Temperature-sensitive acetylesterase activity of haemagglutinin-esterase specified by respiratory bovine coronaviruses

Numerous respiratory bovine coronaviruses (RBCV) were isolated recently fro m nasal swab samples and lung tissues of feedlot cattle with acute respirat ory tract disease. These newly emerging RBCV isolates exhibited distinct ph enotypic features that differentiated them from enteropathogenic bovine cor onaviruses (EBCV), The RBCV strains had a receptor-destroying enzyme functi on mediated by acetylesterase (AE) activity of the haemagglutinin-esterase (HE) glycoprotein, The HE genes of wild-type EBCV strain LY138 and RBCV str ains OK-0514 (OK) and LSU-94LSS-051 (LSU) were cloned, sequenced and transi ently expressed in COS-7 cells. The enzymic properties of HE proteins in CO S-7 cellular extracts and in purified virus preparations were assayed at ro om temperature, 37 degreesC and 39 degreesC by two different assays. One as say used rho -nitrophenyl acetate (PNPA) as substrate and detected serine-e sterase activity; the second assay monitored AE function with bovine submax illary mucin (BSM) as substrate. The PNPA tests confirmed that HE proteins of EBCV and RBCV were functionally expressed in transfected COS-7 cells. Ti me-dependent determination of the AE activity of purified RBCV OK and LSU p articles showed lower AE activity at 39 degreesC than at 37 degreesC, where as the purified EBCV LY particles retained full AE activity at both 37 degr eesC and 39 degreesC, Transiently expressed RBCV HE exhibited a marked redu ction of AE activity after 40 min of assay time at 37 degreesC, In contrast , the AE activity of the transiently expressed EBCV HE remained stable beyo nd 40 min. The deduced amino-acid sequences of the HE proteins specified by the RBCV strains OK and LSU contained specific amino-acid changes in compa rison with the EBCV LY strain, which may be responsible for the observed en zymic differences. These results are consistent with the hypothesis that RB CV strains have evolved to selectively replicate in respiratory tissues and that HE may play a role in this tissue tropism.