Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.

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.

In order to investigate the genome organization of the porcine epidemic diarrhea virus (PEDV) further, cDNA clones covering the region between the nucleocapsid and the spike (S) protein genes were independently constructed and sequenced for the two virulent isolates Br1/87 and CV777. Of the three major ORFs identified, two were found to encode the major and minor coronavirus membrane proteins M and sM. A potentially single ORF, designated ORF3 according to the pattern of the viral subgenomic mRNAs, could be identified between the S and sM genes. A striking variability, essentially generated by short deletions clustered in a few loci, was observed in the ORF3 of both isolates. The largest predicted polypeptide of 223 amino acids showed homology with polypeptides potentially encoded by other members of the same genetic subset, including two shorter polypeptides of human respiratory virus HCV 229E and one of transmissible gastroenteritis virus TGEV. This homology suggests that the two HCV ORFs may have originated from a single precursor. The function of these polypeptides is not known, but the predicted products of the PEDV ORF3 and related ORFs share features suggestive of a membrane-associated protein.

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.Herpesviruses comprise the capsid containing the viral genome, the viral envelope consisting of a lipid bilayer with embedded glycoproteins, and tegument proteins filling the space between capsid and envelope. DNA double strands formed during replication are packed into capsids built of proteins imported from the cytoplasm (32). Capsids are transported to the nuclear periphery. Their pathway through the nucleocytoplasmic barrier and the acquisition of tegument and envelope are yet not fully understood (18). Capsids bud through the inner nuclear membrane into the perinuclear space, concomitantly acquiring an envelope (15) and tegument proteins (43). It is assumed that the envelope derived from budding at the inner nuclear membrane fuses with the outer nuclear membrane, releasing both tegument and capsid into the cytoplasmic matrix (4, 6, 7, 12, 14-16, 20, 42, 46). Capsids are then transported to the trans-Golgi network, where they are wrapped by Golgi membranes leading to an enveloped virion within a transport vacuole. Alternatively, it is speculated that virions escape from the perinuclear space via vesicle formation at the outer nuclear membrane (5,7,11,16,31,37). These vesicles then pass the Golgi complex for final maturation of virions. Contradictory to both the fusion and vesicle formation theory is the fact that fully enveloped virions were found in...

Papillomaviruses appear to be species-specific pathogens, and it was suggested that each animal species might harbour its own set of papillomaviruses. However, all approaches addressing the underlying evolutionary phenomena still suffer from very limited data about animal papillomaviruses. In case of the horse for example, only three equine papillomaviruses (EcPVs) have been identified. To further address the situation in this host, suspected papillomavirusassociated lesions were tested for EcPV DNA. Four novel EcPV types were detected and their genomes entirely cloned and sequenced. They display the characteristic organization, with early (E) and late (L) regions harbouring the seven classical open reading frames divided by non-coding regions. They were named EcPVs 4, 5, 6 and 7, according to their dissimilarity to other papillomaviruses. Most L1 nucleotide identities were shared with EcPV2 in case of EcPV4 (62 %) and EcPV5 (60 %) or with EcPV3 in case of EcPV6 (70 %) and EcPV7 (71 %). Thus, EcPVs 4 and 5 may establish novel species within the genus Dyoiota, while EcPVs 6 and 7 might fit into the genus Dyorho and belong to the same species as EcPV3. They were found in genital plaques (EcPV4), aural plaques (EcPV5, EcPV6) or penile masses (EcPV7). Interestingly, PCR analysis revealed the DNA of EcPV2 and EcPV4 as well as of EcPV3 and EcPV6 together in the same tissue samples, respectively. In conclusion, the DNA of four novel EcPV types was identified and cloned. They cluster with the known types and support broad genetic EcPV diversity in at least two of the known clades. Furthermore, PCR assays also provide evidence for EcPV co-infections in horses.

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.

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.

The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable tetrameric preparation. Circular dichroism spectra of the intact M2 protein in detergent indicate 67% alpha-helix. The uniformly (15)N-labeled M2 protein and both (15)N-Val- and (15)N-Leu-labeled M2 protein have been expressed from defined M9 media. The (1)H-(15)N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d(25) micelles, and the data indicate a homogeneous preparation. The reconstituted M2/DMPC/DMPG proteoliposomes were used for preparing uniformly aligned solid-state NMR samples for (15)N-(1)H dipolar/(15)N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25 degrees, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38 degrees ). In addition, the spectra suggest that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by other research groups based on electrophysiological measurements and substituted cysteine scanning mutagenesis experiments that characterize a tetrameric structure.