Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2).Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-proteinpseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.A distinct coronavirus (SARS-CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS), an acute pulmonary syndrome characterized by an atypical pneumonia that results in progressive respiratory failure and death in close to 10% of infected individuals (8,11,14,15). SARS-CoV is not closely related to any of the three previously defined genetic and serological coronavirus groups, although it may be distantly related to group 2 coronaviruses (21); the SARS-CoV spike (S) protein, a surface glycoprotein that mediates coronavirus entry into receptor-bearing cells, is also distinct from those of other coronaviruses (18,20). Reflecting this difference, SARS-CoV does not utilize any previously identified coronavirus receptors to infect cells. Rather, as our group have recently demonstrated, angiotensin-converting enzyme 2 (ACE2) serves as a functional receptor for this coronavirus (16,24,25).A quantitative system utilizing a well-characterized retroviral vector (1) for measuring SARS-CoV S-protein-mediated infection would obviate the need for specialized biosafety facilities for many studies, including those assessing humoral responses to potential vaccines. Here we show that simian immunodeficiency virus (SIV) pseudotyped with several codon-optimized S-protein variants could efficiently infect Vero E6 cells and HEK293T cells transiently or stably expressing ACE2. One such variant, truncated at its cytoplasmic tail and bearing instead a region of the tail of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (17), was especially efficient at mediating infection. Murine leukemia virus (MLV) pseudotyped with this S-protein variant also infected ACE2-ex...

Severe acute respiratory syndrome (SARS) is a significant emerging infectious disease. Humans infected with the etiological agent, SARS-associated coronavirus (SARS-CoV), primarily present with pneumonitis but may also develop hepatic, gastrointestinal, and renal pathology. We inoculated common marmosets (Callithrix jacchus) with the objective of developing a small nonhuman primate model of SARS. Two groups of C. jacchus were inoculated intratracheally with cell culture supernatant containing SARS-CoV. In a time course pathogenesis study, animals were evaluated at 2, 4, and 7 days after infection for morphological changes and evidence of viral replication. All animals developed a multifocal mononuclear cell interstitial pneumonitis, accompanied by multinucleated syncytial cells, edema, and bronchiolitis in most animals. Viral antigen localized primarily to infected alveolar macrophages and type-1 pneumocytes by immunohistochemistry. Viral RNA was detected in all animals from pulmonary tissue extracts obtained at necropsy. Viral RNA was also detected in tracheobronchial lymph node and myocardium, together with inflammatory changes, in some animals. Hepatic inflammation was observed in most animals, predominantly as a multifocal lymphocytic hepatitis accompanied by necrosis of individual hepatocytes. These findings identify the common marmoset as a promising nonhuman primate to study SARS-CoV pathogenesis.

Previous studies suggested that estrogen administration leads to an increase in circulating immunoreactive PTH (iPTH), thought to be secondary to a slight decrease in serum calcium resulting from inhibition of bone resorption. Using three different RIAs, we measured iPTH in serum from 10 postmenopausal women before and after 14 days of ethinyl estradiol administration. In 2 sensitive RIAs directed at the midregion of the PTH molecule, iPTH values fell or remained unchanged in each subject, with average decreases of 23% (P less than 0.001) and 28% (P less than 0.005) in the two assays. Total urinary cAMP, the tubular maximum for urinary phosphate excretion, and serum iPTH measured with the third RIA did not change after estrogen treatment. Fasting urinary calcium and hydroxyproline and serum calcium, phosphorus, albumin, alkaline phosphatase, and osteocalcin all decreased after treatment, and serum 1,25-dihydroxyvitamin D increased in each subject. In a second cohort of 5 women given ethinyl estradiol for 8 weeks, similar changes were found at 2 weeks, but there was a trend toward increasing serum iPTH, increasing total urinary cAMP excretion, and decreasing the tubular maximum for urinary phosphate excretion by 8 weeks. The increase in serum 1,25-dihydroxyvitamin D and the decrease in serum osteocalcin were again found after 2 weeks of estrogen and did not change further despite continued treatment. These results indicate multiple effects of a 2-week course of estrogen treatment on mineral metabolism in the absence of an increase in serum iPTH or several biological indices of PTH activity.