Recurrent hepatitis occurs in the majority of patients undergoing liver transplantation for hepatitis C virus (HCV) cirrhosis, with progression to cirrhosis in up to 30% after 5 years. Based on these data, a decrease in survival can be anticipated with prolonged follow-up. Furthermore, posttransplantation HCV-fibrosis progression has been shown in recent years to increase. Our aims were (1) to describe the natural history of HCV-infected recipients, particularly to determine whether survival has decreased in recent years; (2) to compare this outcome with that observed in non-HCV-infected cirrhosis controls; and (3) to determine the factors associated with disease severity and survival. Among 522 cirrhotic patients undergoing transplantation between 1991 and 2000, 283 (54%) were infected with HCV. Yearly biopsies were performed in these recipients and at 1 and 5 years in the remainder. With similar follow-up, the percentage of deaths in the HCV(؉) group was significantly higher than in the HCV(؊) group (37% vs. 22%, P < .001), and patient survival was lower (77%, 61%, 55% vs. 87%, 76%, 70% at 1, 5, and 7 years, respectively; P ‫؍‬ .0001). Although survival has increased in the HCV(؊) group in recent years, it has significantly decreased in HCV recipients (P < .0001). The main cause of death among the latter was decompensated graft cirrhosis (n ‫؍‬ 23/105, 22%), whereas that of HCV(؊) patients was infections (n ‫؍‬ 10/52, 19%). Reasons for the recent worse outcome in HCV(؉) recipients include the increased donor age and stronger immunosuppression. In conclusion, patient survival is lower among HCV(؉) recipients than among HCV(؊) ones and has been decreasing in recent years. The aging of donors is a major contributor to this worse outcome. (HEPATOLOGY 2002;36:202-210.) H epatitis C virus (HCV)-cirrhosis is the most frequent diagnosis in patients undergoing liver transplantation. 1 Viral recurrence is universal, 2 with development of histologic hepatitis in the majority of patients 3,4 and progression to cirrhosis in up to 30% after 5 years. 5,6 To date, however, most series have revealed no differences in patient or graft survival compared with uninfected controls. [3][4][5]7 Various reasons may explain these supposed discrepancies, one of which is the existence of different end points when assessing the effect of HCV infection on the graft. An indirect way to assess this effect is by calculating the rate of histologic progression posttransplantation, a task easily performed in the posttransplant setting because of the multiple biopsies generally performed and, through assessment of this rate, estimating the median time to development of graft cirrhosis. In one study, this duration was estimated to be 12 years, a duration significantly shorter than that described for the immunocompetent population. 6 From these data, one can anticipate an increase in HCV-related graft loss in the near future as transplant programs reach their second decade of activity. Our center follows a strict policy of performing annual li...

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While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroy's cat, margay, and ocelot. "Candidatus Mycoplasma haemominutum" was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas "Candidatus Mycoplasma turicensis" was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

BackgroundThe Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. In order to save this species, the Spanish authorities implemented a captive breeding program recruiting lynxes from the wild. In this context, a retrospective survey on prevalence of selected feline pathogens in free-ranging lynxes was initiated.Methodology/ Principal FindingsWe systematically analyzed the prevalence and importance of seven viral, one protozoan (Cytauxzoon felis), and several bacterial (e.g., hemotropic mycoplasma) infections in 77 of approximately 200 remaining free-ranging Iberian lynxes of the Doñana and Sierra Morena areas, in Southern Spain, between 2003 and 2007. With the exception of feline immunodeficiency virus (FIV), evidence of infection by all tested feline pathogens was found in Iberian lynxes. Fourteen lynxes were feline leukemia virus (FeLV) provirus-positive; eleven of these were antigenemic (FeLV p27 positive). All 14 animals tested negative for other viral infections. During a six-month period in 2007, six of the provirus-positive antigenemic lynxes died. Infection with FeLV but not with other infectious agents was associated with mortality (p<0.001). Sequencing of the FeLV surface glycoprotein gene revealed a common origin for ten of the eleven samples. The ten sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Endogenous FeLV sequences were not detected.Conclusions/SignificanceIt was concluded that the FeLV infection most likely originated from domestic cats invading the lynx's habitats. Data available regarding the time frame, co-infections, and outcome of FeLV-infections suggest that, in contrast to the domestic cat, the FeLV strain affecting the lynxes in 2007 is highly virulent to this species. Our data argue strongly for vaccination of lynxes and domestic cats in and around lynx's habitats in order to prevent further spread of the virus as well as reduction the domestic cat population if the lynx population is to be maintained.

The tremendous evolutionary potential of RNA viruses allows them to thrive despite host defense mechanisms and endows them with properties such as emergence, host switching, and virulence. The frequency of mutant viruses after an infectious process results from the interplay between the error rate of the viral replicase, from purifying mechanisms acting after transcription on aberrant RNAs, and from the amplification dynamics of virus RNA positive (1) and negative (-) strands. Two extreme scenarios describing viral RNA amplification are the geometric growth, in which each RNA strand serves as template for the synthesis of complementary strands with the same efficiency, and the stamping machine, where a strand is reiteratively used as template to synthesize multiple copies of the complementary. The resulting mutation frequencies are completely different, being geometric growth largely more mutagenic than stamping machine. In this work we evaluate the contribution of geometric growth and stamping machine to the overall genome amplification of the plant (1)-strand RNA virus turnip mosaic virus (TuMV). By means of transfection experiments of Nicotiana benthamiana protoplasts with a TuMV cDNA infectious clone and by using strand-specific quantitative real-time PCR, we determined the amplification dynamics of viral (1) and (-) RNA during a single-cell infectious process. A mathematical model describing the amplification of each viral strand was fitted to the data. Analyses of the model parameters showed that TuMV (1) and (-) RNA amplification occurs through a mixed strategy with 93% of genomes produced via stamping machine and only 7% resulting from geometric growth.

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.

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