Kurt Tobler

Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence.

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5′ end (no 99–137), and two large, slightly overlapping ORFs, ORF1a (nt 297–12650) and ORF1b (nt 12605–20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.

Impairment of Nuclear Pores in Bovine Herpesvirus 1-Infected MDBK Cells

ABSTRACT Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.

Innate Immune Responses of Calves during Transient Infection with a Noncytopathic Strain of Bovine Viral Diarrhea Virus

ABSTRACT In this study, six immunocompetent calves were experimentally infected with a noncytopathic strain of bovine viral diarrhea virus (BVDV), and the effects of the viral infection on parameters of the innate immune response of the host were analyzed. Clinical and virological data were compared with the temporal activation of the alpha/beta interferon-regulated Mx gene in white blood cells (WBC) and skin as well as the upregulation of the acute-phase serum proteins haptoglobin (Hp) and serum amyloid A (SAA). The viral strain used did provoke transient health impairment, namely, fever and leukopenia that were associated with viremia, viral shedding with nasal secretions, and antiviral seroconversion. Complete recovery was observed within 3 weeks. Elevated levels of SAA and Hp were apparent from days 4 to 13 and 8 to 11, respectively. In WBC, the levels of Mx mRNA and Mx protein were elevated from days 2 to 15. In the context of this study with BVDV, the level of Mx protein expression in WBC provided the most telling diagnostic window to monitor the hosts ongoing innate immune response.

Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo

GREEN fluorescent protein (GFP) is an effective marker for retrovirus and herpes virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. Retrovirus vectors were used to stably transduce several rat and human glioma lines, and a multi-potent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge. Helper virus-free HSV-1 amplicon vectors successfully transferred gfp into non-dividing primary neural cells in culture and in the rat brain. This study describes the versatility of GFP for: (i) labelling of glioma cells in experimental brain tumor models and neural progenitor cells by retrovirus vectors, and (ii) efficient, non-toxic delivery of genes to post mitotic cells of the nervous system using helper-virus free HSV-1 amplicon vectors.

Sequence and classification of FdPV2, a papillomavirus isolated from feline Bowenoid in situ carcinomas

Bowenoid in situ squamous cell carcinoma (BISC) is a rare feline skin disorder, which has been described as often associated with papillomavirus infection. It is clinically characterized by solitary or multiple hyperkeratotic plaques affecting older cats. Papillomavirus (PV) sequences amplified from feline viral plaques, and BISC lesions seldom correspond to FdPV1. The goal of the present study was to investigate three cases of BISC and to carry out initial genomic analysis of the associated viral DNA. Samples of skin biopsies taken from three BISC cats were histologically characterized. DNA was extracted and rolling-circle amplification was performed on the skin samples. Restriction enzyme analysis of the amplified DNA revealed the presence of a putative unknown PV. The whole genome was subsequently sequenced and cloned. Alignments with previously described feline PV sequences were carried out and phylogenetic trees were generated. The circular 7,899 base pair sequence of Felis domesticus PV type 2 (FdPV2) contains a typical noncoding region and characteristic open reading frames (ORF) for six putative viral proteins. Phylogenetic analysis based on the nucleotide alignment of L1 genes or the amino acid alignment of E1 proteins of FdPV2 and 52 other PV types indicates that FdPV2 might represent a new genus.

Isolation of sabin-like polioviruses from wastewater in a country using inactivated polio vaccine.

ABSTRACT From 2001 to 2004, Switzerland switched from routine vaccination with oral polio vaccine (OPV) to inactivated polio vaccine (IPV), using both vaccines in the intervening period. Since IPV is less effective at inducing mucosal immunity than OPV, this change might allow imported poliovirus to circulate undetected more easily in an increasingly IPV-immunized population. Environmental monitoring is a recognized tool for identifying polioviruses in a community. To look for evidence of poliovirus circulation following cessation of OPV use, two sewage treatment plants located in the Zurich area were sampled from 2004 to 2006. Following virus isolation using either RD or L20B cells, enteroviruses and polioviruses were identified by reverse transcription-PCR. A total of 20 out of 174 wastewater samples were positive for 62 Sabin-like isolates. One isolate from each poliovirus-positive sample was analyzed in more detail. Sequencing the complete viral protein 1 (VP1) capsid coding region, as well as intratypic differentiation (ITD), identified 3 Sabin type 1, 13 Sabin type 2, and 4 Sabin type 3 strains. One serotype 1 strain showed a discordant result in the ITD. Three-quarters of the strains showed mutations within the 5′ untranslated region and VP1, known to be associated with reversion to virulence. Moreover, three strains showed heterotypic recombination (S2/S1 and S3/S2/S3). The low number of synonymous mutations and the partial temperature sensitivity are not consistent with extended circulation of these Sabin virus strains. Nevertheless, the continuous introduction of polioviruses into the community emphasizes the necessity for uninterrupted child vaccination to maintain high herd immunity.

Clinically Healthy Skin of Dogs Is a Potential Reservoir for Canine Papillomaviruses

ABSTRACT Papillomaviruses have been linked to several skin disorders in the dog. In order to have a suitable diagnostic tool for canine papillomavirus detection, eight PCRs with published primer combinations were evaluated. The most sensitive PCR was used to demonstrate that papillomavirus DNA can be detected on nonlesional skin of dogs.

Three novel canine papillomaviruses support taxonomic clade formation

More than 100 human papillomaviruses (HPVs) have been identified and had their whole genomes sequenced. Most of these HPVs can be classified into three distinct genera, the alpha-, beta- and gamma-papillomaviruses (PVs). Of note, only one or a small number of PVs have been identified for each individual animal species. However, four canine PVs (CPVs) (COPV, CPV2, CPV3 and CPV4) have been described and their entire genomic sequences have been published. Based on their sequence similarities, they belong to three distinct clades. In the present study, circular viral DNA was amplified from three dogs showing signs of pigmented plaques, endophytic papilloma or in situ squamous cell carcinoma. Analysis of the DNA sequences suggested that these are three novel viruses (CPV5, CPV6 and CPV7) whose genomes comprise all the conserved sequence elements of known PVs. The genomes of these seven CPVs were compared in order properly classify them. Interestingly, phylogenetic analyses, as well as pairwise sequence alignments of the putative amino acid sequences, revealed that CPV5 grouped well with CPV3 and CPV4, whereas CPV7 grouped with CPV2 but neither group fitted with other classified PVs. However, CPV6 grouped with COPV, a lambda-PV. Based on this evidence, allocation of CPVs into three distinct clades could therefore be supported. Thus, similar to HPVs, it might be that the known and currently unknown CPVs are related and form just a few clades or genera.

Exploring the Nuclear Envelope of Herpes Simplex Virus 1-Infected Cells by High-Resolution Microscopy

ABSTRACT Herpesviruses are composed of capsid, tegument, and envelope. Capsids assemble in the nucleus and exit the nucleus by budding at the inner nuclear membrane, acquiring tegument and the envelope. This study focuses on the changes of the nuclear envelope during herpes simplex virus 1 (HSV-1) infection in HeLa and Vero cells by employing preparation techniques at ambient and low temperatures for high-resolution scanning and transmission electron microscopy and confocal laser scanning microscopy. Cryo-field emission scanning electron microscopy of freeze-fractured cells showed for the first time budding of capsids at the nuclear envelope at the third dimension with high activity at 10 h and low activity at 15 h of incubation. The mean number of pores was significantly lower, and the mean interpore distance and the mean interpore area were significantly larger than those for mock-infected cells 15 h after inoculation. Forty-five percent of nuclear pores in HSV-1-infected cells were dilated to more than 140 nm. Nuclear material containing capsids protrude through them into the cytoplasm. Examination of in situ preparations after dry fracturing revealed significant enlargements of the nuclear pore diameter and of the nuclear pore central channel in HSV-1-infected cells compared to mock-infected cells. The demonstration of nucleoporins by confocal microscopy also revealed fewer pores but focal enhancement of fluorescence signals in HSV-1-infected cells, whereas Western blots showed no loss of nucleoporins from cells. The data suggest that infection with HSV-1 alters the number, size, and architecture of nuclear pores without a loss of nucleoporins from altered nuclear pore complexes.

EcPV2 DNA in equine papillomas and in situ and invasive squamous cell carcinomas supports papillomavirus etiology

Equine penile papillomas, in situ carcinomas, and invasive carcinomas are hypothesized to belong to a continuum of papillomavirus-induced diseases. The former ones clinically present as small grey papules, while the latter 2 lesions are more hyperplasic or alternatively ulcerated. To test the hypothesis that these lesions are papillomavirus-induced, samples of 24 horses with characteristic clinical and histologic findings of penile papillomas or in situ or invasive squamous cell carcinomas were collected. As controls, 11 horses with various lesions—namely, Balanoposthitis (6 cases), melanoma (3 cases), follicular cyst (1 case), and amyloidosis (1 case)—were included. DNA was extracted and polymerase chain reaction applied to amplify papillomavirus DNA. The respective primers were designed to amplify DNA of the recently discovered equine papillomavirus EcPV2. All tested papilloma and squamous cell carcinoma samples were found to contain DNA of either of 2 previously published EcPV2 variants. Among the other samples 6 of 11 were found to contain EcPV2 DNA. To further support the findings and to determine where the papillomavirus DNA was located within the lesions, an in situ hybridization for the detection of EcPV2 DNA was established. The samples tested by this technique were found to clearly contain papillomavirus nucleic acid concentrated in the nucleus of the koilocytes. The findings of this study support previous data and the hypothesis that papillomaviruses induce the described penile lesions in horses.