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In JoVE (1)
Other Publications (10)
Articles by Eric Mossel in JoVE
JoVE Immunology and Infection
Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Microbiology, Immunology, and Pathology, Colorado State University
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
Other articles by Eric Mossel on PubMed
Rotavirus Genome Segment 7 (NSP3) is a Determinant of Extraintestinal Spread in the Neonatal Mouse
We used the neonatal mouse model of rotavirus infection to study extraintestinal spread following oral inoculation. Five-day-old pups were inoculated with either SA11-Cl3, SA11-Cl4, SA11-4F, RRV, or B223. By using virus detection in the liver as a proxy determination for extraintestinal spread, rotavirus strains capable of extraintestinal spread at high frequency (rhesus rotavirus [RRV]) and very low frequency (SA11-Cl4) were identified. Both strains productively infected the gastrointestinal tract. Oral inoculation of mice with RRV/ SA11-Cl4 reassortants and determination of virus titers in the gut and liver revealed that the extraintestinal spread phenotype segregated with RRV genome segment 7 to a high level of significance (P = 10(-3)). RRV segment 7 also segregated with the growth of virus in the gut (P = 10(-5)). Although infection of the gut was clearly required for tropism to the liver, there was no correlation between virus titers in the gut and detection of virus in the liver. Five days after intraperitoneal administration to bypass the gut barrier to virus spread, RRV and SA11-Cl4 both were recovered in the liver. However, only RRV was found in the liver following subcutaneous inoculation, suggesting that this peripheral site presented a similar barrier to virus spread as the gut. Sequence analysis of segment 7 from parental RRV and SA11-Cl4 and selected reassortants showed that (i) amino acid differences were distributed throughout the coding sequences and not concentrated in any particular functional motif and (ii) parental sequence was preserved in reassortants. These data support the hypothesis that NSP3, coded for by genome segment 7, plays a significant role in viral growth in the gut and spread to peripheral sites. The mechanism of NSP3-mediated tropism is under investigation.
A Lymphatic Mechanism of Rotavirus Extraintestinal Spread in the Neonatal Mouse
We used the neonatal mouse model of rotavirus infection and virus strains SA11-clone 4 (SA11-Cl4) and Rhesus rotavirus (RRV) to examine the mechanism of the extraintestinal spread of viruses following oral inoculation. The spread-competent viruses, RRV and reassortant R7, demonstrated a temporal progression from the intestine, to the terminal ileum, to the mesenteric lymph nodes (MLN), and to the peripheral tissues. SA11-Cl4 was not found outside the intestine. Reassortant virus S7, which was unable to reach the liver in previous studies (E. C. Mossel and R. F. Ramig, J. Virol. 76:6502-6509, 2002), was recovered from 60% of the MLN, suggesting that there are multiple determinants for the spread of virus from the intestine to the MLN. Phenotypic segregation analysis identified RRV genome segment 6 (VP6) as a secondary determinant of the spread of virus to the MLN (P = 0.02) in reassortant viruses containing segment 7 from the spread-incompetent parent. These data suggest that in the orally infected neonatal mouse, the extraintestinal spread of rotavirus occurs via a lymphatic pathway, and the spread phenotype is primarily determined by NSP3 and can be modified by VP6.
Interferon-beta and Interferon-gamma Synergistically Inhibit the Replication of Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV)
Recent studies have shown that interferon-gamma (IFN-gamma) synergizes with IFN-alpha/beta to inhibit the replication of both RNA and DNA viruses. We investigated the effects of IFNs on the replication of two strains of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). While treatment of Vero E6 cells with 100 U/ml of either IFN-beta or IFN-gamma marginally reduced viral replication, treatment with both IFN-beta and IFN-gamma inhibited SARS-CoV plaque formation by 30-fold and replication by 3000-fold at 24 h and by > 1 x 10(5)-fold at 48 and 72 h post-infection. These studies suggest that combination IFN treatment warrants further investigation as a treatment for SARS.
Severe Acute Respiratory Syndrome Coronavirus 3a Protein is a Viral Structural Protein
The present study showed the association of a severe acute respiratory syndrome coronavirus (SCoV) accessory protein, 3a, with plasma membrane and intracellular SCoV particles in infected cells. 3a protein appeared to undergo posttranslational modifications in infected cells and was incorporated into SCoV particles, establishing that 3a protein was a SCoV structural protein.
Exogenous ACE2 Expression Allows Refractory Cell Lines to Support Severe Acute Respiratory Syndrome Coronavirus Replication
Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression.
Inhibition of Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) Infectivity by Peptides Analogous to the Viral Spike Protein
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002-2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the CoV S2 subunit that may preferentially associate with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-CoV and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pre-transmembrane domain of the S2 subunit inhibited SARS-CoV plaque formation by 40-70% at concentrations of 15-30 microM. Interestingly, peptides analogous to the SARS-CoV or MHV loop region inhibited viral plaque formation by >80% at similar concentrations. The observed effects were dose-dependent (IC50 values of 2-4 microM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the CoV peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.
SARS-CoV Replicates in Primary Human Alveolar Type II Cell Cultures but Not in Type I-like Cells
Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10(5) pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.
Identification of Super-infected Aedes Triseriatus Mosquitoes Collected As Eggs from the Field and Partial Characterization of the Infecting La Crosse Viruses
La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.
Treatment with Cationic Liposome-DNA Complexes (CLDCs) Protects Mice from Lethal Western Equine Encephalitis Virus (WEEV) Challenge
Having recently characterized a CD-1 outbred mouse model of pathogenesis for Western equine encephalitis virus, we examined the possible protective effects of cationic liposome-DNA complexes (CLDCs) against encephalitic arboviral infection. In this investigation, mice were pre-treated, co-treated, or post-treated with CLDC then challenged with a subcutaneous or aerosol dose of the highly virulent WEEV-McMillan strain, lethal in mice 4-5 days after inoculation. Pre-treatment with CLDCs provided a significant protective effect in mice, which was reflected in significantly increased survival rates. Further, in some instances a therapeutic effect of CLDC administration up to 12h after WEEV challenge was observed. Mice treated with CLDC had significantly increased serum IFN-gamma, TNF-alpha, and IL-12, suggesting a strong Th1-biased antiviral activation of the innate immune system. In virus-infected animals, large increases in production of IFN-gamma, TNF-alpha, MCP-1, IL-12, and IL-10 in the brain were observed by 72h after infection, consistent with neuroinvasion and viral replication in the CNS. These results indicate that strong non-specific activation of innate immunity with an immune therapeutic such as CLDC is capable of eliciting significant protective immunity against a rapidly lethal strain of WEEV and suggest a possible prophylactic option for exposed individuals.
