Receptor-destroying Enzyme Activity Research Articles

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.

Genetic and Antigenic Characterization of Newly Isolated Bovine Toroviruses from Japanese Cattle

Torovirus, a member of the Coronaviridae family, is a gastrointestinal infectious agent that has been identified in humans, cattle, pigs, and equines. Toroviruses, except equine torovirus, are difficult to propagate in cell culture; indeed, to date, only the Aichi/2004 strain of bovine torovirus (BToV) has been isolated among the human, bovine, and porcine toroviruses. In the present study, four cytopathogenic BToVs were isolated from diarrheal feces of the cattle using the HRT-18 cell line, and their genetic and antigenic properties were compared. The cytopathogenic features of BToV isolates in HRT-18 cells were similar to those of the Aichi/2004 strain. However, none of the isolates showed cytopathogenic effects in the HRT-18 cells of different origin, suggesting that one significant factor contributing to the cytopathogenicity of BToV depends on properties of the HRT-18 cells themselves. All BToVs isolated were able to agglutinate mouse, but not chicken, erythrocytes, while they lacked receptor-destroying enzyme activity. Analysis of the N terminus of the spike gene showed that three isolates, but not the Gifu-2007TI/E strain, were phylogenetically located in cluster 1 and its analogs and revealed high cross-reactivity with each other, as demonstrated by neutralization (NT) and hemagglutination inhibition (HI) assays. The Gifu-2007TI/E strain was classified close to cluster 2 and exhibited relatively low cross-reactivity with these viruses; however, the difference was not sufficient to classify BToVs into serotypes, suggesting that at least two subtypes distinguishable by the structure of the N terminus of the spike gene and that both NT and HI tests may be exist.

Biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo ( Bubalus bubalis) calves

We describe the isolation, biological and genetic characterization of a host-range variant of bovine coronavirus (BCoV) detected in water buffalo ( Bubalus bubalis). By conventional and real-time RT-PCR assays, the virus was demonstrated in the intestinal contents of two 20-day-old buffalo calves dead of a severe form of enteritis and in the feces of additional 17 buffalo calves with diarrhea. Virus isolation, hemagglutination and receptor-destroying enzyme activity showed that the buffalo coronavirus (BuCoV) is closely related to BCoV but possesses some different biological properties. Sequence and phylogenetic analyses of the 3′ end (9.6 kb) of the BuCoV RNA revealed a genomic organization typical of group 2 coronaviruses. Moreover, the genetic distance between BuCoV and BCoV was proven to be the same or even higher than the distance between other ruminant coronaviruses and BCoV. In conclusion, our data support the existence of a host-range variant of BCoV associated with enteritis in buffaloes.

Biologic, Antigenic, and Full-Length Genomic Characterization of a Bovine-Like Coronavirus Isolated from a Giraffe

Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.

Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon

Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon (Salmo salar) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout (Oncorhynchus mykiss) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus.

Detection and Characterization of Bovine Coronaviruses in Fecal Specimens of Adult Cattle with Diarrhea during the Warmer Seasons

Bovine coronavirus (BCoV) is an etiological agent associated with winter dysentery (WD), prevalent in adult cattle during the winter. Although we previously detected, isolated, and characterized BCoV strains from adult cattle with WD (WD-BCoV strains) during the winter in South Korea, the precise epidemiology, as well as the causative agent of diarrhea in adult cattle in the warmer seasons, has not been examined. We examined 184 diarrheic fecal specimens collected from 75 herds of adult cattle from seven provinces during the spring (warm), autumn (warm), and summer (hot) seasons. Bovine coronavirus-positive reactions were detected for 107 (58.2%) diarrheic fecal samples (in 47/75 herds). Of these 107 positive samples, 90 fecal samples from 33 herds tested positive for BCoV alone and 17 fecal samples from 14 herds also tested positive for other pathogens. Biological comparisons between the 9 BCoV strains isolated in this study and the 10 previously isolated WD-BCoV strains revealed that there was no receptor-destroying enzyme (RDE) activity against mouse erythrocytes in the 9 BCoV strains but the 10 WD-BCoV strains had high RDE activity. Phylogenetic analysis of the spike (S) and hemagglutinin/esterase (HE) proteins revealed that all the Korean BCoVs clustered together regardless of season and were distinct from the other known BCoVs, suggesting a distinct evolutionary pathway for the Korean BCoVs. These and previous results revealed a high prevalence and widespread geographical distribution of BCoV, suggesting that this virus is endemic in adult cattle with diarrhea in all seasons in South Korea.

Infectious salmon anemia virus: causative agent, pathogenesis and immunity.

Infectious salmon anemia (ISA) virus (ISAV), an economically important new pathogen in marine aquaculture, is classified in the family Orthomyxoviridae, genus Isavirus. The main structural properties of this genus include enveloped virions 90-140 nm in diameter with surface projections of a combined receptor-binding hemagglutinin and receptor-destroying enzyme activity demonstrated to be an esterase, hence recently designated HE, and a genome composed of eight segments of linear, single-stranded, negative sense RNA ranging in length from 1.0 to 2.4 kb, with a total size of approximately 14.3 kb. The viral genome encodes at least ten proteins, of which nine are structural and one is non-structural. Examination of more than 160 ISAV isolates has led to the identification of two hemagglutinin subtypes of ISAV, one North American and one European. The immune response against ISAV after infection or vaccination does not provide full protection against the infection. The recent discovery of antibody-mediated uptake and replication of ISAV in macrophage-like fish cell lines suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon (Salmo salar) targets endothelial cells lining blood vessels and macrophage-like cells. Cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0 to 100%. ISAV causes fatal systemic infections in marine-farmed Atlantic salmon and asymptomatic infections in feral fish. Experimentally induced fatal clinical disease in rainbow trout (Oncorhynchus mykiss) has identified a correlate of virulence of ISAV that may explain its emergence as a fish pathogen.

Isolation and partial characterization of a novel paramyxovirus from the gills of diseased seawater-reared Atlantic salmon (Salmo salar L).

A formerly undescribed virus has been isolated from the gills of farmed Atlantic salmon post-smolts in Norway suffering from gill disease. Cytopathic effects appeared in RTgill-W1 cells 9 weeks post-inoculation with gill tissue material. Virus production continued for an extended period thereafter. Light and electron microscopic examination revealed inclusions and replication in the cytoplasm. The viral nucleocapsid consisted of approximately 17 nm thick filaments in a herringbone pattern. Certain areas of the plasma membrane were thickened by the alignment of nucleocapsids on the internal surface and projections of 10 nm long viral glycoprotein spikes on the external surface. Virus assembly and release was achieved by budding through the modified plasma membrane. Negatively stained virions were spherical and partly pleomorphic with a diameter of 150-300 nm as seen by electron microscopy. The virus was sensitive to chloroform, heat and low and high pH, and replication was not inhibited by Br-dU or IdU indicating an RNA genome. Both haemagglutination and receptor-destroying enzyme activity were associated with the virions and the formation of syncytia in infected cultures indicated fusion activity. The receptor-destroying enzyme was identified as neuraminidase. The virus contained five major structural polypeptides with estimated molecular masses of 70, 62, 60, 48 and 37 kDa. Its buoyant density was 1.18-1.19 g ml(-1) in CsCl gradients. From the observed properties we conclude that this new virus belongs to the Paramyxoviridae and suggest the name Atlantic salmon paramyxovirus (ASPV). Furthermore, replication occurred at 6-21 degrees C, suggesting a host range confined to cold-blooded animals.

Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus.

Infectious salmon anaemia virus (ISAV) infects cells via the endocytic pathway and, like many other enveloped viruses, ISAV contains a receptor-destroying enzyme. We have analysed this acetylesterase activity with respect to substrate specificity, enzyme kinetics, inhibitors, temperature and pH stability. The ISAV acetylesterase was inhibited by di-isopropyl fluorophosphate (DFP) in a dose-dependent fashion but not by other known hydrolase inhibitors, suggesting that a serine residue is part of the active site. The pH optimum of the enzyme was in the range 7.5-8.0 and the enzymatic activity was lessened at temperatures above 40 degrees C. The effect of DFP on agglutination/elution of erythrocytes by ISAV demonstrated that the acetylesterase activity is the bona fide receptor-destroying enzyme. A haemadsorption assay was used to analyse whether the esterase was active on the surface of infected cells or not.

Isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses

Abstract Objective To isolate bovine coronaviruses from the respiratory tracts of feedlot cattle and compare antigenic and biological properties of these strains with bovine enteric coronaviruses. Animals 5- to 8-month-old mixed-breed cattle at 4 feedlots. Procedure Samples were obtained from the nasal passages for testing. The 13 samples with the highest magnitude of positive values for bovine coronavirus (BCV) were cultured. Ten strains of bovine respiratory coronavirus (BRCV) were adapted successfully to serial passage. After observation of cytopathic effects (CPE) and confirmation of BRCV by immune electron microscopy and immunofluorescence testing, cell culture-adapted strains were cloned by limiting dilution. These isolates then were compared with a panel of bovine enteric coronaviruses (BECV), using hemagglutination (HA), receptor-destroying enzyme activity (RDE), hemagglutination inhibition (HI), and virus neutralization (VN) assays. Antigenic relatedness values then were calculated. Results The BRCV were detected in 105 of 488 (21.5%) of the cattle tested. Of 13 strains tested, 10 were isolated in cell culture. Six of the BRCV strains were similar to 2 strains obtained from neonatal calves with diarrhea and 2 strains from adult cattle with winter dysentery. The other 4 BRCV isolates had high RDE activity against mouse erythrocytes but differed from other strains of BECV. Nine of 10 BRCV isolates had properties similar to the 2 BECV subtypes. Conclusions and Clinical Relevance The BRCV can be isolated from nasal passages of cattle entering feedlots. Most BRCV were similar to BECV strains, although a few had unique properties. Vaccines developed to protect against enteric strains also may protect against respiratory tract strains. (Am J Vet Res 1999;60:1227–1233)

Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.).

Infectious salmon anemia (ISA) virus is the cause of infectious salmon anemia in farmed Atlantic salmon. The virus has been shown to contain RNA with structural characteristics similar to those of accepted members of the Orthomyxoviridae. Further biochemical, physiochemical, and morphological characterization of ISA virus was undertaken to clarify its taxonomic position. The virus was found to be sensitive to chloroform, heat, and low pH and agglutinated erythrocytes from fish. Erythrocytes from mammals or birds were not agglutinated. Receptor-destroying enzyme activity was detected, and the nature of this enzyme was suggested to be an acetylesterase. The buoyant density of the virus was 1.18 g/ml in sucrose and CsCl gradients. The maximum rate of virus replication was observed at 15 degrees C, while no virus was produced at 25 degrees C. Actinomycin D inhibited viral replication, and viral antigen was detected in nuclei by immunofluorescence. The addition of trypsin to the culture medium during virus replication had a beneficial effect on virus replication. ISA virus contains four major polypeptides with estimated molecular sizes of 71, 53, 43, and 24 kDa. Electron microscopy revealed structures closely resembling the nucleocapsids of influenza virus. Mushroom-shaped surface projections were a distinctive morphological feature, which differed from the rod-shaped hemagglutinin projections of the influenza viruses. The data reported here support the relationship of ISA virus to the Orthomyxoviridae, although ISA virus differs from influenza viruses in some morphological characteristics and in showing restricted hemagglutination, in different specificity of the receptor-destroying enzyme, in different polypeptide profile, in being unable to replicate at temperatures above 25 degrees C, and in host range.

Isolation of coronaviruses antigenically indistinguishable from bovine coronavirus from wild ruminants with diarrhea

Diarrheal feces from three sambar deer and one waterbuck in a wild animal habitat and one white-tailed deer on a wildlife farm in Ohio contained coronavirus particles which were agglutinated by antiserum to bovine coronavirus (BCV) in immune electron microscopy. Three coronavirus strains were isolated in human rectal tumor cells from the feces of the sambar and white-tailed deer and the waterbuck, respectively. Hemagglutination, receptor-destroying enzyme activity, indirect immunofluorescence, hemagglutination inhibition, virus neutralization, and Western blot (immunoblot) tests showed close biological and antigenic relationships among the isolates and with selected BCV strains. Gnotobiotic and colostrum-deprived calves inoculated with each of these isolates developed diarrhea and shed coronavirus in their feces and from their nasal passages. In a serological survey of coronavirus infections among wild deer, 8.7 and 6.6% of sera from mule deer in Wyoming and from white-tailed deer in Ohio, respectively, were seropositive against both of the isolates and selected BCV isolates by indirect immunofluorescence tests. These results confirm the existence of coronaviruses in wild ruminants and suggest that these species may harbor coronavirus strains transmissible to cattle.

The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis

Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-KB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex.

Antigenic and biological comparisons of bovine coronaviruses derived from neonatal calf diarrhea and winter dysentery of adult cattle.

SummaryThe antigenic and biological properties of 6 strains of bovine coronavirus (BCV) derived from neonatal calf diarrhea (CD) and 8 strains of BCV from winter dysentery (WD) of adult cattle, propagated in HRT-18 cells, were compared to determine if CD and WD strains belong to distinct serotypes or subtypes of BCV. All strains hemagglutinated both mouse and chicken erythrocytes at 4 °C, but the ratios of hemagglutination titers with mouse erythrocytes compared to chicken erythrocytes showed diversity for both CD and WD strains. Some CD and WD strains did not hemagglutinate chicken erythrocytes at 37 °C and showed receptor-destroying enzyme activity against chicken erythrocytes. Hyperimmune antisera were produced in guinea pigs against 3 and 7 strains of BCV from CD and WD, respectively. No significant differences in antibody titers against these strains were observed by indirect immunofluorescence tests. However, in virus neutralization tests, antisera to 1 CD and 2 WD strains had 16-fold or lower antibody titers against 3 WD and 1 CD strains than against the homologous strains, and this variation reflected low antigenic relatedness values (R=13–25%), suggesting the presence of different subtypes among BCV. In hemagglutination inhibition tests, some one-way antigenic variations among strains were also observed. These results suggest that some antigenic and biological diversity exists among BCV strains, but these variations were unrelated to the clinical source of the strains; i.e. CD or WD.

Biological and molecular differentiation between coronaviruses associated with neonatal calf diarrhoea and winter dysentery in adult cattle.

Cytopathic coronaviruses were isolated in HRT-18 cells from bloody faecal samples collected from cows in Québec dairy herds with classical winter dysentery (WD). The formation of polykaryons in the infected cell cultures was found to be dependent on the presence of trypsin in the medium. Virus identification was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using rabbit hyperimmune serum, as well as monoclonal antibodies directed against the spike (S) and hemagglutinin-esterase (HE) glycoproteins of the prototype Mebus strain of bovine coronavirus (BCV-Meb). Four WD isolates differed from BCV-Meb by their ability to agglutinate rat erythrocytes at 4 and 37 degrees C, their higher receptor destroying enzyme activity, but lower acetylesterase activity. The WD isolates were serologically indistinguishable from the reference BCV-Meb strain by virus neutralization and Western immunoblotting, but could be differentiated by hemagglutination-inhibition. Sequence analysis of the PCR-amplified HE gene of a plaque-purified WD isolate (BCQ-2590) revealed sufficient number of nucleotide and amino acid substitutions which may explain this antigenic variability.

Comparison of hemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains.

SummaryHemagglutinating and acetylesterase functions as well as the 124 kDa glycoprotein were present in the highly cell-culture adapted, avirulent bovine coronavirus strain BCV-L9, in the Norden vaccine strain derived from it, and in 5 wild-type, virulent strains that multiplied in HRT-18 cells but were restricted in several types of cultured bovine cells. The BCV-L9 and the wild-type strain BCV-LY-138 agglutinated chicken and mouse erythrocytes. The acetylesterase facilitated break-down of the BCV-erythrocyte complex with chicken but only to a minimal extent with mouse erythrocytes in the receptor-destroying enzyme test. Purified preparations of the vaccine and the wild-type strains agglutinated chicken erythrocytes at low titers and mouse erythrocytes at 128 to 256 times higher titers whereas receptor destroying enzyme activity was detectable only with chicken erythrocytes. When wild-type strains were propagated in HRT cells at low passage levels, they produced 5×105 to 4.5×106 plaque forming units per 50 µl which agglutinated erythrocytes from mice but not from chickens. Diisopropylfluoro-phosphate moderately increased the hemagglutination titers, but completely inhibited the receptor destroying enzyme of purified virus of all strains. It had virtually no influence on the plaque-forming infectivity of the different BCV strains. The acetylesterase of strain BCV-L9 reacting in the receptor-destroying enzyme test was stable for 3h at 37 and 42°C. It was inactivated within 30 min at 56°C while the hemagglutinin function of this strain was stable for 3 h at 37, 42, and 56°C, but it was inactivated at 65°C within 1 h.

Characteristics of a persistent infection in Madin-Darby canine kidney cells with influenza C virus.

Influenza C/Ann Arbor/1/50 was used to establish a persistent infection in Madin-Darby canine kidney cells. The persistent state has been stable for more than 4 years (over 66 passages), with infected cells differing from controls in morphology and growth characteristics. In addition, virus recovered from the media of persistent cultures (passage 58) differed from parental wild-type virus C/Ann Arbor/1/50 in (i) antigenicity by the hemagglutination-inhibition test; (ii) its ability to produce plaques in different host cells; (iii) hemagglutination of chicken erythrocytes at different temperatures; (iv) receptor-destroying enzyme activity, and (v) by sensitivity to hemagglutination inhibitors present in rat serum.

An assay for the receptor-destroying activity of influenza C virus.

We have developed a convenient method for assaying the receptor-destroying enzyme (RDE) activity of influenza C virus. This method measures the ability of the RDE to destroy the hemagglutination-inhibition activity of a potent inhibitor present in rat serum. Some physico-chemical properties of the RDE of influenza C virus were investigated by using this method. The temperature optimum for maximal activity of this enzyme was found to be 45 C to 53 C. There was little difference in thermostability between the RDE and hemagglutinating activities of influenza C virus. When influenza C virions were treated with various concentrations of trypsin, the RDE activity decreased in parallel with the decrease in the amount of residual gp88 glycoprotein, suggesting association of RDE with this glycoprotein.

Hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice.

SummaryThe hemagglutination (HA) and receptor destroying enzyme (RDE) activities of a newly isolated mouse enteric coronavirus (designated as DVIM) are described. DVIM agglutinates mouse or rat red blood cells (RBC) at 4° C. At 37° C the agglutination was rapidly reversed. The optimal pH for HA and for RDE activities using mouse red cells were shown to be 6.5 and 7.3 respectively. Hemagglutination by DVIM was not inhibited by pretreatment of RBCs withVibrio cholerae filtrate or by pretreatment with Influenza-A neuraminidase. Therefore, the DVIM receptors on RBCs differ from the receptors of Influenza-A, and the RDE activity of DVIM acts specifically on this receptor.In addition, an analysis of the DVIM polypeptides showed that the virions contain five major, VP1 (M.W. 139,000), VP2 (68,000), VP3 (53,000), VP4 (38,000), VP5 (22,000) and two minor, VP1a (110,000), VP1b (100,000) polypeptides. VP1 and VP1b were digested by bromelain, suggesting that they constitute the surface glycoproteins.

Receptor destroying enzyme (RDE) を作用させる条件について

In a previous paper, some purification studies on the receptor destroying enzyme (RDE) produced in a particular medium containing hog stomac mucin, which allowed the highest production of RDE by V. cholerae, have been reported.For the application of this purified preparation on serological studies of influenza infections and also for detecting the viral hemagglutinins in the organ of animals infected with influenza virus, some systematic researches on the optimum conditions for the action of RDE, seemed advisable to investigate. The results of basic studies obtained along this line are the subject of this communication.1) Using chicken red cells and ovomucin as the substrates, quantitative studies of RDE activity have been performed. The higher the concentration of the substrate, the higher concentration of enzyme was necessary. Linear relationship was obtained between the log concentration of the substrate and of enzyme in order to destroy the activity of the substrate completely after fixed period of incubation.2) Optimum temperature and pH for the action of RDE was 37°C and between 6.5 and 7.0, respectively.3) Ca ion concentration was an important factor for the RDE action and the optimum concentrations laied between M/100 and M/200. Deionization with EDTA, sod. citrate and sod. oxalate worked out to inhibit the RDE action and the effectiveness of these substances was in this order.4) The provisional working standard of determining the titer of both α-inhibitor and RDE has been proposed. Following to this standard, x/4 unit of RDE was necessary to destroy the x unit of α-inhibitor under standard condition of incubation, i.e. at 37°C for 1hr.5) Methods to remove the residual activity of RDE after the completion of the reaction have been examined. Heating was favourable for practical use particularly at the time of serodiagnosis. However, there was the difference of heat stability between purified RDE and just cholerae filtrate. The assay with 2.5% sod. citrate saline, which have been customly used as the routine procedure to remove the residual RDE activity was not so satisfactory when high concentration of RDE was used. 1% aqueous solution of EDTA was preferrable with regard to this respect.The conditions determined above will give us an idea that how much unit of RDE should be used in order to remove the known amount of α-inhibitor contained in the specimens to be tested, and also how to remove the residual activity of used RDE. However, the existence of the other kind of α-inhibitors among the biological specimens which is not impaired by the above procedure was not ruled out.