An experimental study of the survival of turkey coronavirus at room temperature and +4°C.

Turkey coronavirus (TCoV) is a gammacoronavirus (Coronaviridae, Nidovirales) responsible for digestive disorders in young turkeys. TCoV has been associated with poult enteritis complex, a syndrome that severely affects turkey production. No medical prophylaxis exists to control TCoV, therefore sanitary measures such as cleaning and disinfection are essential. It is thus important to evaluate temperatures that allow persistence of TCoV in the environment. Two series of aliquots of a suspension of a French isolate of TCoV (Fr TCoV) were stored at room temperature or +4°C for 0 to 40 days. As TCoV does not grow in cell culture, the presence of residual infectious TCoV in the stored samples was tested by inoculating embryonated specific pathogen free turkey eggs. As TCoV does not induce lesions in the embryo, virus replication in the jejunum and ileum of the embryos was detected 4 days post inoculation, using RNA extraction and a real-time reverse transcriptase-polymerase chain reaction based on the nucleocapsid gene. No surviving virus was detected after 10 days storage at +21.6±1.4°C or after 40 days storage at +4.1±1.6°C, these temperatures being representative of the mean summer and winter temperatures, respectively, in the major French poultry-producing region. The relatively short survival of the virus at room temperature should contribute to limited virus survival during summer months. However, infectious virus was still detected after 20 days storage at the cooler temperatures, a finding that suggests prolonged survival of Fr TCoV and easier transmission between poultry farms in a cool environment are possible.

Assessment of wound healing efficacy and tolerability using CicatrylTM cream based on a suction blister model in healthy subjects

Background: CicatrylTM cream, a topical medical device, is indicated for the treatment of superficial wounds and small skin injuries. Objectives: To assess the efficacy and tolerability of CicatrylTM cream by measuring the recovery of the skin barrier after inducing wounds. Materials & Methods: A suction blister of about 6-mm diameter was induced on the inner side of each forearm of 44 healthy subjects. Using a process of randomisation, CicatrylTM cream was then applied to one wound for a maximum duration of 14 days, while the other wound was left untreated. The primary objective was to evaluate the effect of the test product on wound healing at Day 6, by comparing treated versus untreated wound areas measured by macrophotography. Secondary objectives were to evaluate healing, cutaneous barrier restoration and subjective efficacy of the cream as well as tolerability. Results: The mean wound area (± SD) at Day 6 was significantly smaller for treated wounds compared with untreated wounds (1.76±4.71 vs 15.76±7.61 mm2; p < 0.0001). For treated wounds, wound healing between Days 1 and 6 was 1.6-fold faster compared with untreated wounds (-7.90 vs-4.79 mm2/day; p < 0.0001), and the wounds healed in approximately half the time (6.8 vs 12.2 days for untreated wounds). Cutaneous barrier restoration occurred earlier for treated wounds (Day 6 vs Day 8 for untreated wounds). The cream was well tolerated, and no serious adverse events were observed. Conclusion: CicatrylTM cream improves wound healing, especially within the first six days, if applied in accordance with the manufacturer's instructions.

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes.

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration.

Characterization of a natural heterodimer between MLV genomic RNA and the SD' retroelement generated by alternative splicing.

Murine leukemia virus (MLV) specifically packages both genomic RNA (FL RNA) and a subgenomic RNA, which we call SD'. SD' RNA results from alternative splicing of FL RNA. It is reverse-transcribed, and its DNA copy, integrated into the host genome, constitutes a splice donor-associated retroelement. FL and SD' RNAs share a common 5'-UTR that includes the packaging/dimerization signal (Psi). To investigate whether the mechanism of copackaging of these two RNAs involves RNA heterodimerization, we examined the spontaneous dimerization capacity of the two RNAs as large synthetic RNAs transcribed in vitro. We showed that SD' RNA not only formed homodimers with similar efficiency as the FL RNA, but that FL and SD' RNAs also formed FL/SD' heterodimers via Psi sequences. Comparison of the thermostabilities determined for these different dimeric species and competition experiments with Psi RNA fragments indicate the recruitment of similar dimer-linkage interactions within the Psi region. To validate these results, the dimeric state of the SD' RNA was analyzed in MLV particles. RNA capture assays performed with the FL RNA as bait revealed that SD', and not the host packageable U6 or 7SL RNAs, was associated with the FL RNA in virions. Heterodimerization of SD' RNA with FL RNA may argue for the recent concept of a nuclear dimerization at or near the site of transcription and raises the new hypothesis of RNA dimerization during splicing. Furthermore, FL/SD' heterodimerization may have leukemogenic consequences by influencing the pool of genomic dimers that will undergo recombinogenic template switching by reverse transcriptase.

HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms.

In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5' common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle.

The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA.

The encapsidation signal (Psi) of retroviruses is located in the 5' UTR of the viral genomic unspliced transcript and is highly structured. In the Psi of murine leukaemia virus (MuLV), four stem-loops, called A, B, C and D, promote dimerization and encapsidation of the MuLV unspliced RNA into virions. Through analysis of Psi-deleted transcripts, we found that the AB and CD motifs independently enhanced the cytoplasmic accumulation of RNAs. Furthermore, we showed that over-expression of the Psi sequence in the infected cells led to a competition with the nuclear export of unspliced MuLV transcripts, revealing a new function for these stem-loops in the transport of viral intron-containing RNAs from the nucleus to the cytoplasm.