Octavian Lungu

Typing of human papillomaviruses by polymerase chain reaction amplification with L1 consensus primers and RFLP analysis

Human papillomaviruses (HPV) cause benign and malignant lesions of the epithelial and mucosal surfaces. Certain virus types are associated with cervical carcinomas, while others are associated with benign condylomata. We have developed a rapid method for determining HPV type that is based on restriction fragment length polymorphism (RFLP) analysis within the L1 region of HPVs that is amplified by PCR using the consensus primers described by Manos et al. Analysis of the product generated by PCR amplification of plasmids containing cloned HPV genomes and of 88 clinical specimens, known to contain HPV viral DNA by previous hybridization analysis, revealed that this method is useful for typing HPV sequences amplified from a variety of sources including cervical lavages, fresh tissue, and paraffin-embedded formalin-fixed biopsy material. The method can differentiate between most known types of HPV and discriminate between infections with single, multiple or novel HPV types. A high correlation (86%) was obtained when this method was compared with PCR amplification and Southern blot hybridization analysis of PCR product, or Southern blot hybridization analysis of total genomic DNA. Differences in typing occurred mostly for specimens that contained multiple or new/unknown HPV types. However, RFLP typing easily identified repeated patterns for new HPV types that were not detected by the other methods. In summary, PCR-RFLP typing is a sensitive and specific method to identify and characterize rapidly HPV DNA in clinical specimens from a variety of sources.

Open Reading Frame S/L of Varicella-Zoster Virus Encodes a Cytoplasmic Protein Expressed in Infected Cells

ABSTRACT We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3′ untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.

Essential Role Played by the C-Terminal Domain of Glycoprotein I in Envelopment of Varicella-Zoster Virus in the trans-Golgi Network: Interactions of Glycoproteins with Tegument

ABSTRACT Varicella-zoster virus (VZV) is enveloped in thetrans-Golgi network (TGN). Here we report that glycoprotein I (gI) is required within the TGN for VZV envelopment. Enveloping membranous TGN cisternae were microscopically identified in cells infected with intact VZV. These sacs curved around, and ultimately enclosed, nucleocapsids. Tegument coated the concave face of these sacs, which formed the viral envelope, but the convex surface was tegument-free. TGN cisternae of cells infected with VZV mutants lacking gI (gIΔ) or its C (gIΔC)- or N-terminal (gIΔN)-terminal domains were uniformly tegument coated and adhered to one another, forming bizarre membranous stacks. Viral envelopment was compromised, and no virions were delivered to post-Golgi structures. The TGN was not gI-immunoreactive in cells infected with the gIΔ or gIΔNmutants, but it was in cells infected with gIΔC (because the ectodomains of gI and gE interact). The presence in the TGN of gI lacking a C-terminal domain, therefore, was not sufficient to maintain enveloping cisternae. In cells infected with intact VZV or with gIΔ, gIΔN, or gIΔC mutants, ORF10p immunoreactivity was concentrated on the cytosolic face of TGN membranes, suggesting that it interacts with the cytosolic domains of glycoproteins. Because of the gE-gI interaction, cotransfected cells that expressed gE or gI were able to target truncated forms of the other to the TGN. Our data suggest that the C-terminal domain of gI is required to segregate viral and cellular proteins in enveloping TGN cisternae.

Evaluation of the Hybrid Capture human papillomavirus deoxyribonucleic acid detection test

OBJECTIVE Our purpose was to evaluate the sensitivity and accuracy of a new, nonradioactive human papillomavirus deoxyribonucleic acid detection method. STUDY DESIGN Cervical samples from 520 women were assayed for human papillomavirus deoxyribonucleic acid with both the Hybrid Capture test and polymerase chain reaction. RESULTS Human papillomavirus deoxyribonucleic acid was detected with Hybrid Capture in 106 (42%) of 254 samples from women with no evidence of cervical intraepithelial neoplasia and 211 (79%) of 266 with cervical intraepithelial lesions or cervical cancer. There was a good correlation between Hybrid Capture and polymerase chain reaction. Hybrid Capture correctly identified 92% of samples found to contain a human papillomavirus type with a high or intermediate oncogenic risk with polymerase chain reaction. Although Hybrid Capture can quantify the amount of human papillomavirus deoxyribonucleic acid present in a sample, no correlation was observed between the relative amount of human papillomavirus deoxyribonucleic acid detected with Hybrid Capture and the grade of cervical lesion. CONCLUSION The Hybrid Capture test is a sensitive and accurate method for identifying human papillomavirus types of high and intermediate oncogenic risk in clinical specimens.

Evidence of Latent Varicella-Zoster Virus in Rat Dorsal Root Ganglia

Latent varicella-zoster virus (VZV) was studied in ganglia of rats that had been inoculated subcutaneously with either a high-passaged wild-type, a low-passaged wild-type, or the vaccine strain of virus using in situ hybridization. Nine of 11 rats injected with virus and no control rats developed serum VZV antibodies as demonstrated by fluorescent antibody membrane antigen. Polymerase chain reaction 2 weeks following inoculation did not detect viremia in the rats. VZV was detected by in situ hybridization in ganglia of 10 of the 11 infected rats but not in ganglia of the control rats. The distribution of VZV DNA is identical to that seen in humans; satellite cells and neurons contain VZV DNA. Although all animals received unilateral injections of virus, VZV DNA was in ipsilateral and contralateral ganglia in 6 animals, suggesting that virus replication and viremia had occurred.

Intracellular Transport of Varicella-Zoster Glycoproteins

Previous observations have established that varicella-zoster virus (VZV) is enveloped in the trans-Golgi network (TGN) in cultures infected with VZV and that the glycoprotein gE is targeted to the TGN by a signal sequence (AYRV) and an acidic TGN signal patch in its cytosolic domain. Neither sequence is present in other VZV glycoproteins. Like gE, gI was targeted to the TGN when it was expressed in transfected cells, suggesting that gI also contains TGN targeting information (colocalized with gE and the AP-1 adaptin complex). In contrast, gB, gC, gH, and gL immunoreactivities were not detected in the TGN when they were expressed individually in transfected cells. In VZV-infected cells, gE, gI, gH, and gL were all concentrated in the TGN. Since VZV glycoproteins that lack targeting sequences (gB, gC, gH, and gL) concentrated in the TGN of infected cells, it is proposed that gE and gI, which have such sequences, serve as navigator glycoproteins, forming complexes that direct the signal-deficient glycoproteins to the TGN.

Trafficking of varicella-zoster virus glycoprotein gI: T(338)-dependent retention in the trans-Golgi network, secretion, and mannose 6-phosphate-inhibitable uptake of the ectodomain.

ABSTRACT The trans-Golgi network (TGN) is putatively the site where varicella-zoster virus is enveloped. gE is targeted to the TGN by selective retrieval from the plasmalemma in response to signaling sequences in its endodomain. gI lacks these sequences but forms a complex with gE. We now find that gI is targeted to the TGN and plasma membrane when expressed in Cos-7 cells; nevertheless, surface labeling revealed that gI is not retrieved from the plasma membrane. TGN targeting of gI depended on the T338 of its endodomain and was lost when T338 was deleted or mutated to A, S, or D. The endodomain of gI was sufficient, if it contained T338, to target a fusion protein containing the ectodomain of the human interleukin-2 receptor to the TGN. A truncated protein consisting only of the gI ectodomain was secreted and taken up by nontransfected cells. This uptake of the secreted gI ectodomain was blocked by mannose 6-phosphate. Following cotransfection, both gI and gE were retrieved to the TGN from the plasma membrane in 26.7% of cells, neither gI nor gE was internalized in 18.3%, and gE was retrieved to the TGN while gI remained at the plasma membrane in 55%. We suggest that the T338 of its endodomain is necessary to retain gI in the TGN; moreover, because gI and gE interact, the signaling sequences of each glycoprotein reinforce one another in ensuring that both glycoproteins are concentrated in the TGN yet remain on the cell surface.

Differentiation of Nocardia from rapidly growing Mycobacterium species by PCR-RFLP analysis

The nucleotide sequences from a region of the groEL gene from one Nocardia asteroides and from several species of Mycobacterium were determined and found to be highly homologous. Based on these homologies, we developed a rapid method capable of differentiating between these two genera. The method is based on restriction fragment-length polymorphism (RFLP) analysis of DNA amplified from the groEL gene that is highly conserved between mycobacteria and nocardiae. When the groEL gene from species of these genera is enzymatically amplified by the polymerase chain reaction (PCR), a 422-bp fragment is generated. Correlation of the restriction endonuclease digestion patterns of the amplification products with reference and/or biochemically characterized clinical samples enabled us to establish RFLP profiles for ten species of Mycobacterium and five species of Nocardia. When a portion of the groEL gene from each of these organisms is digested with the restriction endonuclease Hae III, that organism is readily assigned to one of these two genera on the basis of the derived RFLP patterns. The utility of this approach was examined by testing 105 pure cultures from samples previously identified by routine culture techniques for the presence of groEL DNA sequences of mycobacterial or nocardial origin. This analysis correctly identified the organism in all samples tested. In summary, PCR-RFLP analysis provides a rapid and sensitive method for the differentiation of Nocardia species from rapidly growing Mycobacterium species.

Congenital Varicella-Zoster Virus Infection and Barrett's Esophagus

Congenital varicella syndrome is a rare complication of varicella-zoster virus (VZV) infection during pregnancy. An infant was exposed to VZV at 18.5 weeks of gestation and had eye and skin abnormalities at birth and persistent feeding difficulties, prompting esophageal biopsies at 12 days and 20 and 20.5 months of age. Esophageal tissues demonstrated specialized intestinal metaplasia (Barretts esophagus). VZV DNA (in situ hybridization) and proteins (immunohistochemistry and polymerase chain reaction) were found in esophageal epithelial cells adjacent to the Barretts lesion. Immediate-early 63 protein (IE63) of VZV was demonstrated in the day 12 specimen, and IE62 and the late VZV glycoprotein E (gE) were found in the 20-month specimen. Clinical and endoscopic improvement followed fundoplication and acyclovir therapy, but VZV DNA and IE62 persisted in esophageal tissue. These findings associate VZV with specialized intestinal metaplasia within the esophagus and suggest a novel site for either latent or active VZV infection.

In situ hybridization detection of varicella zoster virus in paraffin-embedded skin biopsy samples

BACKGROUND When virologic and molecular diagnostic techniques are unavailable, the diagnosis of varicella zoster virus (VZV) infection depends on clinical criteria and histologic evaluation of skin biopsy specimens or Tzank preparations. These methods can misdiagnose chickenpox and zoster, particularly when the clinical manifestations are atypical. OBJECTIVE To improve diagnosis in these settings, we developed an in situ hybridization technique for the detection of VZV utilizing a fluorescein-labeled oligonucleotide probe visualized with anti-fluorescein alkaline phosphatase-conjugated antibody. STUDY DESIGN We retrospectively examined 26 paraffin-embedded skin biopsy specimens with histologic features consistent with VZV or herpes simplex virus (HSV) infection and 11 control cases by in situ hybridization. In situ hybridization for VZV and HSV-1 was compared with polymerase chain reaction (PCR) for VZV and HSV-1 and clinical and histologic examination. RESULTS Thirteen of the 26 study cases and two of the 11 control cases were positive for VZV by in situ hybridization. When compared with PCR, in situ hybridization was 92% sensitive and 88% specific. When compared with clinical diagnosis, in situ hybridization was 86% sensitive and 87% specific. All cases of chickenpox had VZV-positive inflammatory cells in the dermis but this finding was less frequent among the cases of zoster. CONCLUSIONS This in situ hybridization technique is a sensitive and specific method for the diagnosis of VZV in skin lesions that is applicable to most histopathology laboratory settings. In addition, in situ hybridization reveals individual infected cells and may provide insight into the pathogenesis of VZV skin infection.