Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA

Dengue fever is a mosquito-borne viral disease of global importance with no available antiviral therapy. We assessed the ability of mycophenolic acid (MPA), a drug currently used as an immunosuppressive agent, to inhibit dengue virus (DV) antigen expression, RNA replication, and virus production. Pharmacological concentrations of MPA effectively blocked DV infection, decreasing the percentage of infected cells by 99% and the levels of secreted virus by up to a millionfold. Results were reproduced with four hepatoma cell lines and different flaviviruses, including a recent West Nile virus isolate. Experiments were performed to define the stage in the viral lifecycle at which MPA abrogates infection. Early steps in viral infection, such as viral entry and nucleocapsid uncoating, were not the primary targets of MPA action since its inhibitory effect was retained when naked DV RNA was transfected directly into cells. Biosynthetic labeling experiments showed that MPA did not block the initial phase of viral translation but did interfere with viral protein synthesis in the amplification phase. Quantitative RT-PCR demonstrated that MPA prevented the accumulation of viral positive- and negative-strand RNA as the infection proceeded. We conclude that MPA inhibits flavivirus infection by preventing synthesis and accumulation of viral RNA.     

Internal initiation of translation of bovine viral diarrhea virus RNA.

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon.     

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.     

Interferon induction by rice dwarf virus RNA

Summary Interferon (IF) induction by rice dwarf virus RNA (RDV-RNA) was studied in comparison with that by poly I:C. The IF inducing capacities of both were approximately equivalent in rabbit kidney cells at the same concentration. Interferon was also induced by purified RDV virions. However, IF production by RDV was generally lower than by RDV-RNA. It was observed that marked differences exist in IF response of several species of cells to RDV-RNA. Highest titer of IF was obtained in rabbit kidney cells. The IF inducing capacity of RDV-RNA was shown to be more resistant to RN-ase and UV-irradiation as compared to poly I:C. Treatment of RDV-RNA at 60° C for 15 minutes did not alter its IF inducing capacity while poly I:C lost 50% of the activity.     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.     

抗DEN-2基因核酶的克隆及其体外切割活性的研究

目的：探讨核酶抗登革病毒活性。方法：根据DEN－2基因结构的特点，按照锤头状核酶的结构模型和作用模式，设计，合成针对172－174GUC位点的核酶基因；定向插入质粒pGEM-3Zf( )的Sac I和Sal I位点之间；核酶基因克隆和DEN－2靶基因克隆分别进行体外转录，生成核酶的靶RNA，体外进行切割反应。结果：核酶与登革病毒靶序列体外产物电泳 见预期切割条带。结论：该核酶具有切割相应靶RNA的活性，可进一步用于细胞内核酶抗登革病毒的研究。     

Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

In vitro, the coat protein of brome mosaic virus (BMV) inhibited RNA synthesis by replicase extracted from BMV-infected barley leaves. The inhibition was due to the interaction of coat protein with BMV RNA. Under the same conditions, no inhibition by TMV coat protein or bovine serum albumin was detected. For the complete inhibition of RNA synthesis in vitro, a coat protein:RNA ratio of 620:1 was required, and their preincubation before addition to the reaction mixture was essential. If the coat protein was added to the reaction mixture during incubation, synthesis continued for a few minutes at the level of the control (omission of coat protein), then decreased gradually, and stopped 6 min after the addition of coat protein. These results suggest that the inhibitory effect of coat protein on RNA synthesis in vitro is due to the interference with the binding site of replicase by partial reassembly of nucleoprotein and that this phenomenon may be a cause of cross protection.     

The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system

The small (S) genomic segment of Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to elucidate the function of NSs, we established a plasmid-based minireplicon system using mammalian cells that express large amounts of T7 RNA polymerase. Expression of N, the viral polymerase protein (L), and a minireplicon containing a reporter gene was sufficient to reconstitute functional virus nucleocapsids. Coexpression of NSs, however, led to a dose-dependent decrease in reporter activity without affecting expression of controls. The inhibition could not be reversed by overexpression of N, L or the minireplicon, indicating that the NSs effect was not caused by a reduction in virus gene expression. The NSs proteins of two other members of the Bunyavirus genus, Guaroa virus and Lumbo virus, were also inhibitory in our system. The intracellular localisation of Bunyamwera virus NSs was investigated and found to be predominantly cytoplasmic, but intranuclear inclusion was also detected. Taken together, these data suggest that, in mammalian cells, the bunyavirus NSs protein controls the activity of the viral polymerase by a highly conserved mechanism.     

Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication

The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by approximately 37 and approximately 60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes.     

Interferon inhibits induction of endogenous murine leukemia virus production.

In 5-iododeoxyuridine (IUDR) induction of murine leukemia virus (MLV) in Balb cells transformed by SV40 (Balb SV40) or AKR cells, interferon inhibited virus production. This could have been due to inhibition of the X- and N-tropic virus induced or to inhibition of subsequent reinfection of the mouse cells by the N tropic virus. Cycloheximide, however, activates $\text{KBalb}\text{3T3}$ cells ($\text{Balb}\text{3T3}$ cells transformed by Kirsten sarcoma virus) to produce only an X-tropic virus which could not reinfect mouse cells. In this system, interferon treatment also decreases virus yields and, therefore, an event in the virus induction process.     

Development of a novel mouse model for dengue virus infection.

In the present study, we established an animal model for dengue virus infection using severe combined immunodeficient mice transplanted with a human hepatocarcinoma cell line (HepG2). At 7-8 weeks after transplantation, the HepG2-grafted mice were infected intraperitoneally with dengue virus type 2 (DEN-2). A higher titer of the virus was detected in the liver and serum but not in the brain in the early stage of postinfection. When the mice showed paralysis, the highest titer of virus was detected in the serum and brain. DEN-2 antigens were also found in HepG2 cells of the liver in the early stage and some neurons of the brain in the late stage. Upon clinical examination, thrombocytopenia, prolonged partial thromboplastin time, and increased hematocrit, blood urea nitrogen, and tumor necrosis factor alpha were seen in the paralyzed mice. Moreover, mild hemorrhage in the liver and tarry stool in the small intestine were observed in some mice. Our results show some similarities to human DEN infection and this mouse model might be valuable for studying some aspects of pathogenesis of this disease.     

Acute transverse myelitis following dengue virus infection.

The spinal cord is infrequently affected following dengue virus infection. We report a case of transverse myelitis that developed 2 weeks after acute dengue infection and review the literature to elucidate the pathogenesis of spinal cord involvement in dengue infection. We postulate that temporal factors may play a role in the different clinical manifestations, i.e. that acute parainfectious dengue infection presents with flaccid paralysis, whereas late-stage (postinfectious) dengue infection, presents with spastic weakness. Further studies need to be performed to elucidate the mechanisms of spinal neurological damage in dengue.     

Prevention of transplacental infection of bovine foetus by bovine viral diarrhoea virus through vaccination

Results are presented for an experimental validation of the efficacy of an EU licensed, inactivated bovine viral diarrhoea virus vaccine (Bovilis BVDV). This study was designed to assess the quality of efficacy 6 months after a single course of vaccination (two intramuscular doses a month apart). A natural challenge at about 87 days of gestation by 3 persistently infected carrier heifers rapidly infected all experimental heifers. This resulted in transplacental BVDV infection of all 7 unvaccinated dams whereas 11 immunised dams produced 9 live-born calves and 2 aborted foetuses from which no BVDV could be recovered.