Miriam E. R. Darnell

New Antiviral Pathway That Mediates Hepatitis C Virus Replicon Interferon Sensitivity through ADAR1

ABSTRACT While many clinical hepatitis C virus (HCV) infections are resistant to alpha interferon (IFN-α) therapy, subgenomic in vitro self-replicating HCV RNAs (HCV replicons) are characterized by marked IFN-α sensitivity. IFN-α treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through double-stranded RNA-activated protein kinase (PKR) and also through a new pathway involving RNA editing by an adenosine deaminase that acts on double-stranded RNA (ADAR1). More than 200 genes are induced by IFN-α, and yet only a few are attributed with an antiviral role. We show that inhibition of both PKR and ADAR1 by the addition of adenovirus-associated RNA stimulates replicon expression and reduces the amount of inosine recovered from RNA in replicon cells. Small inhibitory RNA, specific for ADAR1, stimulated the replicon 40-fold, indicating that ADAR1 has a role in limiting replication of the viral RNA. This is the first report of ADARs involvement in a potent antiviral pathway and its action to specifically eliminate HCV RNA through adenosine to inosine editing. These results may explain successful HCV replicon clearance by IFN-α in vitro and may provide a promising new therapeutic strategy for HCV as well as other viral infections.

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Abstract Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254nm, heat treatment of 65°C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV containing materials, that are rendered non-infectious, to be conducted at reduced safety levels.

Severe Acute Respiratory Syndrome Coronavirus Infection in Vaccinated Ferrets

Abstract Background. Development of vaccines to prevent severe acute respiratory syndrome (SARS) is limited by the lack of well-characterized animal models. Previous vaccine reports have noted robust neutralizing antibody and inflammatory responses in ferrets, resulting in enhanced hepatitis. Methods. We evaluated the humoral immune response and pathological end points in ferrets challenged with the Urbani strain of SARS-associated coronavirus (SARS-CoV) after having received formalin-inactivated whole-virus vaccine or mock vaccine. Results. Humoral responses were observed in ferrets that received an inactivated virus vaccine. Histopathological findings in lungs showed that infection of ferrets produced residual lung lesions not seen in both mock and vaccinated ferrets. SARS-CoV infection demonstrated bronchial and bronchiolar hyperplasia and perivascular cuffing in ferret lung tissue, as seen previously in infected mice. No evidence of enhanced disease was observed in any of the ferrets. All of the ferrets cleared the virus by day 14, 1 week earlier if vaccinated. Conclusions. The vaccine provided mild immune protection to the ferrets after challenge; however, there was no evidence of enhanced liver or lung disease induced by the inactivated whole-virus vaccine. The ferret may provide another useful model for evaluating SARS vaccine safety and efficacy.

Evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS‐CoV) has been detected in the blood of infected individuals, which may have the potential to contaminate donated blood and plasma‐derived products in the event of a future outbreak. Effective methods for inactivating the SARS‐CoV in protein solutions are described in this report.

Unilateral nasal infection of cotton rats with respiratory syncytial virus allows assessment of local and systemic immunity

An in vivo model for the study of local and systemic effectors of immunity to respiratory syncytial virus (RSV) is described. Cotton rats (Sigmodon fulviventer) inoculated in one nostril with a small volume (2 microliters) of virus suspension contracted a unilateral nasal infection which did not extend to the contralateral nasal turbinates, nor to the lungs. Immunity to subsequent RSV challenge could be induced by small priming doses ( < 10 p.f.u. per animal), but was dependent upon viral replication, as virus inactivated by UV light was not immunogenic. Immunity occurred in the absence of detectable neutralizing serum antibody. The onset of resistance to viral challenge occurred simultaneously in ipsilateral nasal, contralateral nasal and pulmonary tissues. However, low levels of transient viral replication occurred in contralateral nasal turbinates and in lungs following virus challenge, thus indicating that local components of immunity acting at the ipsilateral site of infection were more effective than systemic components acting at the other sites. Further evidence is provided to suggest that three types of immunological effectors - local, persistent, systemic and transient systemic - participate in the immune response to RSV infection.

Inactivation of respiratory syncytial virus by generic hand dishwashing detergents and antibacterial hand soaps

In an in-vitro test, generic liquid hand dishwashing detergents were as much as 100-fold more effective than proprietary antibacterial soaps in inactivating respiratory syncytial virus (RSV). The use of such detergents for hand washing during annual RSV epidemics, or the incorporation of their antiviral components into antibacterial soaps might be considered to limit nosocomial spread.

Immunoprophylaxis of Group B Respiratory Syncytial Virus Infection in Cotton Rats

Two antigenic groups of respiratory syncytial virus (RSV) have been identified: A (RSV/A) and B (RSV/B). Topical administration of human IgG screened for high titers of antibody to RSV/A (RSVIg) is protective against RSV/A infection in the cotton rat model. The study attempted to determine if topical RSVIg would also be protective against RSV/B. Cotton rats were pretreated intranasally with RSVIg or with monospecific antiserum obtained from animals previously infected with RSV/A or RSV/B (day 0), challenged intranasally with RSV/A or RSV/B (day 1), and sacrificed for virus titration (day 5). Cotton rat antiserum to RSV/B protected against RSV/A and RSV/B, while antiserum to RSV/A protected only against RSV/A. RSVIg, although prepared on the basis of activity against RSV/A, was also protective against RSV/B.