Parvovirus B 19 infections

We report the human parvovirus (HPV) infection cases diagnosed in 1984 and 1985 in the virological laboratory of C.N.T.S. Detection of viral antigen and total anti-HPV antibodies was performed by electroimmunodiffusion, and specific IgM by antibody-capture-radioimmunoassay. Seven viraemias were found in 38,730 sera sent for detection of hepatitis A and B markers. Twenty-two observations of aplastic crisis are described, with underlying haemolytic anaemias in 21 cases. HPV infection was serologically proven in 4 cases out of 22 rubeolelike illness, and, in 17 cases out of 17 erythema infectiosum (fifth disease). Four cases of vascular purpura (one of which was Schonlein-Henoch purpura) were studied, and HPV antigen was isolated in 2 cases. An observation of arthropathies in a young adult is described. Finally, 2 spontaneous abortions were simultaneous to an HPV infection. Our study allows us to underline the following points: HPV is the principal (but not exclusive) agent of the aplastic crisis in chronic haemolytic anaemias; these acute erythroblastopenias can reveal an unknown haemolytic anaemia, in particular in hereditary spherocytosis; the polymorphism of the clinical expression of HPV infection is important. Some viraemias are asymptomatic, while others are accompanied by vascular purpura, or atypic erythema; HPV has never been isolated in the fifth disease. Nonetheless bringing out of specific IgM and association of HPV infection linked manifestations in a same patient or in a same family shows its responsibility in the fifth disease; HPV responsibility in spontaneous abortions and fetal malformations remains to be demonstrated.     

Prevalence of HPV 16 and HPV 18 Lineages in Galicia,Spain

Genetic variants of human papillomavirus types 16 and 18 (HPV16/18) could differ in their cancer risk. We studied the prevalence and association with high-grade cervical lesions of different HPV16/18 variant lineages in a case-control study including 217 cases (cervical intraepithelial neoplasia grade 2 or grade 3 or worse: CIN2 or CIN3+) and 116 controls (no CIN2 or CIN3+ in two-year follow-up). HPV lineages were determined by sequencing the long control region (LCR) and the E6 gene. Phylogenetic analysis of HPV16 confirmed that isolates clustered into previously described lineages: A (260, 87.5%), B (4, 1.3%), C (8, 2.7%), and D (25, 8.4%). Lineage D/lineage A strains were, respectively, detected in 4/82 control patients, 19/126 CIN3+ cases (OR = 3.1, 95%CI: 1.0–12.9, p = 0.04), 6/1 glandular high-grade lesions (OR = 123, 95%CI: 9.7–5713.6, p<0.0001), and 4/5 invasive lesions (OR = 16.4, 95%CI: 2.2–113.7, p = 0.002). HPV18 clustered in lineages A (32, 88.9%) and B (4, 11.1%). Lineage B/lineage A strains were respectively detected in 1/23 control patients and 2/5 CIN3+ cases (OR = 9.2, 95%CI: 0.4–565.4, p = 0.12). In conclusion, lineages A of HPV16/18 were predominant in Spain. Lineage D of HPV16 was associated with increased risk for CIN3+, glandular high-grade lesions, and invasive lesions compared with lineage A. Lineage B of HPV18 may be associated with increased risk for CIN3+ compared with lineage A, but the association was not significant. Large well-designed studies are needed before the application of HPV lineage detection in clinical settings.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

Inhibition of growth, transformation, and expression of human papillomavirus type 16 E7 in human keratinocytes by alpha interferons.

We used a model system of normal human keratinocytes (HKc) and HKc immortalized with human papillomavirus type 16 DNA (HKc/HPV16) to investigate the effects of alpha interferons (IFN-alpha) on the growth of HPV16-immortalized human epithelial cells, on HPV16-mediated immortalization of normal HKc, and on HPV16 gene expression. Normal HKc and HKc/HPV16 were treated with several recombinant human IFN-alpha subtypes (IFN-alpha B, IFN-alpha D, and IFN-alpha B/D). These IFN-alpha subtypes inhibited proliferation of both normal HKc and HKc/HPV16 in a dose-dependent fashion; however, although 1,000 to 10,000 U of IFN-alpha per ml were required to inhibit growth of normal HKc, HKc/HPV16 were substantially growth inhibited by 100 U/ml. In addition, 100 U of IFN-alpha B/D per ml inhibited transformation of normal HKc by HPV16 DNA. Northern (RNA) blot analysis showed no effect of IFN-alpha on the mRNA levels of the HPV16 E6 and E7 open reading frames. However, immunofluorescence studies of the HPV16 E6 and E7 proteins with anti-E6 and anti-E7 monoclonal antibodies showed significant inhibition of E7 protein expression in cells treated with IFN-alpha, whereas E6 protein expression was not altered. The inhibition of E7 protein expression in cells treated with IFN-alpha was further confirmed by Western immunoblot analysis. These results suggest that IFN-alpha may inhibit HPV16-mediated transformation of HKc and proliferation of HKc/HPV16 through an inhibition of HPV16 E7 protein expression.     

Genetic characteristics of human papillomavirus type 16, 18, 52 and 58 in southern China

Persistent infections of high-risk human papillomaviruses (HPVs) are the leading cause of cervical cancers. We collected cervical exfoliated cell samples from females in Changsha city, Hunan Province and obtained 338 viral genomes of four major HPV types, including HPV 16 (n = 82), 18 (n = 35), 52 (n = 121) and 58 (n = 100). The lineage/sublineage distribution of the four HPVs confirmed previous epidemiological reports, with the predominant prevailing sublineage as A4 (50%), A1 (37%) and A3 (13%) for HPV16, A1 (83%) for HPV18, B2 (86%) for HPV52 and A1 (65%), A3 (19%) and A2 (12%) for HPV58. We also identified two potentially novel HPV18 sublineages, i.e. A6 and A7. Virus mutation analysis further revealed the presence of HPV16 and HPV58 sublineages associated with potentially high oncogenicity. These findings expanded our knowledge of the HPV genetic diversity in China, providing valuable evidence to facilitate HPV DNA screening, vaccine effectiveness evaluation and control strategy development.     

兰州地区女性感染HPV及其基因类型分析

目的:了解兰州地区成年女性感染人乳头瘤病毒(humanpapillomavirus,HPV)及其基因类型分布状况,为本地区HPV分子流行病学研究提供理论依据。方法:利用PCR技术分别对100例妇科门诊就诊者进行HPV基因亚型检测。结果:100例样品中,HPV DNA检出率为19%(19/100),其中HPV16 DNA感染率15%(15/100),HPV58 DNA感染率3%(3/100),HPV18 DNA感染率2%(2/100),HPV 16与HPV18双重感染1例。结论:本地区成年女性HPV感染主要以HPV16多见,而HPV16与恶性肿瘤密切相关,因此对HPVDNA阳性者定期随访,有利于宫颈癌的防治。     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.     

Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity

Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.     

Erythema infectiosum following generalized petechial eruption induced by human parvovirus B19

Parvovirus B19 is a DNA virus responsible for a wide spectrum of clinical illnesses. Among dermatological manifestations, the most common is erythema infectiosum, also known as the fifth disease. In 1990 Harms et al first described a papular-purpuric gloves and socks syndrome (PPGSS) due to parvovirus B19. It is an acute acral dermatosis characterized by an eruption of petechiae and small purpuric papules affecting the hands and feet in a gloves-and-socks distribution. Recently it was observed that PPGSS may be associated with involvement of the peri-oral region and the chin (acropetechial syndrome) and other sites provoking unusual presentation of the rash. We describe a patient with an acral purpura with the features of the "acropetechial syndrome" involving the buttocks, genital and axillary regions who subsequently developed a maculopapular eruption with the characteristics of the fifth disease. Parvovirus B19 DNA was detected by polymerase chain reaction (PCR) both in skin vasculitic lesions and in the serum during the petechial eruption, before the onset of antibodies. The immune response coincided with the development of the exanthem, suggesting a direct role of parvovirus B19 in the pathogenesis of endothelial cell injury.     

In Vivo and In Vitro Studies Suggest a Possible Involvement of HPV Infection in the Early Stage of Breast Carcinogenesis via APOBEC3B Induction

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.     

肛门生殖器区肿瘤组织中HPV的快速检测和分型

迄今已发现的人乳头瘤病毒（ＨＰＶ）型已超过７７型,其中至少有３０个型与泌尿生殖道肿瘤有关。所以ＨＰＶ的检测与分型对于泌尿生殖道肿瘤的病因和预后将是非常重要的。ＨＰＶ有很大的异质性,故用常规的诊断技术来测定未知标本中的ＨＰＶ基因型有一定困难。为此,我们应用了反向点杂交法（ＲＯＢ）来检测ＨＰＶ分型。即用７种序列特异性寡核苷酸探针分别对应７型ＨＰＶ（ＨＰＶ６Ｂ、１１、１６、１８、３１、３３、３５）,这些     

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.     

孟鲁司特钠联合双歧杆菌四联活菌片对小儿过敏性紫癜的疗效

目的 对孟鲁司特钠联合双歧杆菌四联活菌片治疗小儿过敏性紫癜的临床疗效进行分析。方法 选取2015年3月1日至2018年3月31日我院收治的96例过敏性紫癜患儿为研究对象,依据随机数字表法分为A组、B组、C组、D组,每组24例。A组患儿施以常规临床治疗,B组在A组基础上给予孟鲁司特钠治疗、C组在A组基础上给予双歧杆菌四联活菌片治疗、D组在A组基础上施以孟鲁司特钠联合双歧杆菌四联活菌片治疗,4组患儿持续用药至出院后2个月。结果 A组患儿治疗有效率为66.67%,皮肤紫癜消失时间为（10.49±3.01）d,关节肿痛消失时间为（5.87±1.56）d,腹痛消失时间为（4.05±1.28）d,Alb水平为（27.11±7.80）mg/L,β2-MG水平为（0.31±0.15）mg/L,治疗后复发率为29.17%。D组患儿治疗有效率为91.67%、皮肤紫癜消失时间为（6.98±2.57）d,关节肿痛消失时间为（3.07±1.04）d,腹痛消失时间为（2.77±1.05）d,Alb水平为（22.00±8.47）mg/L,β2-MG水平为（0.19±0.06）mg/L,治疗后复发率为4.17%。D组患儿各指标与A组比较差异均有统计学意义（均P＜0.05）。B、C组患儿治疗有效率高于A组,但低于D组。B、C组患儿皮肤紫癜消失时间、关节肿痛消失时间、腹痛消失时间与A组比较差异无统计学意义（P＞0.05）,与D组相比差异有统计学意义（P＜0.05）。结论 孟鲁司特钠联合双歧杆菌四联活菌片对小儿过敏性紫癜的疗效显著,能减少患儿不良反应发生,促进患儿恢复健康,值得临床推广。     

人乳头瘤病毒(HPV)31型中和单克隆抗体的制备及性质鉴定

目的:构建获得HPV31构象依赖的中和单抗。方法:采用昆虫细胞表达的HPV31 L1VLP(virus-like particle, VLP)免疫BALB/c小鼠,取免疫鼠脾细胞与SP2/0细胞融合,收集杂交瘤细胞培养上清,首先采用HPV31 VLP-ELISA及HPV31假病毒中和实验筛选分泌HPV31中和单抗的杂交瘤细胞株,然后纯化单克隆抗体,分别采用假病毒中和实验及ELISA实验对纯化后单克隆抗体进行鉴定,包括抗体的亚型及其结合表位的构象特征、针对HPV31的中和IC50及针对HPV16、HPV18、HPV33、HPV45、HPV52、HPV58、HPV6、HPV11的交叉中和活性。结果:筛选获得9株HPV31构象依赖中和单抗,其中3株HPV31特异性中和单抗中有2株的IC5010 ng/mL,分别是XM31-13(0.36,Ig G1)及XM31-23(7.10,Ig G1),6株交叉中和单抗中亦有2株的IC5010ng/mL,分别是XM31-19(7.14,Ig G1)、XM31-20(6.91,Ig G1)。结论:获得的9株HPV31构象依赖的中和单抗,特别是其中4株单抗IC50值10 ng/mL的4株单抗,可用于含HPV31L1VLP多价疫苗的质控疫苗的研究。     

Evaluation of hydrogen peroxide efficacy against AZD1222 chimpanzee adenovirus strain in the recombinant COVID-19 vaccine for application in cleaning validation in a pharmaceutical manufacturing industry

This study aimed to evaluate the performance of hydrogen peroxide vapour (HPV) to inactivate the chimpanzee adenovirus AZD1222 vaccine strain used in the production of recombinant COVID-19 vaccine for application in cleaning validation in pharmaceutical industries production areas. Two matrixes were tested: formulated recombinant COVID-19 vaccine (FCV) and active pharmaceutical ingredient (API). The samples were dried on stainless steel and exposed to HPV in an isolator. One biological indicator with population >10⁶ Geobacillus stearothermophilus spores was used to validate the HPV decontamination cycle as standard. HPV exposure resulted in complete virus inactivation in FVC (≥5·03 log₁₀) and API (≥6·40 log₁₀), showing HPV efficacy for reducing chimpanzee adenovirus AZD1222 vaccine strain. However, the optimum concentration and contact time will vary depending on the type of application. Future decontamination studies scaling up the process to the recombinant COVID-19 vaccine manufacturing areas are necessary to evaluate if the HPV will have the same or better virucidal effectivity in each specific production area. In conclusion, HPV showed efficacy for reducing AZD1222 chimpanzee adenovirus strain and can be a good choice for pharmaceutical industries facilities disinfection during recombinant COVID-19 vaccine production.     

Expression of human papillomavirus types 6b and 16 L1 open reading frames in Escherichia coli: detection of a 56,000-dalton polypeptide containing genus-specific (common) antigens.

The human papillomavirus (HPV) genome contains two large open reading frames (ORFs), designated L1 and L2. To characterize the antigenic properties of the L1 ORF-encoded proteins, we cloned the L1 ORFs of HPV6b and HPV16 in plasmids, and these were expressed in Escherichia coli. First, the HPV6b DNA, representing 85.2% of the L1 ORF, was cloned in pUC19 and expressed in E. coli JM83 and RB791 as a 160,000-molecular-weight (160K) fusion protein with E. coli beta-galactosidase (6bL1/beta-gal). Second, the HPV16 DNA, representing 89.8% of the L1 ORF, was cloned in pKK233-2 and expressed as a 56K protein (16L1) in strain RB791. Both the 6bL1/beta-gal and 16L1 proteins cross-reacted with anti-bovine papillomavirus type 1 (BPV1) antibody raised against disrupted BPV1 particles. An antibody raised against the 6bL1/beta-gal fusion protein reacted with the 16L1 protein and also with native papillomavirus antigens in human genital condyloma and bovine fibropapilloma tissues, as determined by biotin-streptavidin staining. Furthermore, the anti-6bL1/beta-gal antibody recognized a 54K protein which seemed to be a major capsid protein of BPV1 and also a 56K protein of biopsies harboring HPV6 or HPV11. From these results we concluded that the papillomavirus L1 gene product contains genus-specific (common) antigens and that the HPV6 and HPV11 L1 genes specify the 56K capsid protein.     

Human Papillomavirus Type 6 and 11 Genetic Variants Found in 71 Oral and Anogenital Epithelial Samples from Australia

Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types.