WO1988001292A1

WO1988001292A1 - Human enteric coronavirus

Info

Publication number: WO1988001292A1
Application number: PCT/US1987/002034
Authority: WO
Inventors: Silvia Resta (Nmi); James P. Luby
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1986-08-22
Filing date: 1987-08-18
Publication date: 1988-02-25
Also published as: AU7961487A

Abstract

Biologically pure cultures of human enteric coronaviruses A-14 and C-14 have been prepared. The serial cultivation of viruses from fecal samples of infant patients suffering from necrotizing enterocolitis resulted in the obtainment of the biologically pure cultures. This serial cultivation was in an antibiotic-supplemented medium for mammalian cell culture preferably Leibovitz L-15 medium (pH 6.8), which contained fetal human intestinal pieces. Infections by human enteric coronavirus may be immunochemically diagnosed by use of antibodies against the A-14 and C-14 coronaviruses. Such antibodies have been prepared and found to bind strongly to coronavirus A-14 or C-14 and not to OC43, 229E, MHV-A59 and Breda 1 and 2 viruses. Fecal samples from patients suffering from enteric disease can be interacted with antibody and the amount of binding between the antibody and antigens of the sample measured by any of several means well-known in the art. When the binding between the antibody and antigens of a patient's fecal sample is determined to be significantly greater than that found between the antibody and antigens of a fecal sample from a control uninfected individual which usually demonstrate no interaction, a positive diagnosis of infection by human enteric coronavirus is obtained.

Description

HUMAN ENTERIC CORONAVIRUS

The present invention relates to the detection of infection by human enteric coronavirus and to the first isolation and propagation of a human enteric coronavirus.

Coronaviruses are single-stranded RNA viruses with distinctive surface projections from their envelopes. The members of this group cause a variety of infections in humans and in animals. Because human strains are difficult to isolate and cultivate, they are not well studied. Coronaviruses most commonly cause minor upper respiratory infections (colds) and are sometimes capable of affecting the lower respiratory tract. Of the many coronaviruses affecting animals, the best known are those causing avian infectious bronchitis of chickens, mouse hepatitis, and transmissible gastroenteritis of piglets. Other coronaviruses infect rats, calves, dogs, cats, and foals. A number of the animal strains infect the intestinal tract of the host species; analogous human enteric coronaviruses have also been described (Cukor, et al.. Human viral gastroenteritis, Microbiol. Rev. (1984) 48.: 157-179; Kapikan, et al., J. Infect. Dis. (1969) 119: 282-290; and, Monto, Medical reviews: Coronaviruses, Yale J. Biol. Med. (1974) _ _: 234-251), incorporated by refer¬ ence herein) .

Coronaviruses are about 100-150 nm in diameter and tend to be pleomorphic. The coronaviruses are surrounded by a halo of club-shaped projections resembling spikes or petals which suggest the solar corona. These projections are longer (up to 20 nm) and usually more widely spaced than the spikes of orthomyxoviruses, to which the corona- viruses bear a slight morphologic similarity.

All coronaviruses probably share the same basic biophysical and biochemical structure, but much of the information about them derives from studies of animal strains, particularly mouse hepatitis. The internal component of coronavirus consists of RNA and protein. The nucleoprotein is coiled and forms a^* helical structure 9-16 nm wide that seems fragile and easily disrupted. The membrane that encloses the coronavirus particle has protein and lipid components; its club-shaped projections, called peplomers, are proteins. Biochemical analysis of the virions indicates that they contain polypeptides of three main classes. The first, which is phosphorylated but nonglycosylated (mol. wt. about 50,000 daltons), is associated with the internal ribonucleoprotein. The second polypeptide is glycosylated and is associated with the membrane; the third, which is also glycosylated, is associated with the peplomers. The coronaviruses of humans and of various animal species seem to differ in the number, molecular weight, and extent of glycosylation of these polypeptides. The virus particles can be destroyed by lipid solvents (Siddell, et al., J. Gen. Virol. (1983) 64: 761-776, incorporated by reference herein). Coronavirus replication occurs in the cytoplasm of infected cells. The virus matures by budding into vesicles in the cell cytoplasm and is released when the cell lyses.

Although some of the animal coronaviruses can be grown relatively easily in the laboratory, human strains have very strict host cell specificities, and are notoriously difficult to isolate from patients and to adapt to cell cultures. As a result, coronaviruses still pose many unanswered questions. The prototype human respiratory coronavirus, 229E, and strains related to it can be grown in certain tissue cultures. Other human strains, the OC strains, are so called because most of them can be grown only in organ cultures (explants) of differentiated human respiratory epithelium originating from nasal or tracheal tissue of aborted human fetuses,; the presence of virus in the culture requires confirmation by electron microscopy.

Of the human coronaviruses, strains 229E and OC43 have been studied most (Table 1). These two viruses are antigenically distinct. As yet, there is no definition of an antigenic type or group, and it may be difficult to make such a definition if partial sharing of antigens proves to be as common among the human coronaviruses as among coronaviruses causing infections bronchitis of chickens. Subtypes or variants may prove to be epidemio- logically important. Human coronavirus strains isolated in tissue culture all appear to be antigenically related to 229E and may thus constitute one broad type or group, and OC strains constitute at least one other such group. None of the human respiratory coronaviruses tested so far is related antigenically to the infectious bronchitis of chicken, although human strain 229E has been found to be related to certain coronaviruses of pigs, dogs, and cats, and human strain OC43 to coronaviruses of mice (mouse hepatitis), rats, and cattle. The relationship of virus structure to antigenic composition is being investigated. The surface spikes probably elicit neutralizing activity and, when it occurs, hemagglutinating activity, but definite information is sparse.

TABLE 1

TYPES OF HUMAN CORONAVIRUS: IN VITRO CULTIVATION AND SEROLOGIC TESTS

Cultivation in Vitro Serologic Tests Available*

Types of Human Organ Tissue Baby Culture Culture Mige N _C IF HI IEM ELISA

229E types + + + + + + +

OC43 + ±t +t + + + + +

Other OC types + +* +** -I-**

^", neutralization; CF, complement-fixation, IF, immunofluorescence; HI, hemagglutination inhibition IEM, immunoelectron microscopy; ELISA, enzyme-linked immunosorbent assay. t After adaptation.

** Using organ cultures.

-5-

Type OC43 coronavirus has been adapted to grow in brains of infant mice and also (although somewhat poorly) in tissue culture. The mouse-adapted virus agglutinates red blood cells and this property can be used in serologic tests. Serologic properties of the strains that grow in tissue culture may be studied by neutralization or comple¬ ment fixation; neutralization tests can also be done with some difficulty in organ culture. ELISAs with 229E and OC43 antigens have been used successfully and have confirmed the antigenic difference between the two strains. All strains may be examined by the rather cumbersome techniques of fluorescent staining of viral antigen in infected cells or by immune electron micro¬ scopy.

Inoculation of human volunteers with coronaviruses has provided valuable information. Volunteers develop colds, often with profuse coryza, 2-3 days after intranasal inoculation of a coronavirus. The virus evidently multiplies in nasal epithelium and can be recovered form nasal secretions by appropriate tissue cultures or organ cultures. In adult volunteers, infections generally are brief and without pyrexia; asymptomatic infections also may occur. Natural infections with coronaviruses occur in all age groups.

Lower respiratory involvement is presently not thought to be a common feature of the infection, although corona¬ viruses, like rhlnoviruses, have been implicated in exacerbations of asthma and chronic bronchitis.

Coronaviruses can cause chronic or persistent infections in some animals and in tissue cultures, but persistent infection in humans has not been demonstrated conclusively. Coronaviruses or coranaviruslike particles have been observed by electron microscopy in human feces. but the association of these particles with disease has not previously been established. However, coronavirus strains have been associated with diarrheal diseases in lower animals, and there is evidence that these viruses may be involved in human enteric diseases (Stair et al.. Am. J. Vet. Res., 3_3: 1147 (1972); Tajima et al.. Arch. Gesamte Virusforshung, J29., 105 (1975); Garwes et al., J. Gen. Virol. 2__: 25 (1975); Hierholzer et al.. Infect. Immun. .24.: 508 (1979). Most of the data in support of the latter hypothesis result from electron microscopic observations of coronavirus-like particles in stool samples obtained from patients with gastroenteritis or necrotizing enterocolitis (NEC) (Chany et al.. Pediatrics, __ , 209 (1982); Vaucher et al., J. Infect. Dis., 14_5, 27 (1982); Gerna et al., J. Infect. Dis., 150., 618 (1984); Laporte et al., Perspect. Virol, 11, 189 (1981) and; MacNaughton et al.. Arch. Virol., 7_0_, 301 (1981). Attempts to cultivate these particles for antigenic or biochemical analysis have been unrewarding to date.

Coronavirus infections cannot be distinguished clinically from other viral respiratory infections. Laboratory diagnosis usually is made by examining paired sera to detect rising antibody titers. Viral strain 229E is used in complement-fixation or neutralization tests and strain OC43 in complement-fixation or hemagglutination- inhibition or neutralization tests. ELISA methods may supplement these. Rising antibody titers against 229E or OC43 may also be produced by infection with heterologous but related viruses.

Virus may be isolated from nasal swabs or washings by using human embryo nasal or tracheal organ cultures. Strain 229E and related strains sometimes can be isolated directly in human embryo kidney cells, diploid human e bryo fibroblast lines, or other sensitive cells, but isolation may be easier after preliminary passage in organ culture. The cytopathic effect in tissue culture is not distinctive; the presence of coronavirus in the cultures is confirmed by electron microscopy and use of reference antisera. Identification of coronavirus in nasal secretions by ELISA also has been described. Neither vaccination nor chemoprophylaxis is available for human secretions by ELSIA also has been described. Neither vaccination nor chemoprophylaxis is available for human coronavirus infections.

Other workers have reported an association between coronaviruses and NEC or with serious .gastrointestinal disease in nursery infants. These reports were based on ^• observations of virus particles in stools by electron inicroscopy of immune electron microscopy (Chany et al., . Pediatrics 4_9_, 209 (1982); Vaucher et al., J. Infect. Dis. 145, 27 (1982); and Gerna et al., J. Infect Dis. 150., 618 (1984). However, their attempts to cultivate these particles were not successful. The results described herein establish the existence of a human enteric corona¬ virus (HEC) and indicate an association between these virus particles and cases of NEC observed in a Dallas epidemic.

Biologically pure cultures of human enteric corona¬ viruses A-14 and C-14 have been prepared. The serial cultivation of viruses from fecal samples of infant patients suffering from necrotizing enterocolitis resulted in the obtainment of the biologically pure cultures. This serial cultivation was in an antibiotic-supplemented medium for mammalian cell culture preferably Leibovitz L- 15 medium (pH 6.8), which contained fetal human intestinal pieces. Infections by human enteric coronavirus may be immunochemically diagnosed by use of antibodies against the A-14 and C-14 coronaviruses. Such antibodies have been prepared and found to bind strongly to coronavirus A-14 or C-14 and not to OC43, 229E, MHV-A59 and Breda 1 and 2 viruses. Fecal samples from patients suffering from enteric disease can be interacted with antibody and the amount of binding between the antibody and antigens of the sample measured by any of several means well-known in the art. When the binding between the antibody and antigens of a patient's fecal sample is determined to be signi¬ ficantly greater than that found between the antibody and antigens of a fecal sample from a control uninfected individual which usually demonstrate no interaction, a positive diagnosis of infection by human enteric corona¬ virus is obtained.-

An immunological response of a patient to human enteric coronavirus infection may be determined by a process of the present invention. Antigens obtained from coronavirus A-15 or C-15, when brought into contact with a patient's serum containing antibody against those corona¬ viruses, result in well-known and recognized patterns characteristic of antigen-antibody interactions. The presence of such antibodies are indicative of a present or past immunological response of the patient to human enteric coronavirus infection.

The present invention comprises a human enteric coronavirus (HEC) associated with necrotizing enteroco¬ litis (NEC) in ailing infants which has been isolated, identified and characterized as described herein. Pure cultures of the virus (A-14 and C-14) have been obtained from fecal samples of two infants. The A-14 and C-14 viruses of these cultures appeared to be morphologically and antigenically identical to each other and different from any previously described coronavirus.

An immunological response to HEC was noted in infants suffering from NEC and was generated in guinea pigs by injection of whole HEC. The diagnosis of NEC when involving HEC may be facilitated by a determination of the presence of an antibody against HEC in serum samples from a patient. This presence may be determined with many standard immunoassay techniques by using the unique HEC described herein or characteristic antigens thereof.

Immunoprophylaxis has been a successful approach to the control of viral diseases. Such immunoprophylaxis may involve passive or active immunization. Passive immuniza¬ tion with preparations comprising antibodies specific for HEC may be used for the short-term treatment of acute

— conditions involving HEC. Polyclonal or monoclonal antibodies specific for HEC A-14 or HEC C-14 or antigens thereof may be prepared by contemporary techniques of immunology. Currently pooled plasma preparations containing antibodies against Varicella zoster and the viruses causing rabies and hepatitis B are commercially available for clinical use.

Immunity to HEC may be generated by an. active immunization process. Currently available for general use are numerous anti-viral vaccines containing live attenuated virus, killed virus or viral antigens. These vaccines include those to generate immunity against smallpox, rabies, yellow fever, influenza, poliomyelitis, measles, mumps and rubella. Active immunization against HEC may be accomplished by the administration of an attenuated A-14 or C-14 virus. The development of a viable non-toxic attenuated HEC will require extensive experimentation and testing. Immunization with killed HEC or HEC antigens should be more readily accomplished than the development of attenuated virus strains. The isolated HEC A-14 or C-14 described herein may be killed, fragmented or modified by chemical or physical means and used as non-infectious antigens by methods well known in the immunological arts. Parenteral administration of such antigens mixed with a pharmaceutically acceptable carrier such as a sterile isotonic salt solution, with or without an immunological adjuvant, comprises use as a vaccine to preclude the onset of infectious NEC or any other infection by a coronavirus having antigenic commonality with HEC C-14 or HEC A-14.

These examples are presented to describe preferred embodiments and utilities of the present invention and are not meant to limit the present .invention unless otherwise stated in the claims-appended hereto.

EXAMPLE 1

Isolation of Human Enteric Coronavirus

An epidemic of NEC occurred in a hospital special care nursery in Dallas, Texas in 1982-83. All of the patients showed established criteria for NEC (Bell et al.. Arch. Int. Med. 198, 1 (1978)) -intolerance to food, abdominal distension, occult or gross blood in stool, and radiologic evidence of pneumatosis intestinalis. Stool samples from patients revealed coronavirus-like particles. During the epidemic, stool specimens and sera were obtained from controls and from patients with NEC and diarrhea at acute and convalescent stages of disease. All stool specimens were screened for the presence of coronavirus-like particles by electron microscopy before inoculation in cultures of human fetal intestinal organ. The cultures were prepared as earlier described (Horn et al., J. Exp. Pathol., __\._t 109 (1965); Rubenstein et al., J. Exp. Pathol., 51, 210 (1970); Dolin et al., J. Infect. Dis., 122, 227 (1970); and Antrup et al., Gastroenterology, 74, 1248 (1978)), incorporated by reference herein) . Briefly, the intestines were opened longitudinally with microscissors, and 2 by 2 mm pieces, with the intestinal villi oriented upward, were placed in tissue culture dishes. At each passage, the organ cultures were incubated in Leiboviz L-15 medium (pH 6.8) supplemented with antibiotics in a humidified environment at 37°C with a 5 percent C0₂ atmosphere, ϋninoculated organ cultures, prepared as controls, were maintained under the same conditions.

Seven stool samples, showing positive results by electron microscopy, and eight showing negative results were cultured. None of the negative inocula resulted in the growth of coronavirus-like particles after being passaged five times and assayed by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two of seven positive specimens led to the isolation and propagation of coronavirus-like particles and these have been maintained for 14 passages (HEC A-14 and HEC C-14). After the first blind passages (3 to 5), the two strains produced destruction of the brush border of the intestinal epithelium and degeneration of the villi. For passages 8 to 14, trypsin, which is thought to have an enhancing effect on coronavirus replication, was added to the growth medium at 5 ug/ml. Cultures infected in the absence of trypsin were maintained in parallel. Treatment of the inocula with chloroform or heat (56°C for 15 minutes) nullified the effect of the inocula on the organ cultures—that is, there was no cytopathic effect, virus-like particles were not seen by electron microscopy, and the protein band profile characteristic of viral growth was not revealed by SDS-PAGE. Control infected cultures not treated with chloroform or heat continued to show evidence of the presence of HEC. Filtration of the inocula through filters of 0.45 urn and 0.22 urn pore did not prevent infection of the organ cultures.

The supernatants and tissue extracts of the infected cultures from passages 6 to 14 were purified on a glycerol-potassium tartrate gradient (Hierholzer, Virology 75, 155 (1976); and Obijeski et al. «, J. Gen. Virol. 22, 21 (1974)). The peak of spectrophotometric activity at 280nm

3 wavelength corresponded to a density of 1.18 g/cm„.

Observation of the band collected from the gradient revealed particles with morphology typical of coronaviruses—namely, club-shaped spikes, a diameter of 100 to 150 nm, a pleomorphic appearance, and an erythrocyte-like profile. The purified particles were tested for hemagglutination with goose, chicken, rat, guinea pig, rabbit, and human 0 erythrocytes at 4°, 25°, and 37°C. No hemagglutinating activity was observed.

EXAMPLE 2

Characteristics of the Isolated Coronavirus Particles

During single radial hemolysis (SRH) assay, the purified particles of Example 1 were reacted with convalescent-stage sera from six patients with NEC; four of the patients showed seroconversion. Sera from control infants showed no reactivity against the antigens (Table 2). Neither the purified HEC antigens nor the infants' sera revealed cross-reactivity with antisera to OC43 and 229E and with OC43 and 229Ξ antigens, respectively.

Single radial hemolysis (SRH) assay was performed according to a previously described technique (Hierholzer et al., J. Clin. Microbiol. 5., 613 (1977). Before being used, all sera were adsorbed overnight (4°C) with human fetal intestinal homogenate. All sera were tested after heating at 56°C for 30 minutes (1, first serum; 2, second serum) . A serum was considered positive when the reaction caused a halo of hemolysis at least 3 mm in diameter (well diameter, 1mm). The first or acute-stage serum was collected 4 to 8 weeks after the onset of illness. Controls were infants in the nursery without necrotizing enterocolitis or diarrhea. A6, A9, A-14 and C6, C9, C-14 designate the passage level of -the isolates.- Abbreviations: NCS, newborn calf serum; FCS, fetal calf serum; BSA, bovine serum albumin; SRBC; sheep red blood cells; ND, not done.

TABLE 2 SINGLE RADIAL HEMOLYSIS ASSAY

Diameter of HEC sample (mm) Diameter of HEC sample (mm)

Pa¬ tients A6 A9 A14 C6 C9 C14 Controls A6 A9 A14 C6 C9 C14

H-l 0 0 0 0 0 0 R 0 0 0 0 0 0

H-2 4.3 3.4 4.5* 4.2 3.5 4.0* McM-1 0 0 0 0 0 0

M-l 0 0 0 0 0 0 c -2 0 0 0 0 0 0

McC-1 0 0 0 0 0 0* P 0 0 0 0 0 0

McC-2 0 0 5.0* 0 0 4.8* Pc 0 0 0 0 0 0

E-2 ND ND 3.5 ND ND 3.5 T 0 0 0 0 0 0

D-l 0 0 0* 0 0 0* Lo 0 0 0 0 0 0

D-2 3.5 3.5 4.2* 3.5 3.5 -4.0* Hi 0 0 0 0 0 0

Hn-1 0 0 0* 0 0 0* NCS 0 0 0 0 0 0

Hn-2 3.0 3.5 4.5* 3.0 3.0 4.3* FCS 0 0 0 0 0 0

Mo-1 0 3.5 3.5* 0 3.5 3.5* Anti- -OC43 0 0 0 0 0 0

Mo-2 4.0 4.0 4.5* 3.8 4.0 4.3* Anti- -229E 0 0 0 0 0 0 Anti- ^•BSA 0 0 0 0 0 0 Anti- -SRBC 10 10 10 10 10 10

* Sample diluted 1:5; all other sera were used undiluted except the NCS, FCS, anti-BSA, and anti-SRBC, which were diluted 1:10.

Several in vitro systems, such as primary human embryonic kidney cells, human embryonic kidney cells human embryonic lung fibroblasts, HEP-2, Vero, and BHK cells, did not support the growth of the viral particles. Although attempts have been made to adapt the virus to a cellular substrate that can be more easily managed, human fetal intestinal organ culture is the only reproducible system discovered at present. Treatment of the cultures, with trypsin appeared to facilitate the infection, since the treated cultures gave rise to higher yields of viral particles, as seen on electron microscopy, than did untrypsinized cultures.

In tests to date, the two strains isolated appear to be identical. Immunologic tests with specific antisera should allow verification of this finding and enable the establishment of possible antigenic relationships with other coronaviruses. Both strains: 1.) Were obtained from infants in the same epidemic, 2. ) Had identical physical characteristics (density, sensitivity to heat and chloroform, 3.) Had identical appearances by electron microscopy, 4.) Had identical protein patterns by SDS- PAGE, and 5.) Had antisera to each virus interact in the same way with the other virus, i.e., antiserum to A-14 reacts with A-14 proteins in an identical manner to C-14 proteins in a Western blot and vice versa.

EXAMPLE 3

ImmunoloQical HEC Antigen Characterization

Antigens (from purified HEC A-14 and C-14, OC43, 229E, and human fetal intestinal homogenates) were tested by enzyme-linked immunosorbent assay (ELISA) against the infants' sera and against antisera to OC43, 229E, MHV-A59, and Breda 1 and 2 viruses. For sources of other viruses see Woode et al., Vet. Microbiol. 1,221 (1982); Weiss et al., J. Gen. Virol. S± 1849 (1983); Horzineck et al., J. Gen Virol., 6_5, 1849 (1983); and Beards et al.. Lancet 1984-1, 1050 (1984), incorporated by reference herein.

These interactions were also tested by standard techniques (Voller et al. in Manual of Clinical Immunology, King et al., eds., Amer. Soc. for Microbiol., Wash. D.C. (1976) p 506; and Yolken et al.. Lancet 1977-11, 261 (1977). The working dilutions of serum were 1:10 and 1:100, and each determination was made in triplicate. Appropriate controls for all of the reagents used were included in each assay. All of the sera were adsorbed overnight (4°C) with human fetal intestinal homogenates before being used. Briefly, both the infants' sera and the other antisera were assayed by binding the antigen directly to the wells of the microtiter plates. After incubation of the test serum samples, alkaline phosphatase-conjugated immuno- globulins (anti-human, anti-rabbit, and anti-guinea pig) were incubated in the wells. The enzyme substrate (p- nitrophenyl phosphate in diethanolamine buffer) was added after washing, and the reaction was stopped and evaluated for absorbance (AB) in a spectrophotometer. Antisera to OC43, 229E. MHV-A59, and Breda 1 and 2 viruses were also assayed by means of a capturing antibody-coated well assay, with specific guinea pig antisera to HEC A-14 and C-14, OC43, and human intestinal homogenates, test anti¬ sera, conjugated immunoglobulins, and enzyme substrate were consecutively incubated in the wells. Each incubation step was followed by washing three times with standard buffer solutions. The AB value of each serum- control antigen reaction was subtracted from the corres¬ ponding AB value of the serum-viral antigen reaction to obtain the value of the test sample. A threshold cutoff point was determined on the basis of the highest values obtained on a group of negative controls. Positive sera always had an AB value greater than 3 standard deviations above the mean of a group of negative control sera. The ELISA test confirmed the results obtained by the SRH assay. Convalescent-stage sera from five of the infants with NEC showed titers of 1:100 or more; two infants showed seroconversion; and six control sera had titers less than 1:20. The infants' sera did not react with OC43 and 229E antigens. No reactions were demonstrable between A-14 and C-14 antigens and antisera to OC43, 229E, MHV- A59, and Breda 1 and 2 viruses. '

Gradient (5 to 17 percent) SDS-PAGE of the purified particles revealed the presence of at least five major bands corresponding to molecular sizes ranging from 190 to 23 kilodaltons. Electrophoretically separated proteins (HEC A-14 and C-14, OC43 and 229E viruses, and human fetal intestinal homogenates) were blotted onto nitrocellulose paper for Western immunoblotting. The blotted proteins were allowed to react with dilutions (1:50 or 1:100) of the acute-and convalescent-stage sera from patients and serum samples from controls obtained during the Dallas NEC epidemic. Seven of seven convalescent-stage sera and two of 11 control sera reacted against HEC A-14 and C-14. Two patients showed seroconversion. The convalescent-stage sera did not react against OC43 and 229E viruses. Reactions occurred mainly with proteins corresponding to molecular sizes of 190, 120, and 50kD. A reaction was seen with the 23-kD protein for some of the samples. EXAMPLE 4

Preparation of Guinea Pig Antisera to HEC

Each of two guinea pigs were given two intramuscular injections of whole HEC A-14 virus or whole HEC C-14 virus in complete Freund's adjuvant at 4 week intervals. At two week intervals each guinea pig was administered four additional doses of the virus (in normal saline), the first two doses by intramuscular injection and the last two by subcutaneous injection. As a later final booster each guinea pig was given, one week before blood was withdrawn, a subcutaneous injection of virus in normal saline. Blood was drawn, allowed to clot, and antisera stored in a conventional manner.

x x x x x x x x

Changes may be made in the components described herein or in the steps or the sequence of steps of the methds described herein without departing from the concept and scope of the invention as defined in the following claims.

Claims

CLAIMS :

1. A biologically pure culture of HEC A-14,

2. A biologically pure culture of HEC C-14.

3. A biologically pure culture of human enteric corona- virus, said coronavirus being obtainable by serial cultivation of viruses from fecal samples of patients having necrotizing enteric coronavirus infection, said cultivation being in an antibiotic-supplemented medium for mammalian cell culture with human fetal intestinal pieces.

4. The biologically pure culture of claim 3--wherein the medium is pH 6.8 Liebowitz L-15 medium.

5. A method for the immunochemical diagnosis of infection with human enteric coronavirus in a patient, the method comprising:

contacting a fecal sample from the patient with an antibody binding more strongly to HEC A-14; or HEC C-14 than to OC43, 229E, MHV-A59 and Breda 1 and 2 viruses;

measuring the amount of binding between antigens of the sample and said antibody; and

determining, for a positive diagnosis, whether the amount of binding is significantly greater than that found between the antibody and antigens of a fecal sample from a control uninfected individual.

6. A method for the detection of an immunological response of a patient to infection by human enteric coronavirus, the method comprising:

obtaining a serum sample from the patient; and

contacting the serum sample with antigen from HEC A- 14 or HEC C-14 to ascertain whether an antibody specific for said antige-n is present in the serum, presence of such antibody being indicative of .-a present or past immunological response of the patient to human enteric corona¬ virus infection.

7. A composition comprising an antibody, the antibody being characterized by binding more strongly to HEC A-14 or HEC C-14 than to OC43, 229E, MHV-A59 and Breda 1 and 2 viruses.

8. A method for producing a vaccine capable of inducing an immunological respor.se against HEC, the method comprising preparing an antigen from HEC A-14 or HEC C-14 and mixing said antigen with a pharmaceutical acceptable carrier or an immunological adjuvant and a pharmaceuti¬ cally acceptable carrier.

9. A method for inducing immunity to HEC in an individual, the method comprising parenteral administra¬ tion of antigens from HEC A-14 or HEC C-14 to said individual.

10. A composition comprising a pharmaceutically accept¬ able carrier and an antigen from HEC A-14 or HEC C-14.

11. A method for passive immunization to protect an individual from HEC infection, the method comprising parenteral administration to said individual of a preparation comprising antibodies specific for HEC A-14 or HEC C-14.

12. The method of claim 11 wherein the antibodies are defined further as binding more strongly to HEC A-14 or HEC C-14 than to OC43, 229E, MHV-A59 and Breda 1 and 2 viruses.