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.

Influenza viruses and rhinoviruses are responsible for a large number of acute respiratory viral infections in human populations and are detected as copathogens within hosts. Clinical and epidemiological studies suggest that coinfection by rhinovirus and influenza virus may reduce disease severity and that they may also interfere with each other's spread within a host population. To determine how coinfection by these two unrelated respiratory viruses affects pathogenesis, we established a mouse model using a minor serogroup rhinovirus (rhinovirus strain 1B [RV1B]) and mouse-adapted influenza A virus (A/Puerto Rico/8/1934 [PR8]). Infection of mice with RV1B 2 days before PR8 reduced the severity of infection by a low or medium, but not high, dose of PR8. Disease attenuation was associated with an early inflammatory response in the lungs and enhanced clearance of PR8. However, coinfection by RV1B did not reduce PR8 viral loads early in infection or inhibit replication of PR8 within respiratory epithelia or Inflammation in coinfected mice remained focal compared to diffuse inflammation and damage in the lungs of mice infected by PR8. The timing of RV1B coinfection was a critical determinant of protection, suggesting that sufficient time is needed to induce this response. Finally, disease attenuation was not unique to RV1B: dose-dependent coinfection by a murine coronavirus (mouse hepatitis virus strain 1 [MHV-1]) also reduced the severity of PR8 infection. Unlike RV1B, coinfection with MHV-1 reduced early PR8 replication, which was associated with upregulation of beta interferon (IFN-β) expression. This model is critical for understanding the mechanisms responsible for influenza disease attenuation during coinfection by unrelated respiratory viruses. Viral infections in the respiratory tract can cause severe disease and are responsible for a majority of pediatric hospitalizations. Molecular diagnostics have revealed that approximately 20% of these patients are infected by more than one unrelated viral pathogen. To understand how viral coinfection affects disease severity, we inoculated mice with a mild viral pathogen (rhinovirus or murine coronavirus), followed 2 days later by a virulent viral pathogen (influenza A virus). This model demonstrated that rhinovirus can reduce the severity of influenza A virus, which corresponded with an early but controlled inflammatory response in the lungs and early clearance of influenza A virus. We further determined the dose and timing parameters that were important for effective disease attenuation and showed that influenza disease is also reduced by coinfection with a murine coronavirus. These findings demonstrate that coinfecting viruses can alter immune responses and pathogenesis in the respiratory tract.

The MHV-JHM strain of the murine coronavirus mouse hepatitis virus is much more neurovirulent than the MHV-A59 strain, although both strains use murine CEACAM1a (mCEACAM1a) as the receptor to infect murine cells. We previously showed that Ceacam1a in Ceacam1a ؊/؊ mice. This study shows that infection and spread of MHV-JHM in the brain are dependent upon the viral spike glycoprotein. RJHM can initiate infection in the brains of Ceacam1a ؊/؊ mice, but expression of mCEACAM1a increases susceptibility to infection. The spread of infection in the brain is mCEACAM1a independent. Thus, the ability of the MHV-JHM spike to mediate mCEACAM1a-independent spread in the brain is likely an important factor in the severe neurovirulence of MHV-JHM in wild-type mice.Mouse hepatitis virus (MHV), an enveloped virus with a positive-sense RNA genome in the Coronaviridae family, has been studied widely as a model of viral pathogenesis. Strains of MHV differ in tissue tropism and virulence in mice. While MHV-A59 is both hepatotropic and neurotropic, MHV-JHM is primarily neurotropic and is much more neurovirulent than MHV-A59 (4, 22). When inoculated intracerebrally (i.c.), MHV-A59 and MHV-JHM both replicate in the central nervous systems (CNS) of susceptible mice, yet these viruses differ markedly in pathogenesis (18,34,38). MHV-A59 causes hepatitis, mild encephalitis, and subacute demyelination, whereas MHV-JHM causes acute, fatal encephalitis and, in mice that survive acute infection, chronic demyelination. The spike glycoprotein of MHV-JHM is a major determinant of neurovirulence, although other viral genes also contribute to neurovirulence (18,34,35,38). The spike glycoprotein of coronaviruses is responsible for viral attachment to the cellular receptor and fusion of the viral and cellular membranes, resulting in virus entry. In addition, the spike glycoprotein is expressed on the surface of infected cells, where it mediates cell-to-cell fusion, resulting in spread of virus by syncytium formation.

The 2014 Ebola virus (EBOV) outbreak in West Africa is the largest in recorded history and resulted in over 11,000 deaths. It is essential that strategies for treatment and containment be developed to avoid future epidemics of this magnitude. With the development of vaccines and antibody-based therapies using the envelope glycoprotein (GP) of the 1976 Mayinga strain, one important strategy is to anticipate how the evolution of EBOV might compromise these efforts. In this study we have initiated a watch list of potential antibody escape mutations of EBOV by modeling interactions between GP and the antibody KZ52. The watch list was generated using molecular modeling to estimate stability changes due to mutation. Every possible mutation of GP was considered and the list was generated from those that are predicted to disrupt GP-KZ52 binding but not to disrupt the ability of GP to fold and to form trimers. The resulting watch list contains 34 mutations (one of which has already been seen in humans) at six sites in the GP2 subunit. Should mutations from the watch list appear and spread during an epidemic, it warrants attention as these mutations may reflect an evolutionary response from the virus that could reduce the effectiveness of interventions such as vaccination. However, this watch list is incomplete and emphasizes the need for more experimental structures of EBOV interacting with antibodies in order to expand the watch list to other epitopes. We hope that this work provokes experimental research on evolutionary escape in both Ebola and other viral pathogens.