New horizons in translational medicine最新文献

Walleye dermal sarcoma virus is a complex retrovirus that causes seasonal tumors in walleye fish. RV-cyclin is one accessory protein encoded by the virus, and is one of only two viral proteins expressed during tumor development. Therefore, role of RV-cyclin in tumor development was explored. RV-cyclin interacts with host cyclin dependent kinase8 (CDK8). CDK8 has oncogenic like properties in colon cancer and melanoma, and one target of CDK8 kinase activity is the carboxy terminal domain of RNA Pol II. Here qRT-PCR analysis demonstrates RV-cyclin’s direct interaction with CDK8 increases transcript levels of another set of oncogenes—the serum-response genes (Fos, EGR1, and Jun). Nuclear run-on experiments, and chromatin immunoprecipitation experiments with an antibody to RNA Pol II, show that RV-cyclin enhances transcription elongation along the EGR1 gene locus. This enhancement correlates with increased recruitment of CDK8 to the EGR1 gene locus. In addition to increasing CDK8 occupancy at the EGR1 gene, in vitro kinase experiments demonstrate RV-cyclin increases the amount of CDK8-phosphorylation on the CTD of RNA Pol II. In conclusion, not only does RV-cyclin direct CDK8 to specific genes during tumor development, RV-cyclin enhances CDK8 kinase activity while it is there. The end result of the CDK8-RV-cyclin interaction is a rise in the mRNA levels of another pool of oncogenes, the serum-response genes. This is one mechanism by which RV-cyclin could contribute to the development of walleye dermal sarcoma.

Mutation, recombination, and reassortment generate virus particles with variable genotypes, some of which may be better adapted to infect a new host, resist drug treatment, or escape immune pressure. The arenaviruses are a family of viruses that package a large (L) and small (S) genome segment. Arenaviruses infect mammals and snakes and are associated with fatal disease in both groups of animals. Although recombination and reassortment are well documented in some virus families, neither process has been observed in natural arenavirus infections.

In this study, we documented a surprising degree of genetic diversity in arenavirus-infected snakes. Instead of one or two viral species or quasispecies, individual animals harbored complex populations of viral genotypes composed of up to 5 S and 11 L genotypes, which replicated as stable ensembles in culture. S and L segment genotype accumulation was not balanced and a particular S segment genotype dominated, both in individual animals and at a population level. Numerous instances of recombination and reassortment were detected. Some recombinant segments had unusual organizations with 2 intergenic regions.

This genetic fluidity is closer to that observed in influenza viruses than to the relatively placid genetics of mammalian arenavirus. However, the observed imbalance between the S and L segments and the intrahost accumulation and persistence of multiple genotypes is previously undocumented. Overall, this provides an opportunity to study basic mechanisms of viral adaptation and stretches the idea of what it means to be infected by “a virus”.

Modoc virus (MODV) is a flavivirus that was first isolated from deer mice (Peromyscus maniculatus) in Modoc County, California during a 1958 surveillance study for novel viruses. Although many flaviviruses are arthropod-borne, MODV has no known intermediate. Subsequent to its initial isolation, MODV was detected in deer mice found in other regions of the United States, including northeastern Colorado. These findings suggested that deer mice may be a reservoir host of MODV. We intramuscularly inoculated 18 deer mice with 10⁵ TCID₅₀ of MODV strain M544 for susceptibility testing. Groups of three deer mice were euthanized for necropsy and tissue collection on days 2, 4, 7, 11, 21, and 31. No conspicuous signs of disease occurred in the deer mice; however, minor pulmonary multifocal vasculitis and hemorrhages, multifocal portal hepatitis and splenic lymphoid hyperplasia with hemosiderosis were detected in several deer mice. No virus was detected in sera, suggesting viremia did not occur. Neutralizing antibody was detected as early as day 7-post inoculation, and thereafter all deer were seropositive. MODV RNA was detected by PCR in organs of deer mice euthanized between days 2 and 4, with lung tissue of one deer mouse euthanized on day 7 also indicating the presence of MODV RNA. Viral RNA was detected in most spleens but less frequently in the kidneys and hearts. These data indicate deer mice are susceptible to MODV without signs of disease, although mild pathology occurs in some organs. Clearance of the virus is suggested by the PCR results since no virus was detected in any organ tissue beyond 7 days, which is contrary to the expectation of a natural reservoir host.

Chikungunya virus (CHIKV) is an arbovirus distributed widely in tropical regions of the world that causes a febrile and often painful disease in adults and children. Recent outbreaks of CHIKV infection in the Caribbean have raised concerns about establishment of this virus in North America. A significant question about the transmission cycle of CHIKV is whether non-human reservoir hosts are important in maintenance or transmission of the virus. We conducted experimental infections with CHIKV and discovered that several reptiles and amphibians developed viremia of sufficient magnitude to possibly serve as reservoir hosts. One or two strains of CHIKV were inoculated into a variety of ball pythons, Burmese pythons, Northern garter snakes, American alligators, green iguanas, painted turtles, leopard frogs, Bufo species toads and cane toads. Viremia was not detected in alligators or cane toads but all other species developed viremia at variable magnitude. Peak viremia in the other species varied from 2.8 (Burmese pythons) to 4.7 (leopard frogs) log10 pfu/ml. We also conducted experiment to evaluate the effect of ambient temperature changes to monitor the “over wintering” capabilities of CHIKV in snakes. Northern garter snakes were inoculated a South African strain of CHIKV at temperatures of 16 C versus 26 C and tested for viremia. The snakes kept at 26 C developed a short term viremia, whereas in snakes kept at 16 C, the virus was maintained for a longer period and viremia titers as high as 7.2 log10 pfu/ml were achieved when animals were subsequently slowly warmed to 26 C.

Prions are infectious agents composed entirely of protein. Prion activity results from the conversion of soluble proteins into an insoluble, self-templating amyloid form. Nine different amyloid-based prions have been identified in yeast. All but one contain a glutamine/asparagine (Q/N) rich region that is responsible for prion activity. Similar Q/N-rich regions are over-represented in eukaryotic genomes. In humans, aggregation-causing mutations in Q/N-rich proteins have been linked to various degenerative diseases, including ALS. Our lab previously developed a prediction algorithm, PAPA (Prion Aggregation Prediction Algorithm), to predict the aggregation propensity of Q/N-rich proteins, and to predict the effects of mutations on aggregation propensity. Here, we tested the ability of PAPA to design mutations to turn non-prion proteins into prions. We identified four yeast Q/N-rich protein fragments that lacked any detectable aggregation or prion activity. In each case, a small number of designed mutations were sufficient to cause these domains to aggregate, and in two cases, to create bona fide prion activity. We then tested whether simply generating tandem repeats of short, aggregation-prone segments within these domains would likewise be sufficient to create prion activity. We found that such segment duplications consistently increased prion activity in a length-dependent manner. This suggests that duplication of aggregation-prone segments might represent a simple mechanism for evolving new prion domains, potentially explaining why oligopeptide repeats are frequently found in prion proteins.

A difficulty in studying hemorrhagic arenavirus pathogenesis is a lack of animal models that recapitulate human disease and which can be manipulated at BSL-3 or lower. Pirital virus (PIRV), a South American arenavirus hosted by Alston’s cotton rats (Sigmodon alstoni), has not been shown to cause disease in humans and is considered a BSL-3 agent. Infection of Syrian golden hamsters (Mesocricetus auratus) causes a disease that shares many features of the South American hemorrhagic fevers and Lassa fever. Significantly, neurological manifestations occur, a feature commonly found in Argentine hemorrhagic fever and which is absent in most other models of arenavirus disease. We examined the pathogenesis of PIRV infection by clinical and molecular methods, with a focus on gene expression of the antiviral type I interferon response using RNA-Seq. More than 3,000 genes were differentially expressed in the livers of infected hamsters, of which 86 are involved with antiviral responses. Several of these genes participate in apoptosis and autophagy, which may suggest a mechanism of pathogenesis observed in damaged livers.

Included among the many current world problems are viral diseases of considerable significance and threat. This talk could not possible cover them all but will discuss the emergence of Middle East respiratory syndrome in Saudi Arabia and nearby countries, ebolavirus hemorrhagic fever in West Africa, chikungunya in the Western Hemisphere, and the re-emergence of poliomyelitis.

Transmissible spongiform encephalopathies (TSEs), or prions, cause a fatal neurodegenerative disease affecting mammals including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, variant Creutzfeldt-Jakob disease in humans and chronic wasting disease (CWD) in deer, elk and moose. CWD, the only prion disease to infect a native free-ranging population, has now been detected in 22 American states, 2 Canadian provinces and South Korea. While horizontal transmission is credited for much of the spread of CWD, few studies have monitored the potential for vertical/maternal transmission with an emphasis on lactation. Using a small, polyestrous cervid— the Reeves’ muntjac deer— we are addressing this issue by supplementing naïve Reeve’s muntjac fawns (n=5) with milk collected from CWD-inoculated, pre-clinical and clinical muntjac doe. Blood, saliva, feces, urine and lymphoid biopsies will be collected from milk-exposed fawns at 10d, 21d, 40d, 3mo, 6mo, 12 and 18 mo pi to aid in CWD diagnosis. Similar samples, with the addition of mammary biopsy, will be collected from each mother doe at 3 months intervals to monitor CWD status. CWD fawn and mother doe CWD status will be monitored by immunohistochemistry, real time quaking induced conversion assay (RT-QuIC), protein misfolding cyclic amplification (PMCA) and clinical disease progression The results of this study will establish: 1) if there are sufficient infectious prions in the milk of lactating doe to transmit disease to offspring and 2) if mother to offspring transmission plays a role in the high efficiency with which CWD is transmitted in nature.

We recently identified and sequenced a novel herpesvirus of domestic cats, Felis catus gammaherpesvirus 1 (FcaGHV1). FcaGHV1 is a member of the gammaherpesvirus subfamily which also includes the human cancer-associated herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). As a first step toward developing a serologic assay to detect exposure to FcaGHV1, we are seeking to determine which viral proteins elicit an antibody response in naturally occurring domestic cat infections. We cloned selected FcaGHV1 genes into a mammalian expression vector and performed transfections of a feline cell line for expression of recombinant FcaGHV1 proteins. We fixed cells with paraformaldehyde and methanol-acetone and tested reactivity to serum from cats naturally infected with FcaGHV1 using immunofluorescent antibody staining. An FIV immunoflouresence test was developed as a positive control for transfection and assay function. Serum from specific pathogen-free laboratory cats served as negative controls. Preliminary data from 9 cats with FcaGHV1 infection indicates that capsid protein ORF 65 and tegument protein ORF38 may elicit antibodies during naturally occurring FcaGHV1 infection. Results of this study will suggest which FcaGHV1 proteins are immunodominant during natural infection. With this information we plan to develop a serologic assay and further evaluate FcaGHV1 as a model for EBV and KSHV.

The Middle East respiratory syndrome coronavirus (MERS CoV) is a novel coronavirus first recognized in 2012 and is associated with severe respiratory disease in humans. Virus has been isolated from dromedary camels in endemic areas, and many camels also have neutralizing antibodies against the virus, suggesting that they are likely a reservoir host. In order to better understand the role of camels in virus transmission we experimentally infected 3 adult, male dromedary camels with a human isolate of MERS CoV. All animals developed a transient, upper respiratory tract infection associated with very minor clinical disease. Large quantities of infectious virus were isolated from nasal secretions from each animal through 7 days post-inoculation, and viral RNA was detected much longer. Although our study design was limited to 3 animals, these data indicate that MERS CoV readily infects camels, which shed large amounts of virus and likely can efficiently transmit virus to other camels and humans.