Byron E. E. Martina

Dengue Virus Pathogenesis: an Integrated View

SUMMARY Much remains to be learned about the pathogenesis of the different manifestations of dengue virus (DENV) infections in humans. They may range from subclinical infection to dengue fever, dengue hemorrhagic fever (DHF), and eventually dengue shock syndrome (DSS). As both cell tropism and tissue tropism of DENV are considered major determinants in the pathogenesis of dengue, there is a critical need for adequate tropism assays, animal models, and human autopsy data. More than 50 years of research on dengue has resulted in a host of literature, which strongly suggests that the pathogenesis of DHF and DSS involves viral virulence factors and detrimental host responses, collectively resulting in abnormal hemostasis and increased vascular permeability. Differential targeting of specific vascular beds is likely to trigger the localized vascular hyperpermeability underlying DSS. A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk for development of severe disease.

Virology: SARS virus infection of cats and ferrets.

The reservoir of the coronavirus isolated from patients with severe acute respiratory syndrome (SARS) is still unknown, but is suspected to have been a wild animal species. Here we show that ferrets (Mustela furo) and domestic cats (Felis domesticus) are susceptible to infection by SARS coronavirus (SCV) and that they can efficiently transmit the virus to previously uninfected animals that are housed with them. The observation that these two distantly related carnivores can so easily be infected with the virus indicates that the reservoir for this pathogen may involve a range of animal species.

Pegylated interferon-|[alpha]| protects type 1 pneumocytes against SARS coronavirus infection in macaques

The primary cause of severe acute respiratory syndrome (SARS) is a newly discovered coronavirus. Replication of this SARS coronavirus (SCV) occurs mainly in the lower respiratory tract, and causes diffuse alveolar damage. Lack of understanding of the pathogenesis of SARS has prevented the rational development of a therapy against this disease. Here we show extensive SCV antigen expression in type 1 pneumocytes of experimentally infected cynomolgus macaques (Macaca fascicularis) at 4 d postinfection (d.p.i.), indicating that this cell type is the primary target for SCV infection early in the disease, and explaining the subsequent pulmonary damage. We also show that prophylactic treatment of SCV-infected macaques with the antiviral agent pegylated interferon-α (IFN-α) significantly reduces viral replication and excretion, viral antigen expression by type 1 pneumocytes and pulmonary damage, compared with untreated macaques. Postexposure treatment with pegylated IFN-α yielded intermediate results. We therefore suggest that pegylated IFN-α protects type 1 pneumocytes from SCV infection, and should be considered a candidate drug for SARS therapy

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.

Summary SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3·3 logs (95% Cl 2·6–4·0 logs; p<0·001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0·013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.

Vaccine-induced enhancement of viral infections.

Abstract Examples of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis have been documented for infections by members of different virus families. Several mechanisms, many of which still are poorly understood, are at the basis of this phenomenon. Vaccine development for lentivirus infections in general, and for HIV/AIDS in particular, has been little successful. Certain experimental lentiviral vaccines even proved to be counterproductive: they rendered vaccinated subjects more susceptible to infection rather than protecting them. For vaccine-induced enhanced susceptibility to infection with certain viruses like feline coronavirus, Dengue virus, and feline immunodeficiency virus, it has been shown that antibody-dependent enhancement (ADE) plays an important role. Other mechanisms may, either in the absence of or in combination with ADE, be involved. Consequently, vaccine-induced enhancement has been a major stumble block in the development of certain flavi-, corona-, paramyxo-, and lentivirus vaccines. Also recent failures in the development of a vaccine against HIV may at least in part be attributed to induction of enhanced susceptibility to infection. There may well be a delicate balance between the induction of protective immunity on the one hand and the induction of enhanced susceptibility on the other. The present paper reviews the currently known mechanisms of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis.

Morbillivirus in monk seal mass mortality

We have identified a morbillivirus in organs of Mediterranean monk seals (Monachus monachus), lost during a recent mass die-off. About half of the population of this highly endangered species, which inhabits the Mauritanian coast of West Africa (Cap Blanc), were killed. The outbreak is reminiscent of several recent morbillivirus outbreaks in aquatic mammals.

Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral

OBJECTIVES T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus. METHODS The anti-CHIKV effect of favipiravir was examined in cell culture and in a mouse model of lethal infection. A five-step protocol was used to select for CHIKV variants with reduced susceptibility to favipiravir. The resistant phenotype was confirmed in cell culture and the whole genome was sequenced. The identified mutations were reverse-engineered into an infectious clone to confirm their impact on the antiviral efficacy of favipiravir. RESULTS Favipiravir inhibits the replication of laboratory strains and clinical isolates of CHIKV, as well as of a panel of other alphaviruses. Several favipiravir-resistant CHIKV variants were independently selected and all of them in particular acquired the unique K291R mutation in the RNA-dependent RNA polymerase (RdRp). Reverse-engineering of this K291R mutation into an infectious clone of CHIKV confirmed the link between the mutant genotype and the resistant phenotype. Interestingly, this particular lysine is also highly conserved in the RdRp of positive-stranded RNA viruses in general. CONCLUSIONS This study provides an important insight into the precise molecular mechanism by which favipiravir exerts its antiviral activity against (alpha)viruses, which may be of help in designing other potent broad-spectrum antivirals.

A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice.

BackgroundSince its first appearance in the USA in 1999, West Nile virus (WNV) has spread in the Western hemisphere and continues to represent an important public health concern. In the absence of effective treatment, there is a medical need for the development of a safe and efficient vaccine. Live attenuated WNV vaccines have shown promise in preclinical and clinical studies but might carry inherent risks due to the possibility of reversion to more virulent forms. Subunit vaccines based on the large envelope (E) glycoprotein of WNV have therefore been explored as an alternative approach. Although these vaccines were shown to protect from disease in animal models, multiple injections and/or strong adjuvants were required to reach efficacy, underscoring the need for more immunogenic, yet safe DIII-based vaccines.ResultsWe produced a conjugate vaccine against WNV consisting of recombinantly expressed domain III (DIII) of the E glycoprotein chemically cross-linked to virus-like particles derived from the recently discovered bacteriophage AP205. In contrast to isolated DIII protein, which required three administrations to induce detectable antibody titers in mice, high titers of DIII-specific antibodies were induced after a single injection of the conjugate vaccine. These antibodies were able to neutralize the virus in vitro and provided partial protection from a challenge with a lethal dose of WNV. Three injections of the vaccine induced high titers of virus-neutralizing antibodies, and completely protected mice from WNV infection.ConclusionsThe immunogenicity of DIII can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. The superior immunogenicity of the conjugate vaccine with respect to other DIII-based subunit vaccines, its anticipated favourable safety profile and low production costs highlight its potential as an efficacious and cost-effective prophylaxis against WNV.

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

BackgroundChikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.ResultsExpression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf 21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.ConclusionsChikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.