Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

We have previously detected a group of human papillomaviruses originally found in skin lesions of epidermodysplasia verruciformis (EV) patients in skin cancers from renal transplant recipients and from non-immunosuppressed patients. The reservoir of EV-HPVs is still unknown. In the current study we investigated whether EV-HPV DNA can be detected in plucked hairs from renal transplant recipients and healthy volunteers. Hairs were plucked from eyebrows, scalp, arms, and/or legs and DNA was subsequently isolated. To detect EV-HPV, we used nested PCR with degenerate primers located in the HPV L1 open reading frame. HPV DNA was detected in hairs from one or more sites in all 26 renal transplant recipients tested. Forty-five of 49 samples (92%) from these 26 patients were positive. The HPV type was successfully determined by sequencing in 38 samples, and all types belonged to the EV-HPVs. In ten of 22 healthy volunteers (45%), EV-HPV DNA was also detected in hairs from one or more sites. Twenty of 38 samples (53%) were positive, of which 17 samples were typed as EV-HPV types. These findings indicate that EV-HPV is subclinically present in the skin of the general population. Immunosuppression may lead to activation of the virus, explaining the finding that the apparent prevalence of EV-HPV in plucked hairs from renal transplant patients is higher than in those from the volunteers. If a dose-response situation exists for the carcinogenic potential of HPV infection, this finding may be relevant to the increased risk of skin cancer in this group of patients.

The epidermodysplasia verruciformis (EV)-associated human papillomaviruses (HPVs) constitute a group of HPV genotypes isolated mostly from the cutaneous lesions of patients with the genetic disorder of EV. Broad-spectrum detection of EV HPVs in cutaneous lesions of non-EV patients was previously difficult because no EV HPV consensus PCR was available. We describe a nested PCR that enables the detection of all known EV HPV types at relatively low-copy-number levels. The deduced sequences of a 92-amino-acid stretch of the L1 open reading frames of all types are shown for convenient typing. The technique proved very valuable in viral studies of skin cancers from renal transplant recipients. A high prevalence (81%) of EV HPV types was found in skin cancer biopsies. A wide spectrum of EV HPV types that differed from HPV-5 and-8 was found to be involved. The technique also proved useful in detecting potentially novel EV HPV types in skin cancers. The relationship of these new types to known HPV types is demonstrated by phylogenetic tree analysis.

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Based on immunologic and epidemiologic data, it is plausible that skin cancer in renal transplant recipients is associated with human papillomaviruses (HPV). At present, conflicting evidence exists concerning the presence of HPV DNA in these cancers. We recently described a nested polymerase chain reaction method that enables the detection of all previously isolated epidermodysplasia verruciformis (EV)-associated HPVs. We now describe the detection of EV-associated HPV DNA in 49 (80%) of 61 biopsies from squamous cell carcinomas, in four (50%) of eight basal cell carcinomas, in 14 (93%) of 15 actinic keratoses, in two (40%) of five cases of Bowen's disease, and in four (57%) of seven keratoacanthomas. HPV DNA typing revealed that all detected HPV types belonged to the EV-associated HPV types. A wide spectrum of EV-associated HPVs was found, including six putative new HPV types. In a high percentage of the lesions more than one HPV type was detected. We often found the same HPV types in different skin biopsies from both malignant and premalignant lesions from the same patient. The high frequency of detection of EV-associated HPV types in biopsies from malignant and premalignant lesions is in agreement with the hypothesis that EV-associated HPVs are involved in the pathogenesis of skin cancer in renal transplant recipients.