Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

A new method was developed for detection of human papillomavirus (HPV) by loop-mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real-time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type-specific. In order to evaluate the reliability of HPV type-specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV-6 DNA and HPV-11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV-16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real-time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV-11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA.     

Hepatitis B virus (HBV) reactivation—The potential role of direct‐acting agents for hepatitis C virus (HCV)

Hepatitis C virus (HCV) is known to inhibit hepatitis B virus (HBV) replication in patients with HBV/HCV coinfection. Reactivation of HBV in patients treated for HCV with direct‐acting agents (DAAs) has emerged recently as an important clinical consideration. A growing number of case reports and case series support the association between new HCV treatments and HBV reactivation. Yet, very little is known about the specific viral characteristics that facilitate reactivation as functional characterization of the reactivated HBV has been conducted only rarely. This review provides the most recent data on HBV reactivation in the context of DAA initiation and highlights the existing viral genomic data from reactivating viruses. Current functional studies of HBV reactivation are largely limited by the retrospective identification of cases, no standardization of genomic regions that are studied with respect to HBV reactivation, and the lack of inclusion of nonreactivating controls to establish specific viral mutations that are associated with HBV reactivation. Importantly, none of these sequencing studies included cases of HBV reactivation after initiation of DAAs. While new HCV treatments have revolutionized care for HCV infected patients, HBV reactivation will likely increase in frequency, as DAAs are more commonly prescribed. Pretreatment determination of HBV status and thoughtful management of HBV coinfections will be necessary and lead to improved patient safety and yield optimal treatment results.     

Molecular detection and sequencing of "Norwalk-like viruses" in outbreaks and sporadic cases of gastroenteritis in Ireland

Norwalk-like viruses (NLVs) are now established as the most important causative agents of epidemic gastroenteritis worldwide. The overall objective of this study was to determine the molecular epidemiology of Irish NLV isolates for the first time by obtaining sequence data from specimens originating from outbreaks and sporadic cases of gastroenteritis. Eight samples from sporadic cases of gastroenteritis and nine isolates from separate NLV outbreaks were examined. Of the sporadic isolates, six were shown to be genogroup 2 (G2) by RT-PCR, while two were G1. All of the outbreak isolates were G2. All isolates were partially sequenced within a highly conserved region of ORF1 (RNA-dependent RNA polymerase gene). Sequence data were aligned and a dendogram was constructed. The results indicated that the majority of G2 isolates were seen to cluster with Bristol and Lordsdale virus, while the two G1 specimens were related most closely to Southampton virus. Further downstream sequence analysis of a number of the isolates confirmed this result. It is concluded that the majority of NLV isolates circulating in Ireland belong to the Bristol/Lordsdale clade.     

Hepatitis B virus (HBV) X gene diversity and evidence of recombination in HBV/HIV co-infected persons

The high frequency of mutation during hepatitis B virus (HBV) infection has resulted in 8 genotypes (A-H) with varying effects on disease severity and treatment efficacy. However, analysis of intrapatient HBV diversity is limited, especially during HIV co-infection. Therefore, a preliminary study was performed to analyze HBV X gene diversity in 17 HBV/HIV co-infected individuals. Phylogenetic analysis revealed HBV genotype A in 13 individuals (76.5%) or genotype E in 1 individual (5.9%). Additionally, 3 individuals were dually infected with HBV genotypes A and G (17.6%). Overall, higher genetic distance and entropy were observed in the X region and overlapping polymerase (Pol(X)) regions when compared to the PreS, S, and overlapping polymerase (Pol(PS) and Pol(S)) regions analyzed in the same patients as part of a previous study. In addition, multiple viral variants from 2 individuals with dual HBV infection did not group with either genotype A or G by phylogenetic analysis, indicating possible recombination. SimPlot bootscan analysis confirmed recombination breakpoints within the X gene in both individuals. Recombination between HBV genotypes may represent an important evolutionary strategy that enhances overall pathogenic potential and/or alters the downstream effects of the HBV X protein.     

乙型肝炎病毒DNA在肝癌患者基因组中的检测

目的：为了研究乙型肝炎病毒ＤＮＡ在人类基因组中的整合作用与肝癌发生之间的关系。方法：采用聚合酶链反应技术,对肝癌细胞基因组中的乙肝病毒ＤＮＡ第Ｖ 区段进行了测定。结果：１２ 例肝癌细胞基因组８ 例为阳性,１０ 例肝癌患者外周血细胞基因组均为阴性。结论：乙型肝炎病毒ＤＮＡ 的整合与肝癌发生有密切关系。     

乙型肝炎病毒基因分型荧光PCR检测及临床应用

目的:了解乙型肝炎病毒基因分型、分布及其与肝功能损害、病毒复制水平的关系.方法:采集69例乙肝患者血样,采用荧光聚合酶链反应(PCR)检测乙肝病毒基因分型,通过荧光探针识别基因型特异性序列,采集分析荧光信号确定病毒基因型,分别检测谷丙转氨酶(ALT)水平、乙肝病毒e抗原(HBeAg)和HBV DNA含量.结果:69例乙肝患者中B型26例,C型43例.B型和C型患者的ALT水平分别为(453.54±447.93)U/L和(330.23±306.90)U/L(P＞0.05);HBeAg阳性数分别为11例和23例(P＞0.05);HBV DNA含量分别为(38.74±36.49)×104拷贝/ml和(34.53±34.09)×104拷贝/ml(P＞0.05);不同临床类型乙肝患者中两种基因型的分布没有显著差异(P＞0.05).结论:本地区乙肝患者病毒基因型主要为B型和C型;二种基因型ALT水平、HBeAg表达水平、病毒复制水平以及肝炎病情均无显著性差异.     

慢性乙肝患者HBV核心基因一组单核苷酸多态性与血清HBV DNA水平的相关性研究

目的 研究慢性乙肝患者HBV核心基因的单核苷酸多态性(SNP)与血清HBV DNA水平的相关关系.方法 采用PCR-RFLP和限制性内切酶Tsp509I榆测HBV核心基因的SNP,采用双脱氧终止法对核心基因进行序列测定,实时荧光PCR技术用于HBVDNA水平的定量检测.结果 慢性乙肝人群中发现5种典型的PCR-RFLP图谱,分别是RFLP-C、RFLP-D、RFLP-E、RFLP-G和RFLP-C/G,各种RFLP图谱的分布频率依次为61.5%、2.6%、9.6%、16.7%和9.6%.A165T、A336C、A336T、T337C和T385C等5种SNP与RFLP图谱的变化有关,但是只有SNP A336C和A336T会导致HBcAg第83位的Glu被Asp所替代.RFLP-C组患者的血清HBV DNA水平显著高于RFLP-G组(P=0.02)和RFLP-C/G组(P=0 006),而且RFLP-C/G组患者血清ALT的阳性率显著低于RFLP-C、RFLP-E和RFLP-G组;当HBeAg阳性时,RFLP-C组患者的血清HBV DNA水平显著高于RFLP-G组(P=0.015)和RFLP-C/G组(P=0.008).结论 本研究中使用的PCR-RFLP能够用于检测核心基因的SNP,RFLP-C组患者的血清HBV DNA水平显著高于RFLP-G组和RFLP-C/G组,可能与Glu83 Asp突变有关.

Abstract：

Objective To investigate the relation between a set of single nucleotide polymorphisms (SNP) in core gene of HBV in chronic hepatitis B patients and HBV DNA levels. Methods PCR restriction fragment length polymorphism(PCR-RFLP) assay and restriction enzyme Tsp509I were adopted to determine HBV SNP in HBV core gene. Nucleotide sequences of core gene were determined using the dideoxy chain termination method. HBV DNA levels were quantitated with real-time PCR. Results Five typical RFLP patterns, RFLP-C, RFLP-D, RFLP-E, RFLP-G and RFLP-C/G mixture were found and the distribution of HBV RFLP patterns was as follows: C, 61. 5% ; D, 2. 6% ; E, 9.6%; G, 16.7%; C/G mixture, 9.6%. Five SNPs, A165T, A336C, A336T, T337C and T385C, were found to be associated with RFLP patterns change and only SNP A336C or A336T caused the substitution of Glu-83 with Asp in HBcAg. The serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P =0. 02) and RFLP-C/G group(P = 0. 006) , respectively, furthermore, the positive rate of serum ALT in RFLP-C/G group was lower than that in RFLP-C, RFLP-E and RFLP-G group, respectively. Under the condition of HBeAg-positive, the serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P = 0. 015) and RFLP-C/G group(P =0.008) , respectively. Conclusion PCR-RFLP used in this study can be adopted to determine HBV SNPs, not genotypes in Chinese patients with chronic hepatitis B. The serum HBV DNA level in RFLP-C group higher than that in RFLP-G or RFLP-C/G group maybe associated with amino acid mutation, Glu83 Asp.     

不同基因型HBV感染患者血清Lp(a)检测及其临床意义分析

目的 探讨乙型肝炎病毒(hepatitis B virus,HBV)感染患者不同基因型与健康人血清脂蛋白(a)[lipoprotein (a),LP(a)]水平相关性的临床意义.方法 分别取HBV感染患者A基因型35例,B基因型57例,C基因型56例,D基因型52例和健康对照64例血清,采用全自动生化分析仪对其脂蛋白(a)进行检测.结果 A,B,C,D四个基因型HBV感染患者的Lp(a)均低于健康对照组(P＜0.05).而且不同基因型HBV感染患者的最终治疗转归率和治愈率不同.结论 HBV能抑制脂蛋白(a)的表达,且其血清中表达水平与乙肝的基因型相关.     

Prevention of perinatal transmission of hepatitis B virus (HBV): a comparison of two prophylactic schedules

Perinatal transmission of hepatitis B virus (HBV) from HBsAg carrier mothers who were HBeAg+, antiHBe+, or negative for both HBe markers, was interrupted using either 4 doses of vaccine, or one dose of hepatitis B immunoglobulin (HBIG) at birth, combined with 4 doses of vaccine. In those infants who received HBIG at birth, the antiHBs titre was significantly higher at 1 and 2 months old, but at 6, 9, and 18 months old, there was no significant difference. Among the infants of carrier mothers who did not display HBeAg (i.e., were antiHBe+, or negative for both HBe markers), a transient subclinical infection would have been expected in around 10% had there been no intervention. No evidence of such infection was detected, and no difference in outcome was found between the two treatment groups. Amongst infants born to HBeAg+ carrier mothers, infection occurred in 1 out of 8 who had received HBIG and vaccine, and in 3 of 8 who had received vaccine only. The difference in outcome was not statistically significant, but the numbers analysed were small. The infections which occurred in spite of prophylaxis may be attributable to in utero infection, poor response to vaccine by the infant, or to the mother having a particularly high HBV-DNA level. HBIG given at birth to infants of HBeAg+ carrier mothers may enhance the protection of infants who are destined to be poor responders to vaccine.     

Heat shock protein 90 facilitates formation of the HBV capsid via interacting with the HBV core protein dimers

The mechanism by which host factors contribute to hepatitis B virus (HBV) capsid formation during the viral life cycle remains unclear. This study analyzed the interaction between heat shock protein 90 (Hsp90), a host factor, and the HBV core protein. Hsp90 was found to bind to HBV core protein dimers, which was then encapsidated into the HBV capsid. Furthermore, activated Hsp90 may facilitate the formation of the human HBV capsid by catalyzing core assembly and reducing the degree of capsid dissociation at various temperatures, both in vitro and in vivo, and when subjected to detergent treatments in vitro. In addition, inhibition or downregulation of Hsp90 reduced HBV production in HepG2.2.15 cells. These results showed that Hsp90 plays an important role in HBV capsid stabilization and HBV formation.     

乙型肝炎患者血清中庚肝病毒核酸的检测

目的:了解乙型肝炎患者HGV感染情况,探讨HGV感染与HBV感染的关系.方法:应用巢氏PCR对1104例乙型肝炎患者进行了血清HGV-RNA检测,以251名正常人为对照.结果:共检出血清HGV-RNA阳性者34例,阳性率3.08%.乙型肝炎患者血清HGV-RNA阳性率(3.17%)与正常人群(2.79%)相比无显著差异(P＞0.05).慢性乙肝患者血清HGV-RNA阳性率(4.78%)显著高于急性乙肝患者(0.96%)(P＜0.05).结论:HGV感染可表现为病毒携带状态、亚临床型和不同临床类型,乙型肝炎患者并不比正常人群更易感染HGV,HGV与HBV重叠感染可能与病情发展和慢性化的形成有关.     

Development of sensitive methods for detection of porcine endogenous retrovirus-C (PERV-C) in the genome of pigs

Porcine endogenous retroviruses (PERV) represent a risk for xenotransplantation using pig cells, tissues or organs. PERV-A and PERV-B are present in the genome of all pigs and both infect human cells in vitro. PERV-C infects only pig cells and it is integrated in the genome of most, but not all pigs. Recombinants between PERV-A and PERV-C were described that infect human cells and replicate at high titres. To avoid such recombinations, PERV-C positive animals should not be used for breeding animals suited for xenotransplantation.In order to detect PERV-C positive pigs, different methods were developed such as specific PCRs using different primers, a highly sensitive nested PCR and a real-time PCR allowing measurement of proviral copy numbers. The real-time PCR was found to be useful to discriminate between contamination and actual provirus copies. The PCRs were optimized and their sensitivity was determined. Screening can be started with PCR1, if the result is negative, PCR2 to PCR5 or the nested PCR should be used, if the result is positive, the real-time PCR should be used to exclude contaminations. All methods were used to evaluate the prevalence of PERV-C and to identify PERV-C free animals. Due to the risk of contamination with cells from other animals testing should be performed with blood cells, not with ear biopsies.     

HBV subtype as a marker of the clinical course of chronic HBV infection in Japanese patients

Hepatitis B virus (HBV) genotype C is predominant in Japan. However, many HBV subtypes are involved in each genotype, and the clinical manifestations in the patients associated with each subtype remain unknown. Therefore, we investigated the relationship between HBV subtype and clinical aspects of chronic HBV infection. The subtype of 237 patients with chronic HBV infection, including 74 asymptomatic carriers, was determined. The subtypes of 110 HBV carriers undergoing long-term follow-up management were determined twice to detect subtypic changes. The clinical features of the patients were also studied with regard to presence or absence of subtypic change. The subtypic distribution in the 237 HBV carriers was as follows: subtype adr, 161 (68%); subtype adw, 25 (11%); subtype adwr, 12 (5%); subtype ar, 24 (10%); subtype adyr, 4 (2%); and unclassified, 8 (3%). The proportion of asymptomatic carriers in patients with subtype adw was significantly higher than those in patients with subtype adr (56% vs. 28%, P < 0.05). In addition, the proportion of HCC in patients with subtype adwr was significantly higher than those in patients with subtype adr (25% vs. 6%, P < 0.05). The prevalence of subtype adr in 74 asymptomatic carriers tended to decrease with age (82% in carriers aged < or =35 years vs 43% in those aged > or =61 years, P < 0.05). The subtypic change and the course of chronic HBV infection had no significant correlation. These results suggest that HBV subtypes are associated with the clinical course of chronic HBV infection.