Detection of genotype-specific antibody responses to glycoproteins B and H in primary and non-primary human cytomegalovirus infections by peptide-based ELISA

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6/7) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB

Human Cytomegalovirus UL130 Protein Promotes Endothelial Cell Infection through a Producer Cell Modification of the Virion

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC

L1 gene sequence of a putative novel type human papillomavirus in an immunocompetent patient with glans lichen sclerosus

6nononeThe identification of a putative novel type human papillomaviruses (HPV) strain related to HPV-RTRX3 in a subject with penile skin warts and glans lichen sclerosus is reported. A beta-HPV-RTRX3-like strain was detected in a immunocompetent patient with glans lichen sclerosus. HPV screening was performed by PCR in L1 gene. The MY fragment showed 99% nt identity with HPV-RTRX3 and 64.5% nt identity with HPV-37. The remaining part of the L1 gene showed similarity with HPV 80, 15, 17, and 37. Based on the presence of penile lichen sclerosus and the HPV-RTRX3-like strain found in our patient, a potential correlation was hypothesized.Paolucci, Stefania; Fiorina, Loretta; Piralla, Antonio; Novati, Stefano; Zerbinati, Nicola; Baldanti, FaustoPaolucci, Stefania; Fiorina, Loretta; Piralla, Antonio; Novati, Stefano; Zerbinati, Nicola; Baldanti, Faust

Development and persistence of DAA resistance associated mutations in patients failing HCV treatment

BACKGROUND: Direct-acting antiviral agents (DAAs) combined with pegylated-interferon (PegIFN) and ribavirin (RBV) are still a standard treatment in patients with genotype 1HCV infection. However, virologic response could be impaired by baseline or early selection of resistant HCV strains. OBJECTIVES: The aim of this study was to determine the onset and persistence of resistance-associated mutations (RAMs) in the NS3 and NS5B genes of DAA-naïve patients failing treatment. STUDY DESIGN: Direct sequencing of HCV NS3 was performed in 49 DAA-naïve patients with HCV genotype 1 infection. RESULTS: Eight out of 23 patients (34.7%) failed PegIFN/RBV/telaprevir during the 12-weeks of therapy. Treatment failure was associated with the development of RAMs at amino-acids 36,54,80 and 155 of the HCV protease in 6/8 patients (75%). Among patients treated with PegIFN/RBV/boceprevir treatment, 4/18 (22.2%) failed therapy. Of these, 2 (50%) carried virus strains which developed a RAM at amino-acids 54 and 155. Among HCV strains with RAMs, 7 belonged to genotype 1a and 1 to 1b. Finally, in 6/10 (60%) patients, drug-resistant variants could still be detected for up to 3-7 months after stopping therapy. CONCLUSIONS: A higher rate (p=0.49) of treatment failure was observed in patients receiving telaprevir- compared to the boceprevir-based combination. In addition, compared with genotype 1b, genotype 1a was associated with higher rates (p=0.01) of treatment failure due to virus resistant strains. Resistance testing at baseline and during DAA treatment should be taken into consideration when treating patients with new HCV combination therapies