214例患者均对LAM产生应答,在治疗12．18个月后血清HBV-DNA载量稳定在较低水平。（2）病毒株在NAs治疗前已经出现耐药性突变（18％）,用药加速了HBV病毒的耐药进化,18个月后耐药病毒数在214例样本中均超过10％的检测标准。结论：LAM治疗在9．18个月获得良好的治疗效果,建议24个月后停药更换临床处方防止耐药病毒株的进化。     

Genetic divergence with emergence of novel phenotypic variants of equine arteritis virus during persistent infection of stallions

The persistently infected carrier stallion is the critical natural reservoir of equine arteritis virus (EAV), as venereal infection of mares frequently occurs after breeding to such stallions. Two Thoroughbred stallions that were infected during the 1984 outbreak of equine viral arteritis in central Kentucky subsequently became long-term EAV carriers. EAV genomes amplified from the semen of these two stallions were compared by sequence analysis of the six 3' open reading frames (ORFs 2 through 7), which encode the four known structural proteins and two uncharacterized glycoproteins. The major variants of the EAV population that sequentially arose within the reproductive tract of each carrier stallion varied by approximately 1% per year, and the heterogeneity of the viral quasispecies increased during the course of long-term persistent infection. The various ORFs of the dominant EAV variants evolved independently, and there was apparently strong selective pressure on the uncharacterized GP3 protein during persistent infection. Amino acid changes also occurred in the V1 variable region of the GL protein. This region has been previously identified as a crucial neutralization domain, and selective pressures exerted on the V1 region during persistent EAV infection led to the emergence of virus variants with distinct neutralization properties. Thus, evolution of the EAV quasispecies that occurs during persistent infection of the stallion clearly can influence viral phenotypic properties such as neutralization and perhaps virulence.     

Characterization of Acute and Chronic Hepatitis B Virus Genotypes in Canada

Objective

The prevalence and distribution of hepatitis B virus (HBV) genotypes in Canada is not known. Genotypic analysis may contribute to a better understanding of HBV strain distribution and transmission risk.

Methods

HBV surface antigen (HBsAg) positive samples of acute (n = 152) and chronic (n = 1533) HBV submitted for strain analysis or reference genotype testing between 2006 and 2012 were analyzed. The HBsAg coding region was amplified to determine the HBV genotype by INNO-LiPA assay or sequence analysis. Single and multivariate analyses were used to describe genotypes’ associations with known demographic and behavioral risk factors for 126 linked cases of acute HBV.

Results

Nine genotypes were detected (A to I), including mixed infections. Genotype C (HBV/C) dominated within chronic infections while HBV/D and A prevailed among acute HBV cases. History of incarceration and residing with a chronic HBV carrier or injection drug user were the most frequently reported risks for acute HBV infection. Over time, HBV/A increased among both acute and chronic infections, and HBV/C and HBV/D decreased among chronic infections.

Conclusion

Chronic and acute HBV genotypes in Canada differ in the relative distribution and their associations with known risk factors, suggesting different routes of transmission and clinical progression of infection.     

Isolation of CD4-independent primary human immunodeficiency virus type 1 isolates that are syncytium inducing and acutely cytopathic for CD8+ lymphocytes

Previous studies have established the existence of CD4-independent simian immunodeficiency virus, human immunodeficiency virus type 2 (HIV-2), and laboratory strains of HIV-1. However, whether CD4-independent viruses may also exist in HIV-1-infected patients has remained unclear. We have recently reported the isolation of viruses from an AIDS patient that were able to infect CD8(+) cells independent of CD4, using CD8 as a receptor. Using a similar approach, here we examined viruses from 12 randomly selected patients (obtained from the AIDS Research and Reference Program, National Institutes of Health) for the presence of CD4-independent HIV-1. CD4-independent variants were isolated from infected CD8(+) cells from the viral quasispecies of 7 of 12 patients. The CD4-independent isolates were able to infect primary CD8(+) cells as well as a CD4(-) CD8(+) T-cell line. Soluble CD4 and blocking anti-CD4 or -CD8 antibody had no effect on infection of CD8(+) cells. Remarkably, two of the seven CD4-independent isolates, but not their parental bulk viruses, induced syncytia and caused acute death of infected CD8(+) cells. Some of the CD4-independent variants were also able to infect U87 cells that were negative for CD4, CD8, and common HIV coreceptors, suggesting a novel entry mechanism for these isolates. The CD4-independent isolates were derived from adults and children infected with subtypes A, B, and D. Although no common motif for CD4 independence was found, novel sequence changes were observed in critical areas of the envelopes of the CD4-independent viruses. These results demonstrate that HIV-1-infected patients can frequently harbor viruses that are able to mediate CD4-independent infection of CD8(+) cells. In addition, this study also provides evidence of primary HIV-1 variants that are syncytium inducing and acutely cytopathic for CD8(+) lymphocytes.     

Influence of the mutant spectrum in viral evolution: focused selection of antigenic variants in a reconstructed viral quasispecies

RNA viruses replicate as complex mutant distributions termed viral quasispecies. Despite this, studies on virus populations subjected to positive selection have generally been performed and analyzed as if the viral population consisted of a defined genomic nucleotide sequence; such a simplification may not reflect accurately the molecular events underlying the selection process. In the present study, we have reconstructed a foot-and-mouth disease virus quasispecies with multiple, low-frequency, genetically distinguishable mutants that can escape neutralization by a monoclonal antibody. Some of the mutants included an amino acid substitution that affected an integrin recognition motif that overlaps with the antibody-binding site, whereas other mutants included an amino acid substitution that affected antibody binding but not integrin recognition. We have monitored consensus and clonal nucleotide sequences of populations passaged either in the absence or the presence of the neutralizing antibody. In both cases, the populations focused toward a specific mutant that was surrounded by a cloud of mutants with different antigenic and cell recognition specificities. In the absence of antibody selection, an antigenic variant that maintained integrin recognition became dominant, but the mutant cloud included as one of its minority components a variant with altered integrin recognition. Conversely, in the presence of antibody selection, a variant with altered integrin recognition motif became dominant, but it was surrounded by a cloud of antigenic variants that maintained integrin recognition. The results have documented that a mutant spectrum can exert an influence on a viral population subjected to a sustained positive selection pressure and have unveiled a mechanism of antigenic flexibility in viral populations, consisting in the presence in the selected quasispecies of mutants with different antigenic and cell recognition specificities.     

Hepatitis e virus quasispecies and the outcome of acute hepatitis e in solid-organ transplant patients

Hepatitis E virus (HEV) infections are responsible for chronic hepatitis in immunocompromised patients, and this can evolve to cirrhosis. Like all RNA viruses, HEV exists as a mixture of heterogeneous viruses defining quasispecies. The relationship between the genetic heterogeneity described as a quasispecies, cytokine secretion, and the outcome of acute hepatitis in immunocompromised patients remains to be elucidated. We cloned and sequenced the region encoding the M and P capsid domains of HEV from eight solid-organ transplant (SOT) patients with acute HEV infection who subsequently cleared the virus and from eight SOT patients whose infection became chronic. We analyzed the cytokines and chemokines in the sera of these SOT patients by multianalyte profiling. The nucleotide sequence entropy and genetic distances were greater in patients whose infections became chronic. A lower K(a)/K(s) ratio was associated with the persistence of HEV. The patients who developed chronic infection had lower serum concentrations of interleukin-1 (IL-1) receptor antagonist and soluble IL-2 receptor. Increased concentrations of the chemokines implicated in leukocyte recruitment to the liver were associated with persistent infection. Those patients with chronic HEV infection and progressing liver fibrosis had less quasispecies diversification during the first year than patients without liver fibrosis progression. Great quasispecies heterogeneity, a weak inflammatory response, and high serum concentrations of the chemokines involved in leukocyte recruitment to the liver in the acute phase were associated with persistent HEV infection. Slow quasispecies diversification during the first year was associated with rapidly developing liver fibrosis.