Shin-je Ghim

Characterization of a Novel Close-to-Root Papillomavirus from a Florida Manatee by Using Multiply Primed Rolling-Circle Amplification: Trichechus manatus latirostris Papillomavirus Type 1

ABSTRACT By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.

HPV-1 L1 protein expressed in cos cells displays conformational epitopes found on intact virions.

Seven polyclonal and monoclonal antibodies were characterized for their ability to react specifically with either conformational or nonconformational epitopes of the HPV-1 virion. Using these antibodies, it was shown that the HPV-1 L1 protein (when expressed by an SV40 vector in cos cells) displayed conformational epitopes characteristic of intact viral particles. In addition, the L1 capsid protein was translocated normally into cell nuclei, was of appropriate size (57 kDa), and could be isolated in native form by immunoprecipitation techniques. Most importantly, the screening of expressed papillomavirus capsid proteins for reactivity with conformation-dependent antibodies represents a new, general methodology for ensuring that such proteins will be suitable for use in vaccine development or in the serologic detection/typing of human papillomavirus infections.

The Role of Human Papilloma Virus in Lung Cancer: A Review of the Evidence

Papillomaviruses are small nonenveloped DNA viruses that infect squamous epithelial cells. These viruses have been found in many organisms. Human papillomaviruses (HPVs) give rise to a large spectrum of epithelial lesions, mainly benign hyperplasia (eg, warts and papillomas) with low malignant potential. There is a subgroup of HPV, the “high-risk” HPV, which is associated with precancerous and cancerous lesions. A small fraction of people infected with high-risk HPV will develop cancers that usually arise many years after the initial infection (Psyrri and Dimaio, Nat Clin Pract Oncol. 2008;5:24–31). Nonsmall cell lung cancer is a heterogeneous disease. The most common histologic subtypes include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Despite different histologies, nonsmall cell lung cancers are often classified together because of similarities in approach and management of the disease. In this article, we reviewed the current literature on lung cancer and HPV. On the basis of this data, we suggested a possible mechanism of carcinogenesis induced by HPV.

Genomic characterization of novel dolphin papillomaviruses provides indications for recombination within the Papillomaviridae

Phylogenetic analysis of novel dolphin (Tursiops truncatus) papillomavirus sequences, TtPV1, -2, and -3, indicates that the early and late protein coding regions of their genomes differ in evolutionary history. Sliding window bootscan analysis showed a significant a change in phylogenetic clustering, in which the grouped sequences of TtPV1 and -3 move from a cluster with the Phocoena spinipinnis PsPV1 in the early region to a cluster with TtPV2 in the late region. This provides indications for a possible recombination event near the end of E2/beginning of L2. A second possible recombination site could be located near the end of L1, in the upstream regulatory region. Selection analysis by using maximum likelihood models of codon substitutions ruled out the possibility of intense selective pressure, acting asymmetrically on the viral genomes, as an alternative explanation for the observed difference in evolutionary history between the early and late genomic regions of these cetacean papillomaviruses.

Genomic analysis of the first laboratory-mouse papillomavirus

A papillomavirus (PV) that naturally infects laboratory mice will provide an extremely valuable tool for PV research. We describe here the isolation, cloning and molecular analysis of the first novel laboratory-mouse PV, designated MusPV. This agent, recently identified in the tissues from florid and asymmetrical papillomas on the face of nude mice (NMRI-Foxn1(nu)/Foxn1(nu)), was demonstrated to be transmissible to immunocompetent mice (Ingle et al., 2010). The MusPV genome is 7510 bp in length, is organized similarly to those of other PVs and has at least seven ORFs (E1, E2, E4, E6, E7, L1 and L2). Phylogenetic analysis indicates that MusPV belongs to the π genus together with four other rodent PVs (McPV2, MaPV1, MmiPV and RnPV1). Of the rodent PVs, MusPV appears most closely related to Mastomys coucha PV (McPV2), with 65 % genomic homogeneity and 80 % L1 amino acid similarity. Rodent PVs, except for MnPV1, do not contain any identifiable retinoblastoma protein (RB) binding sites. MusPV has one putative RB-binding site on the E6 protein but not on the E7 protein. Non-coding regions (NCRs) of PVs maintain multiple binding sites for transcription factors (TFs). The NCR of MusPV has numerous sites for TF binding, of which at least 13 TFs are common to all PVs in the π genus. MusPV provides a potentially valuable, novel mouse model to study mechanisms of infection, oncology and novel preventive and therapeutic approaches in mice that can be translated to diseases caused by human PVs.

Orogenital Neoplasia in Atlantic Bottlenose Dolphins (Tursiops truncatus)

This study describes lingual papillomas and squa- mous cell carcinomas (n = 11) and genital pap- illomas (n = 4) in Atlantic bottlenose dolphins (Tursiops truncatus) evaluated from January 2000 to January 2005. Tumors were found primarily in adult dolphins of both sexes living in free-ranging and captive conditions. Three dolphins had mul- tiple lingual tumors of mixed histological type, consisting of papillomas and squamous cell car- cinomas, suggesting malignant transformation of the benign papillomatous lesions. To our knowl- edge, this is the first report of oral papillomas in bottlenose dolphins and concurrent oral neoplasia that included both sessile papilloma and squamous cell carcinoma in the same dolphin. Additionally, it is the first known report of genital papillomas in free-ranging bottlenose dolphins from Atlantic coastal waters. The unusually high occurrence of related benign and malignant orogenital epithelial neoplastic lesions in a short period suggests that the lesions may represent one or more emerging diseases. Preliminary evidence suggests that these tumors may be of infectious etiology, possibly having an orogenital route of transmission.

Recombinant vaccines for the prevention of human papillomavirus infection and cervical cancer

Carcinogenic human papillomaviruses (HPVs) that cause cervical cancer preferentially infect basal, metaplastic squamous cells of the transformation zone. If infection persists, and a vegetative infection ensues, a premalignant lesion may develop with the potential to progress into an invasive squamous cell carcinoma. Papillomavirus prophylactic vaccines target the systemic immune system for induction of neutralizing antibodies that protect the basal cells against infection. Because the carcinogenic HPVs are susceptible to neutralization by antibodies for 9-48 h after reaching the basal cells, both low and high titered HPV type-specific antibodies induced by HPV L1 and L2-based vaccines are highly efficacious. The greatest burden of HPV-associated cancers occurs in poor areas of the world where women do not have access to routine gynecological care. The burden of HIV/AIDS in these same regions of the world has added to the burden of HPV-associated disease. There is an urgent need for a cost-effective, broad-spectrum HPV prophylactic vaccine in developing countries, which necessitates substantial cost subsidization of the virus-like particle (VLP) based vaccines licensed in industrialized countries or an alternative approach with second-generation vaccines that are specifically designed for delivery to women in resource-poor communities.

Immune Status, Strain Background, and Anatomic Site of Inoculation Affect Mouse Papillomavirus (MmuPV1) Induction of Exophytic Papillomas or Endophytic Trichoblastomas

Papillomaviruses (PVs) induce papillomas, premalignant lesions, and carcinomas in a wide variety of species. PVs are classified first based on their host and tissue tropism and then their genomic diversities. A laboratory mouse papillomavirus, MmuPV1 (formerly MusPV), was horizontally transmitted within an inbred colony of NMRI-Foxn1nu/Foxn1nu (nude; T cell deficient) mice of an unknown period of time. A ground-up, filtered papilloma inoculum was not capable of infecting C57BL/6J wild-type mice; however, immunocompetent, alopecic, S/RV/Cri-ba/ba (bare) mice developed small papillomas at injection sites that regressed. NMRI-Foxn1nu and B6.Cg-Foxn1nu, but not NU/J-Foxn1nu, mice were susceptible to MmuPV1 infection. B6 congenic strains, but not other congenic strains carrying the same allelic mutations, lacking B- and T-cells, but not B-cells alone, were susceptible to infection, indicating that mouse strain and T-cell deficiency are critical to tumor formation. Lesions initially observed were exophytic papillomas around the muzzle, exophytic papillomas on the tail, and condylomas of the vaginal lining which could be induced by separate scarification or simultaneous scarification of MmuPV1 at all four sites. On the dorsal skin, locally invasive, poorly differentiated tumors developed with features similar to human trichoblastomas. Transcriptome analysis revealed significant differences between the normal skin in these anatomic sites and in papillomas versus trichoblastomas. The primarily dysregulated genes involved molecular pathways associated with cancer, cellular development, cellular growth and proliferation, cell morphology, and connective tissue development and function. Although trichoepitheliomas are benign, aggressive tumors, few of the genes commonly associated with basal cell carcinoma or squamous cells carcinoma were highly dysregulated.

Molecular diagnosis of a laboratory mouse papillomavirus (MusPV).

MusPV, a novel papillomavirus (PV) that naturally infects laboratory mice, was isolated and characterized from a colony of NMRI-Foxn1(nu)/Foxn1(nu) (nude) mice in India. Because MusPV may have been missed during routine pathogen screening of mice in colonies worldwide, a variety of detection methods are described to detect MusPV. The clinical and histologic lesions of productive MusPV infections fit PV-associated features, including papillomas, koilocytes within the stratum granulosum of the hyperplastic/acanthotic papillomatous epithelium, and the presence of intranuclear virus particles in koilocytotic cells visualized by electron microscopy. Antiserum against disrupted PV virions, isolated from another species (canine), identified conserved viral antigens in productively infected cells by immunohistochemistry. A rolling circle technique was used to amplify viral circular DNAs followed by endonuclease restriction enzyme digestion to determine the correct size of PV DNA. Consensus PV degenerative primers, My09/11, commonly used to detect many different types of PVs by polymerase chain reaction (PCR), particularly mucosotropic HPVs, also identified MusPV and all rodent PVs tested. Since there was one nucleotide mismatch between the My09/11 primer set and the MusPV template, a new primer set, MusPV-My09/11, was designed to specifically detect MusPV in latent infections and spontaneous MusPV-induced papillomas. Southern blot analysis verified the presence of full size PV DNA in infected tissues. Virus-like particles (VLPs), generated from MusPV L1 genes, provided a substrate for serological testing of naturally and experimentally infected mice. In summary, a series of diagnostic assays were developed and validated to detect MusPV infection in skin tumors and serological response in laboratory mice.

Antigenicity of bovine papillomavirus type 1 (BPV-1) L1 virus-like particles compared with that of intact BPV-1 virions.

Virus-like-particles (VLPs) of various papillomavirus (PV) types have been produced by expressing recombinant L1 proteins in eukaryotic cells. Although VLPs have the same ultrastructural appearance as native virions and their immunogenicity appears to be similar, their antigenicity has not been carefully evaluated. For this reason, the antigenicity of intact bovine PV type 1 (BPV-1) virions was compared with that of BPV-1 recombinant L1 VLPs by ELISA using a well-characterized panel of polyclonal and monoclonal antibodies generated against intact and denatured BPV-1 particles. The structural integrity of the authentic virions and recombinant VLPs was verified by electron microscopy. The specificity of antibodies raised against intact BPV-1 virions and their reactivity with VLPs revealed that the immunodominant, type-specific, conformational epitopes of intact virions were reproduced on VLPs. However, many monoclonal antibodies that define cross-reactive, non-conformational (linear) epitopes cryptic to the authentic BPV-1 virion tested positively when reacted with intact VLPs. One monoclonal antibody, which recognizes a BPV-1 and deer PV surface conformational epitope, did not react with VLPs. Therefore, although VLPs can be used to immunize animals against infection, the external exposure of broadly cross-reactive epitopes of intact L1 VLPs suggests that the use of L1 VLPs in antigenicity studies such as serological screening should be done with caution.