Severe acute respiratory syndrome (SARS), which is caused by a novel coronavirus (CoV), is a highly communicable disease with the lungs as the major pathological target. Although SARS likely stems from overexuberant host inflammatory responses, the exact mechanism leading to the detrimental outcome in patients remains unknown. Pulmonary macrophages (M), airway epithelium, and dendritic cells (DC) are key cellular elements of the host innate defenses against respiratory infections. While pulmonary M are situated at the luminal epithelial surface, DC reside abundantly underneath the epithelium. Such strategic locations of these cells within the airways make it relevant to investigate their likely impact on SARS pathogenesis subsequent to their interaction with infected lung epithelial cells. To study this, we established highly polarized human lung epithelial Calu-3 cells by using the Transwell culture system. Here we report that supernatants harvested from the apical and basolateral domains of infected Calu-3 cells are potent in modulating the intrinsic functions of M and DC, respectively. They prompted the production of cytokines by both M and DC and selectively induced CD40 and CD86 expression only on DC. However, they compromised the abilities of the DC and M in priming naïve T cells and phagocytosis, respectively. We also identified interleukin-6 (IL-6) and IL-8 as key SARS-CoV-induced epithelial cytokines capable of inhibiting the T-cellpriming ability of DC. Taken together, our results provide insights into the molecular and cellular bases of the host antiviral innate immunity within the lungs that eventually lead to an exacerbated inflammatory cascades and severe tissue damage in SARS patients.

Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NF)κB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-β, -λs, inflammatory mediators, and many IFN-stimulated genes (ISGs). We also showed, for the first time, that IFN-β and IFN-λs were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by >3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipaviruslike viruses. IMPORTANCENipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; th...

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. IMPORTANCERift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.

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f Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-␣) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-␣ nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

BackgroundJunín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses.Methodology/Principal FindingsTo evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1–2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33–40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection.Conclusions/SignificanceThe remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses.

We previously reported that transgenic (Tg) mice expressing human angiotensin-converting enzyme 2 (hACE2), the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), were highly susceptible to SARS-CoV infection, which resulted in the development of disease of various severity and even death in some lineages. In this study, we further characterized and compared the pathogeneses of SARS-CoV infection in two of the most stable Tg lineages, AC70 and AC22, representing those susceptible and resistant to the lethal SARS-CoV infection, respectively. The kinetics of virus replication and the inflammatory responses within the lungs and brains, as well as the clinical and pathological outcomes, were assessed in each lineage. In addition, we generated information on lymphocyte subsets and mitogen-mediated proliferation of splenocytes. We found that while both lineages were permissive to SARS-CoV infection, causing elevated secretion of many inflammatory mediators within the lungs and brains, viral infection appeared to be more intense in AC70 than in AC22 mice, especially in the brain. Moreover, such infection was accompanied by a more profound immune suppression in the former, as evidenced by the extensive loss of T cells, compromised responses to concanavalin A stimulation, and absence of inflammatory infiltrates within the brain. We also found that CD8 ؉ T cells were partially effective in attenuating the pathogenesis of SARS-CoV infection in lethality-resistant AC22 mice. Collectively, our data revealed a more intense viral infection and immunosuppression in AC70 mice than in AC22 mice, thereby providing us with an immunopathogenic basis for the fatal outcome of SARS-CoV infection in the AC70 mice.