Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans

Advanced age has repeatedly been identified as an independent correlate of adverse outcome and a predictor of mortality in cases of severe acute respiratory syndrome (SARS). SARS-associated mortality may exceed 50% for persons aged 60 years or older. Heightened susceptibility of the elderly to severe SARS and the ability of SARS coronavirus to replicate in mice led us to examine whether aged mice might be susceptible to disease. We report here that viral replication in aged mice was associated with clinical illness and pneumonia, demonstrating an age-related susceptibility to SARS disease in animals that parallels the human experience.     

Co-infection of respiratory bacterium with severe acute respiratory syndrome coronavirus induces an exacerbated pneumonia in mice

SARS-CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS-CoV infection in cultured cells, which suggests that SARS development may be due to elastase-mediated, enhanced SARS-CoV infection in the lungs. To explore this possibility, we examined whether co-infection of mice with SARS-CoV and Pp, a low-pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co-infected with SARS-CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33~90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co-infection with SARS-CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.     

严重急性呼吸综合征、中东呼吸综合征和2019冠状病毒病治疗研究进展

严重急性呼吸综合征2019(sever acute respiratory syndrome,SARS)、中东呼吸综合征(Middle East respiratory syndrome,MERS)和2019冠状病毒病(corona virus disease 2019,COVID-19)对全世界人民造成了严重的经济损...     

重症急性呼吸综合征病原学研究进展

SARS即重症急性呼吸综合征,是一种急性呼吸道传染病,对人类健康已构成巨大威胁。本就其病原寻找、病原基本特点、病原进化和变异、病原诊断、病原来源等方面对SARS病原学研究进展作一简要介绍。     

Cytokine responses in porcine respiratory coronavirus-infected pigs treated with corticosteroids as a model for severe acute respiratory syndrome

The effectiveness and potential immunosuppressive effects of anti-inflammatory glucocorticoids in the lungs of severe acute respiratory syndrome (SARS) patients are undefined. We treated porcine respiratory coronavirus (PRCV)-infected conventional pigs with the corticosteroid dexamethasone (DEX) as a model for SARS. Innate and Th1 cytokines in bronchoalveolar lavage (BAL) and serum were elevated in PRCV-infected pigs compared to controls, but were decreased after DEX treatment in the PRCV-infected, DEX-treated (PRCV/DEX) pigs. Although decreased in BAL, Th2 cytokine levels were higher in serum after DEX treatment. Levels of the proinflammatory cytokine interleukin-6 in BAL and serum were decreased in PRCV/DEX pigs early but increased later compared to those in phosphate-buffered saline-treated, PRCV-infected pigs, corresponding to a similar trend for lung lesions. PRCV infection increased T-cell frequencies in BAL, but DEX treatment of PRCV-infected pigs reduced frequencies of T cells; interestingly B and SWC3a(+) (monocytes/macrophages/granulocytes) cell frequencies were increased. DEX reduced numbers of PRCV-stimulated Th1 gamma interferon-secreting cells in spleen, tracheobroncheolar lymph nodes, and blood. Our findings suggest that future glucocorticoid treatment of SARS patients should be reconsidered in the context of potential local immunosuppression of immune responses in lung and systemic Th1 cytokine-biased suppression.     

Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets

Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.     

The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.     

Experimental infection of macaques with the human reovirus BYD1 strain: an animal model for the study of the severe acute respiratory syndrome

Experimental studies were performed to determine the role of a newly isolated reovirus (ReoV) from a severe acute respiratory syndrome (SARS) patient in the etiology of this newly described serious respiratory syndrome. Four cynomologus macaques were inoculated with this reovirus (BYD1) in an attempt to replicate the infection and pathology observed in SARS. The body temperature of the infected monkeys was monitored three times a day, and blood and fecal samples were periodically collected for specific immunology determinations. On days 7 and 33 after inoculation, necropsies for pathological accessment and pathogen isolation were performed. The four infected macaques developed a fever on days 3 and 4 after inoculation, and maintainted a febrile state for 4-6 days. The highest temperature in the animals recorded was 40.4 degrees C. After a recovery phase, the macaques developed a second febrile condition. Antibody titers against the reovirus injected by the intravenous route occurred in higher number than those in the nasal cavity. Four macaque monkeys demonstrated diffuse alveolar damage, characterized by hemorrhagic pneumonia, serosanguineous exudates, formation of hyaline membranes, and type II pneumocyte hyperplasia, which were similar to those that have been noted in SARS patients. Lymphocytes decreased in the cortex of the lymph node and in the white pulp of the spleen. ReoV was detected in pneumonic tissue by virus isolation and RT-PCR. The macaques infected with the newly isolated reovirus developed a fever, diffuse alveolar damage and pulmonary interstitial inflammation similar to that noted in SARS patients. This evidence demonstrates that ReoV might have a primary role in the etiology of SARS.     

Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.     

Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus

Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP(3014)) or wild-type VEE glycoproteins (VRP(3000)) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP(3000)-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP(3014)-based vaccines were not. The superior protection for the aged observed with VRP(3000)-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP(3000) vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP(3014) platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.     

Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. Since its associated morbidity and mortality have been postulated to be due to immune dysregulation, we investigated which of the viral proteins is responsible for chemokine overexpression. To delineate the viral and cellular factor interactions, the role of four SARS coronavirus proteins, including nonstructural protein 1 (nsp-1), nsp-5, envelope, and membrane, were examined in terms of cytokine induction. Our results showed that the SARS coronavirus nsp-1 plays an important role in CCL5, CXCL10, and CCL3 expression in human lung epithelial cells via the activation of NF-kappaB.     

Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice

To analyze the immune responses of DNA vaccine encoded different gene fragments of severe acute respiratory syndrome coronavirus (SARS-Cov), SARS-Cov gene fragments of membrane (M), nucleocapsid (N), spike a (Sa), and spike b (Sb) proteins were cloned into pcDNA3.1 (Invitrogen) vector to form plasmids pcDNAM, pcDNAN, pcDNASa, and pcDNASb, respectively. After mice were immunized intramuscularly with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmid, blood was collected and serum was separated. Humoral immune response was detected with the enzyme-linked immunosorbent assay, and cellular immune response of SARS-Cov DNA vaccines was detected with lymphoproliferation assay and cytotoxic T lymphocyte assay. Results show that cellular and humoral immune responses can be detected after immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmids in BALB/c mice. However, pcDNAM stimulated the highest cellular immune response than other plasmids, and pcDNASa-pcDNASb stimulated the highest humoral immune response in week 12. The present results not only suggest that DNA immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also to illustrate that gene immunization with these SARS DNA vaccines different immune response characters.     

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus

Severe acute respiratory syndrome (SARS) brought aglobal outbreak in spring of 2003 [1–3], and more andmore attention has been paid on it when a new caseresurfaced in Singapore last September [4]. By the endof May in 2003, WHO reported a cumulative total of 8202infected cases with 725 deaths from 28 countries.Because of the high transmission and morality rate ofSARS, scientists in many countries have made theirefforts in studying SARS coronavirus (SARS-CoV)[5, 6]. Several genomes of…     

Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.     

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry

Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.     

Orchitis: a complication of severe acute respiratory syndrome (SARS)

Severe acute respiratory syndrome (SARS) coronavirus has been known to damage multiple organs; however, little is known about its impact on the reproductive system. In the present study, we analyzed the pathological changes of testes from six patients who died of SARS. Results suggested that SARS caused orchitis. All SARS testes displayed widespread germ cell destruction, few or no spermatozoon in the seminiferous tubule, thickened basement membrane, and leukocyte infiltration. The numbers of CD3+ T lymphocytes and CD68+ macrophages increased significantly in the interstitial tissue compared with the control group (P < 0.05). SARS viral genomic sequences were not detected in the testes by in situ hybridization. Immunohistochemistry demonstrated abundant IgG precipitation in the seminiferous epithelium of SARS testes, indicating possible immune response as the cause for the damage. Our findings indicated that orchitis is a complication of SARS. It further suggests that the reproductive functions should be followed and evaluated in recovered male SARS patients.     

Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB activation

Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS-CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK), and significantly enhanced interleukin 8 (IL-8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up-regulated in SARS-CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.     

Curtailing transmission of severe acute respiratory syndrome within a community and its hospital

Severe acute respiratory syndrome (SARS) has been transmitted extensively within hospitals, and healthcare workers (HCWs) have comprised a large proportion of SARS cases worldwide. We present a stochastic model of a SARS outbreak in a community and its hospital. For a range of basic reproductive numbers (R(0)) corresponding to conditions in different cities (but with emphasis on R(0) approximately 3 as reported for Hong Kong and Singapore), we evaluate contact precautions and case management (quarantine and isolation) as containment measures. Hospital-based contact precautions emerge as the most potent measures, with hospital-wide measures being particularly important if screening of HCWs is inadequate. For R(0) = 3, case isolation alone can control a SARS outbreak only if isolation reduces transmission by at least a factor of four and the mean symptom-onset-to-isolation time is less than 3 days. Delays of a few days in contact tracing and case identification severely degrade the utility of quarantine and isolation, particularly in high-transmission settings. Still more detrimental are delays between the onset of an outbreak and the implementation of control measures; for given control scenarios, our model identifies windows of opportunity beyond which the efficacy of containment efforts is reduced greatly. By considering pathways of transmission in our system, we show that if hospital-based transmission is not halted, measures that reduce community-HCW contact are vital to preventing a widespread epidemic. The implications of our results for future emerging pathogens are discussed.