Joseph K.-K. Li

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

Abstract Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572–3577). Ribavirin given at 75mg/kg 4h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1α, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus

The cDNA clone lambda ML-1 encoding one of the extracellular lignin peroxidases from the white rot fungus, Phanerochaete chrysosporium, was heterologously expressed in an active form using a recombinant baculovirus system. The glycosylated extracellular form of the recombinant protein contained the ferriprotoporphyrin IX moiety and was capable of oxidizing both iodide and the model lignin compound, veratryl alcohol. In comparative peroxidase assays using guaiacol and Mn(II), the recombinant lignin peroxidase did not appear to be Mn(II) dependent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the heterologously expressed peroxidase had an apparent molecular weight similar to that of the native fungal isozyme H8. The elution profile of the active recombinant enzyme derived by ion-exchange chromatography and immunoblot analysis using an anti-H8 monoclonal antibody provided further evidence that the lambda ML-1 DNA encodes the lignin peroxidase H8.

Single-dose intranasal administration with mDEF201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal SARS-CoV BALB/c mouse model.

Abstract Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p <0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 106 to 108 PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection (p <0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.

Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus

Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification.

Selective in vitro cytotoxic effect of human cancer cells by Bluetongue virus-10

Bluetongue viruses (BTVs) infect primarily domestic cattle and wild ruminants but have never been shown to infect normal human cells. Thus, humans are sero-negative towards BTVs. The selective and differential effects of BTV serotype 10 (BTV-10) infection were investigated with five cell lines including primary human embryo lung fibroblast (HEL) and primary murine embryos fibroblast(MEF), human hepatic carcinoma 3B cell line (Hep-3B), human lung carcinoma cell line (A549) and mouse fibroblast cell line (NIH 3T3). In this study, comparative analyses of differential cytopathic effects (CPEs), survival rates using different Multiplicities of Infection (MOI), ultra-structural changes by transmission electron microscopy, and the preferential cell cycle changes of infected cells by flow cytometry were made among these cells. Detection of the presence of BTV genome and kinetic analysis of virus titers in TCID50 were also made. We provided the first analytical demonstration and evidence that BTV-10 could selectively infect and degrade human cancer cells but not cultured primary normal cells. No CPE or viral mRNAs could be detected within these normal cells, while various degrees of CPE could be found in Hep-3B and A549, as well as in NIH 3T3 under similar conditions. Before death, BTV-infected human cancer cells were directly arrested in the sub-G1 phase and the diversity of BTV infection as shown by the MTT method had significant difference (F=95.635, p<0.01). Above results suggested that this viral dose-dependent cytotoxic effect is caused by both effective virion amplification and induced apoptosis. Cellular distinctive transformation status may contribute to the selectivity. Thus, selective degradation of human cancer cells but not normal diploid cells by the newly discovered oncolytic potential of BTV would provide a very attractive approach for cancer therapy in the future.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

Abstract Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1±0.4μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575PFU) of virus for 4h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0mg/kg/day for 4 days. Treatment with UDA at 5mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p <0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p <0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells.

Characterization of Wolbachia postica, the cause of reproductive incompatibility among alfalfa weevil strains

Abstract Wolbachia postica, which causes reproductive incompatibility between male western alfalfa weevils and female eastern or Egyptian weevils, was isolated by Renografin density gradients. Hybridization of DNA from this isolated microorganism with a mycoplasma-specific gene probe confirmed it is not a mycoplasma but a wolbachia. Polyclonal antibodies produced against inactivated Wolbachia postica showed high sensitivity in fluorescent light microscopy and immunoelectron microscopy. This antibody was specific for Wolbachia-infected adult weevil by immunoblots which were reliable and reproducible, and more sensitive than the Giemsa stain procedure. A 135-kDa protein detected in all weevils infected with wolbachiae appeared to be a good marker for the microorganism. This technique facilitates the detection of wolbachiae-infected male weevils. Dissection of the ovary was not required to analyze the female weevils.

Selective separation of virus proteins and double-stranded RNAs by SDS-KCl precipitation

The total viral structural polypeptides and the double-stranded genomic RNAs of bluetongue virus can be selectively separated by a single SDS-KCl precipitation step. This simple, rapid and highly reproducible method enables greater than 95% recovery and purity of both viral proteins and dsRNAs within 30 min. The serotypic identity of the separated dsRNAs can be analyzed by SDS-PAGE electrophorogram immediately. After a single phenol/chloroform extraction, the dsRNA can also be used as hybridization probes, templates for molecular cloning and direct RNA sequencing. The SDS-KCl-precipitated viral proteins could be used readily for peptide mapping and as immunogens. Polyclonal and monoclonal antibodies raised against SDS-KCl-precipitated viral structural polypeptides were useful in Western immunoblots.

A New Finding and Range Extension of Bacilliform Virus in the Freshwater Red Claw Crayfish in Utah, USA

Abstract Nuclear pathology of digestive tubules in the hepatopancreas was detected in tissues collected during an inspection of red claw crayfish Cherax quadricarinatus (also known as the tropical blue crayfish) at a farm in the state of Utah, USA. Signs of clinical disease in all age-classes were absent. Foci with hypertrophic, eosinophilic nuclei were detected in tissues from the adult, but the intensity of nuclear abnormalities was low. Histological preparations of hepatopancreas from juveniles showed a greater intensity of nuclear lesions than did preparations of tissues from the adult. Virus was not observed by use of electron microscopy on the adult crayfish; however, rod-shaped virions were detected within nuclei of the hepatopancreatic epithelium in juveniles. Virions had a rod-shaped nucleocapsid surrounded by a loose envelope and a faintly distinct tail-like structure. Mean virion size was 224 ± 14 nm × 74 ± 4 nm, and nucleocapsids measured 180 ± 9 nm × 38 ± 3 nm (mean ± SD). The virus is morpho...

Epitopic mapping of linear and conformation-dependent antigenic determinants on GP5 of five U.S. bluetongue viruses

Two distinct antigenic determinants of the major outer capsid protein, GP5, of five U.S. bluetongue viruses have been identified and mapped using monoclonal and oligoclonal antibodies. One antigenic site, identified by oligoclonal antibody AK-15, was found to be common and conserved in all five U.S. BTV serotypes. This linear epitope was located between amino acid residues 175 and 189 (ALQREAAERSEDEIK). The second determinant identified by monoclonal antibody 34.7 was present in BTV-2, -10, -11, and -17 but absent in BTV-13. The binding of this monoclonal antibody to GP5 could be blocked specifically by one of three short synthetic peptides located among amino acid residues 33-42 (KAAERFAESE), 159-168 (EKILKEEDSK), and 206-215 (EIERDGMQEE), indicating that this antigenic determinant is conformation-dependent. Oligoclonal antibody (AK-15) reacted with denatured GP5 immobilized on nitrocellulose membrane after Western transfer as well as with native GP5 present on the surface of purified BTV virions. Monoclonal antibody (34.7) reacted only with denatured GP5 but not native GP5 using an ELISA assay. However, these two antigenic epitopes alone did not elicit detectable neutralizing antibodies as determined by plaque reduction assay.