- Volume 86, Issue 6, 2005

The aim of this study was to identify recurrent respiratory papillomatosis patients who may benefit from interferon (IFN)-α treatment and to determine the means of IFN-α action. The presence of human papillomavirus (HPV) and viral load and proliferation rate in pre-, ongoing and post-treatment respiratory papillomatosis biopsies were examined retrospectively in 25 patients, 18 of whom were IFN-α treated and seven of whom were IFN-α non-treated. Using PCR, HPV was found to be present in 20/25 respiratory papillomatosis patients and HPV type was determined for 18/25 patients (12 HPV6 and six HPV11). Eighteen of the patients were treated with IFN-α, 14 of whom were HPV positive (eight HPV6, five HPV11 and one undefined HPV). Response to IFN-α therapy was observed in 12 patients (7/8 HPV6, 3/5 HPV11, 1/1 undefined HPV and 1/4 HPV negative), while six patients (1/8 HPV6, 2/5 HPV11 and 3/4 HPV negative) did not respond to therapy. Viral load, determined by quantitative real-time PCR (between 0·03 and 533 HPV copies per cell), and proliferation rate, determined as the percentage of Ki-67-positive cells (between 8 and 54 %), were similar in IFN-α-treated and non-treated patients and were generally unaffected by IFN-α treatment. In summary, most (12/18) IFN-α-treated patients responded to therapy. Moreover, there was a tendency for patients with HPV6-positive (7/8) respiratory papillomatosis to respond more frequently to IFN-α therapy than patients with HPV11 (3/5) or HPV-negative (1/4) respiratory papillomatosis. Finally, the presence of HPV and viral load and proliferation in respiratory papillomatosis biopsies was similar in patients treated or not with IFN-α and were in general unaffected by IFN-α treatment.

Possible human infection with simian virus 40 (SV40) has been of great concern ever since SV40 was discovered in polio vaccines. Human populations are SV40-seropositive, but because of serological cross-reactivity between SV40 and the human polyomaviruses BK virus (BKV) and JC virus (JCV), it is debatable whether these antibodies are specific. An SV40-specific serological assay was established, based on purified virus-like particles (VLPs), where the SV40 VLPs were blocked with hyperimmune sera to BKV and JCV. Competition with SV40 hyperimmune sera was used as a confirmatory test. Among 288 Swedish children of between 1 and 13 years of age, 7·6 % had SV40-specific antibodies. SV40 seroprevalence reached a peak of 14 % at 7–9 years of age. Among 100 control patients with benign tumours, 9 % were SV40-seropositive. However, SV40 DNA was not detectable in corresponding buffy-coat samples. In serial samples taken up to 5 years apart from 141 Finnish women participating in the population-based serological screening for congenital infections, only two of 141 women were SV40-seropositive in both samples. Six women seroconverted and eight women had a loss of antibodies over time. None of the SV40-seropositive samples contained detectable SV40 DNA. In conclusion, there is a low prevalence of SV40-specific antibodies in the Nordic population. The SV40 antibodies appear to have a low stability over time and their origin is not clear.

The complete nucleotide sequence of the genomic RNA of the new virus Nemesia ring necrosis virus (NeRNV), which is widespread in various ornamental plant species belonging to the Scrophulariaceae and Verbenaceae, has been determined. Based on its gene content, the folding properties of its 5′-untranslated region and in vitro translation experiments, NeRNV RNA is a typical tymovirus RNA. Its 3′ end, however, differs greatly from those of the valine-specific tymoviral RNAs that have been analysed previously. It can be folded into an upstream pseudoknot domain and a histidine-specific tRNA-like structure, a combination that, so far, has been found only in tobamoviral RNAs. The identity elements found in NeRNV RNA for recognition by yeast histidyl-tRNA synthetase are more similar to those of yeast tRNAHis than the ones found in tobacco mosaic virus RNA. As a result NeRNV RNA can be charged with histidine even more efficiently than tobacco mosaic virus RNA.

Comparative sequence analysis suggests that the left terminal domain of Potato spindle tuber viroid (PSTVd) and other large pospiviroids may assume a branched tertiary structure containing two pseudoknots. To search for evidence of such a structure in vivo, the nucleotide sequences proposed to interact were mutagenized, tomato seedlings were inoculated with mixtures of potentially infectious PSTVd RNA transcripts and the resulting progeny were screened for compensatory sequence changes. Positions 6–11 and 330–335 tolerated only limited sequence variation, and compensatory changes consistent with formation of an intact pseudoknot were observed in only two of the plants examined. No variation was detected at positions 14–16 or 29–31. Passage of selected variants in Rutgers tomato led to an increase in virulence only upon reversion to wild-type PSTVd_Intermediate. The ability of the left terminal domain to assume a branched conformation containing pseudoknots does not appear to be an important determinant of PSTVd fitness.

The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5′-cap structure, but no 3′-poly(A) tail. Binding of CP to the 3′ end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3′-terminal sequences consisting of the 3′ untranslated region of AMV RNA 3 (Luc–AMV), a poly(A) tail of 50 residues [Luc–poly(A)] or a short vector-derived sequence (Luc–control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc–poly(A) or Luc–control, but strongly stimulated Luc expression from Luc–AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc–AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3′ AMV sequence, the cap structure was required for CP-mediated stimulation of Luc–AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.

Predicted promoter regions of Milk vetch dwarf virus (MDV) components (C1–C11) were isolated and fused with a β-glucuronidase (GUS) reporter gene and the characteristics of the promoters were examined. In transgenic tobacco calli, promoters of MDV C4 (encoding a cell-cycle link protein), C5 and C7 (both encoding unknown proteins), C6 (encoding a nuclear-shuttle protein) and C8 (encoding a movement protein) generated a stronger level of GUS expression than the Cauliflower mosaic virus 35S RNA promoter (P35S). In leaves of transgenic tobacco plants, the promoters of C5 and C8 conferred a level of GUS activity comparable to that of P35S. Histochemical GUS analysis showed that the promoters of C4–C9, the latter encoding a capsid protein, were active in phloem and meristematic tissue. The promoter of C8 was also active in mesophyll and cortex cell types. A low level of activity was found for the promoters of C11, which encodes a master replication-initiator protein (Rep), and C1, C2, C3 and C10, which encode additional Reps, in both transgenic tobacco calli and plants.

In light of the finding of a previously unknown coronavirus as the aetiology of the severe acute respiratory syndrome (SARS), it is probable that other coronaviruses, than those recognized to date, are circulating in animal populations. Here, the results of a screening for coronavirus are presented, using a universal coronavirus RT-PCR, of the bird species graylag goose (Anser anser), feral pigeon (Columbia livia) and mallard (Anas platyrhynchos). Coronaviruses were found in cloacal swab samples from all the three bird species. In the graylag goose, 40 of 163 sampled birds were coronavirus positive, whereas two of 100 sampled pigeons and one of five sampled mallards tested positive. The infected graylag geese showed lower body weights compared with virus-negative birds, suggesting clinical significance of the infection. Phylogenetic analyses performed on the replicase gene and nucleocapsid protein sequences, indicated that the novel coronaviruses described in the present study all branch off from group III coronaviruses. All the novel avian coronaviruses harboured the conserved s2m RNA structure in their 3′ untranslated region, like other previously described group III coronaviruses, and like the SARS coronavirus. Sequencing of the complete nucleocapsid gene and downstream regions of goose and pigeon coronaviruses, evidenced the presence of two additional open reading frames for the goose coronavirus with no sequence similarity to known proteins, but with predicted transmembrane domains for one of the encoded proteins, and one additional open reading frame for the pigeon coronavirus, with a predicted transmembrane domain, downstream of the nucleocapsid gene.

Rotavirus RRV gene 11 encodes two non-structural proteins, NSP5 and NSP6. NSP5 is a phosphorylated non-structural protein that binds single- and double-stranded RNA in a non-specific manner. Transient expression of this protein in uninfected cells has provided evidence for its participation in the formation of electron-dense cytoplasmic structures, known as viroplasms, which are thought to be key structures for the replication of the virus. NSP6 is a protein of unknown function that seems not to be essential for virus replication in cell culture. To study the function of NSP5 in the context of a viral infection, the expression of RRV gene 11 was silenced by RNA interference. Reduction in the synthesis of NSP5, as shown by immunoblot and immunofluorescence assays, correlated with a reduction in the number and size of viroplasms and with an altered intracellular distribution of other viroplasm-associated proteins. Silencing of gene 11 also resulted in a reduced synthesis of viral RNA(+) and double-stranded RNA and of all viral proteins, as well as in a decreased production of infectious virus. A similar phenotype was observed when the NSP5 coding gene of the lapine rotavirus strain Alabama was silenced. The fact that the NSP5 gene of rotavirus Alabama lacks the AUG initiator codon for a complete NSP6 protein, suggests that the described phenotype in gene 11-silenced cells is mostly due to the absence of NSP5. The data presented in this work suggest that NSP5 is a key protein during the replication cycle of rotaviruses.