An Adenovirus Isolated from the Feces of Mice

Experimental infection of an inbred strain of DK1 mice was carried out with a mouse adenovirus strain K87, isolated from the feces of apparently healthy DK1 mice. Strain K87 was orally administered to four-week-old mice and the virus was recovered from their feces for at least 3 weeks. The highest virus titers in the feces were observed between 1 and 2 weeks after inoculation. In 7-week-old mice the period of virus excretion was about one week shorter. When neonatal or 2-week-old mice were administered virus orally, somewhat irregular results but similar to those from the 4 or 7-week-old mice were obtained. In these infected mice, virus growth was detected in the intestinal tract but not in oropharyngeal washings, nasal tissue, lung, spleen or urine. After inoculation through several parenteral routes, the virus was also detected in the feces and virus growth seemed to be limited mainly to the intestinal tract. No clinical manifestations were observed in any infected mice. When the virus was almost undetectable in the mice infected either orally or parenterally, the virus was readministered orally, but no further virus was recovered in the feces. Neutralizing antibody in the serum was detected 3 weeks after primary inoculation. Induction of immunological tolerance was examined by inoculating mice in utero 2–4 days before birth, but no evidence of induced tolerance was obtained. The use of the adenovirus strain K87-mouse system as a model system for the study of the infectious process and immune mechanisms is suggested because strain K87 has a tissue tropism analogous to many human adenoviruses.     

Intestinal Resistance in the Experimental Enteric Infection of Mice with a Mouse Adenovirus

Investigation was made on the process of enteric infection with mouse adenovirus strain K87 in inbred DK1 mice and the intestinal resistance acquired through infection. The cells containing viral antigens were enumerated in most parts of the infected intestinal tract by a fluorescent antibody technique, and the infectivity titer of the virus in each part was examined in mouse kidney tissue culture. The virus was observed to grow in 3~14 days (sometimos 3~21 days) after oral challenge, and infectivity titers reached their peak after 7~14 days, when a number of viral antigen-containing cells and cells with nuclear inclusions were detected. In the mice rechallenged 28 days after the initial challenge, the virus did not grow, and no viral antigen-containing cells were found. From these results it was concluded that the main sites where the virus grows in mice are the cells which are scattered in the epithelial layer of the mucous membrane of the small intestine, and which seem to be the usual epithelial cells and not Paneth's or goblet cells. As for intestinal resistance, experiments with inactivated vaccine and with passive transfer of serum-antibodies were performed in order to find out whether neutralizing antibodies in the serum had any influence on the growth of virus in the intestinal wall, and no influences were indicated. Eighteen days or more after challenge, K87 virus-neutralizing substances were detected in the intestinal wall and in the intestinal contents of the infected mice, but not in the serum-transferred mice, though both groups of mice had equal levels of serum antibodies. The substance continued to be found until 15 weeks after challenge in the intestinal contents, and until later than 34 weeks in the intestinal walls. The nature and the possible role of the substance is discussed, but actual data will be reported in subsequent papers.     

Serological relationship between mouse adenovirus strains FL and K87

Three-week-old outbred mice were inoculated intraperitoneally or orally and intranasally with the FL or K87 strains of mouse adenovirus and bled at intervals after infection. Serum was tested by both the complement fixation and indirect immunofluorescence tests for reactivity with either virus antigen. A unilateral relationship was detected between FL and K87 strains. Serum from mice given the FL strain of virus reacted in both tests with FL and K87 antigens. Serum from mice given the K87 strain reacted only with the homologous antigen. Serum antibody titers were generally higher in the immunofluorescence test than in the complement fixation test. These observations stress the need to use both FL and K87 antigens for specific serologic diagnosis of adenovirus infection in mouse colonies.     

Hybrid of Monkey and Human Adenoviruses

Following joint replication of monkey SA7 adenovirus (C8 strain) and human adenovirus type 2 in green monkey kidney tissue culture, a virus possessing the properties of a hybrid was obtained. It was designated Ad2C8. Ad2C8 preparations contained two types of viral particles: human adenovirus type 2, and hybrid particles. The hybrid virions multiplied in green monkey kidney cells in the presence of human adenovirus types 1, 2, and 3, but not 3 and 7, and acquired the capsid of the helper adenovirus. The hybrid can serve as a helper for human adenoviruses. It can apparently induce T antigen of the C8 virus but, in contrast to the latter, does not induce tumors in hamsters.     

Isolation and properties of reovirus from cattle in an outbreak of acute respiratory disease.

A cytopathogenic virus was isolated in the primary culture of bovine kidney cells from a nasal swab of affected calves in an outbreak of acute respiratory disease in Japan in 1971. It agglutinated human type O erythrocytes and produced cytoplasmic inclusion bodies. Viral replication was inhibited by 5-iodo-2'-deoxyuridine, indicating that the viral nucleic acid was RNA. The virus was resistant to ether, chloroform, sodium deoxycholate, and acid, and passed readily through Sartorius' membrane filter 100 nm in pore size, but not through the filter 50 nm in pore size. Electron microscopy showed many spherical particles 60 approximately 75 nm in diameter with a double-layered capsid in a sample taken at a buoyant density of 1.34 produced by CaCl equilibrium centrifugation. The virus suspended in 1M MgCl2 solution was stable against heating at 50 degrees C for 30 minutes, but not against freezing at -20 degrees C for 60 minutes. The virus was resistant to, and increased in infectivity after, treatment with 0.063 approximately 1.0% trypsin. These properties were consistent with those established for the reoviruses. Most affected cattle showed a significant rise of antibody titer against reovirus and bovine respiratory syncytial virus, whereas only a few of them presented a serological evidence for recent infection with parainfluenza virus type 3, bovine adenovirus type 7, and bovine parovirus.     

A New Candidate Type of Human Enterovirus (Drilon Strain) Isolated in the Philippines

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3-month-old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl₂ against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect in primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI-38 or HEp-2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.     

Cytopathogenic effect of E. coli cells containing heterogeneous human O(H) type antigen on human cells in culture]

Interaction of two enteropathogenic strains E. coli O55-K59 and human Hela cells containing O(H) isoantigen was studied. When E. coli strain No. 5789 containing heterologic antigen O(H) was added to HeLa cell culture the cytopathogenic effect with the microbial doses of 2 X 10(10), 2 X 10(5), 2X 10(4) was revealed on the third day of the interaction. A dose of 2 X 10(3) of E. coli microbes gave no such effect. Strain No. 3827 containing no heterologic antigen of ABO type failed to exert any cytopathogenic effect with maximal, mean, and minimal doses of the microbes. It is assumed that the cytopathogenic effect of strain No. 5789 is connected with the presence of the strain antigen identical to the group antigen of the human cell culture under study.     

Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture

The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented. The viral strain was isolated from a M. fascicularis suffering from spontaneous hepatitis. Before inoculating the cell culture the virus was passaged twice in the M. arctoides capable of reproducing hepatitis. FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation. Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered. There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture. The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.     

A comparison of two antigen strains of each of mouse hepatitis virus and mouse adenovirus for detection of complement fixation antibody in mouse and rat sera

Detection rates of complement fixation antibodies in mice and rats were compared between two antigen strains of each of mouse hepatitis virus (MHV) and mouse adenovirus (MAV). Among 66 and 47 naturally infected MHV-positive sera of mice (18 facilities) and rats (16 facilities) respectively, 17 mouse and 21 rat sera reacted with both Nu-67 and MHV-2 strains, but 49 mouse and 25 rat sera were positive to Nu-67 strain alone. Only one rat serum reacted with MHV-2 strain alone. In comparison with K87 and FL strains of MAV, all 8 positive mouse sera (3 facilities) reacted with K87 strain alone whereas out of 53 positive rat sera (20 facilities), 43, 6 and 4 sera reacted with K87 strain alone, with FL strain alone and with both the two strains, respectively.     

A naturally occurring intestinal mouse adenovirus infection associated with negative serologic findings

Large basophilic intranuclear inclusions were observed in the intestinal epithelium of clinically normal mice. Electron micrographs of the inclusions showed them to be caused by an adenovirus, even though serological testing for mouse adenoviruses in a complement fixation test using the FL strain of mouse adenovirus yielded no titers. A diagnosis of an adenovirus infection resembling that caused by the K87 strain was made.     

Murine virus susceptibility of cell cultures of mouse, rat, hamster, monkey, and human origin.

Studies were initiated to determine the practicality of using various tissue cultures for the propagation of murine viruses isolated from laboratory animals. The cytopathogenic effects of 10 murine viruses known to cause disease in laboratory rodents were compared in monolayer cultures of L929, BHK-21, WI-38, BSC-1, and Vero cells. The susceptibility of primary hamster embryo, hamster kidney, mouse embryo, mouse kidney, and rat embryo cell cultures was also tested. Seven of the viruses produced effects in at least 1 of the cell substrates. The remaining 3 viruses, namely H-1, K, and mouse hepatitis, produced no effects in the cell cultures tested.     

Replication of ovine adenoviruses.

The replication of ovine adenoviruses shows intertypic and intratypic variations, e.g. the extracellular and intracellular virus yield of ORT/111, a strain related to bovine adenovirus type 2, reached its peak 40 to 46 hr postinfection, in contrast to GY/14, a strain classified as ovine adenovirus type 1, which required 52 to 58 hr to reach the highest yield. The replication cycle was not appreciably influenced either by rolling of the tube cultures or the age (between 0 and 7 days) of the supporting ovine cell culture. All the strains under study replicated at 40 degree C more rapidly than at 34 degree C or 37 degree C. There was some intratypic variation in the replication of strains at different temperatures. Ovine adenoviruses replicated well after three consecutive passages in cultured lamb testicle or secondary heterologous cells such as calf kidney, calf testicle or pig kidney cell cultures as well as the MDBK cell line and a cell line from the calf trachea. The tissue spectra of strains showed intratypic variations. As examined by direct immunofluorescence, fluorescing adenovirus antigen appeared first at 8 hr postinfection and wharacter of fluorescence are described.     

Isolation and biological characterization of an adenovirus of rhesus macaques.

The etiology of a disease in rhesus monkeys the main clinical manifestation of which was acute conjunctivitis of an epizootic character has been studied. The cytopathogenic agent well propagating in primarily trypsinized kidney cells of monkeys has been isolated when investigating the affected eye mucosa. It was not pathogenic for laboratory animals. The mean diameter of the virions is 75 nm, the buoyant density in CsCl is 1.34 g/cm3, the viral DNA density is 1.706 g/cm3. The biological properties and findings of physicochemical, electron-microscopic, and serologic investigations allow one to allocate the isolated agent to the SV-37 strain, a representative of the adenovirus group.     

Comparative Effects of β-Propiolactone on Mice, Mouse-derived Cell Cultures, and Venezuelan Equine Encephalomyelitis Virus

Studies were made comparing the toxicity of β-propiolactone (BPL) for mammalian (mouse) cells in vitro and for mice and for Venezuelan equine encephalomyelitis (VEE) virus which is highly cytopathogenic for each. The mammalian cells grown in tissue culture were found to be adversely affected by BPL in concentrations ranging from 0.001 to 0.1 mg/ml of supernatant fluid. The difference in response was influenced by the menstruum in which the BPL was suspended and the difference in cell types tested. Tenfold less BPL appeared to be required to destroy the cells when it was suspended in a balanced salt solution than when it was suspended in protein-containing solutions such as beef heart infusion broth or medium 199 plus 20% horse serum. Secondary embryonic mouse lung cells seemed slightly more adversely affected by BPL than the established embryonic lung or L cells. BPL given to mice by intranasal instillation and by intracerebral injection was lethal to half of the animals within 2 days at doses of 0.31 and 0.39 mg, respectively. Higher concentrations of BPL were required to rapidly inactivate the virus in vitro than were required to kill mice or to cause a toxic effect on cells in culture. It required 10 mg/ml of BPL to completely inactivate a high-titered VEE virus preparation in 5 min and 1 mg/ml to inactivate most, but not all, of the virus in 15 min. A concentration of 0.1 mg/ml of BPL had only a slight effect on the virus after a period as long as 60 min. Evidence is presented indicating that simultaneous inactivation of all of the properties of the VEE virus particles by BPL aerosols did not occur at the same time but that, after treatment, the virus possessed a limited ability to immunize mice despite a loss in infectivity.     

An infectious agent causing “spontaneous” degeneration of swine cells in vitro

Summary “Spontaneous” cell degeneration occurred in clone C-19 of the IB-RS-2 swine kidney cell line at around the 20th passage after each cloning. An infectious cytopathogenic agent designated AgC-19 was isolated from such degenerating cultures; the agent was studied through its inoculation into cultures of the cell clone C-12 derived from the same line. AgC-19 was identified as a medium size ribonucleic acid lipovirus belonging to the Togavirus group, and it was shown to be closely related to hog cholera virus (HCV). The evidence indicated that, most likely, AgC-19 is a cytopathogenic mutant of an attenuated HCV that persistently infects the IB-RS-2 cell cultures. After extensive destruction of the cell monolayers, a few viable cells were able to restore the cultures. Such recuperated cultures were resistant to a reinfection with AgC-19. Therefore, the persistent non-cytopathogenic infection is not able to interfere with AgC-19. On the other hand, in relation to the resistant cell cultures, either AgC-19 had selected genetically resistant cells or it had induced resistance through homologous interference.     

Further Studies on the Effect of 5-Iododeoxyuridine on Polyoma Virus Multiplication in Mouse Cells

5-Iododeoxyuridine (IUDR) inhibited production of infectious polyoma virus in mouse embryo cells and mouse kidney cells in culture. Deoxythymidine reversed its effect. IUDR did not inactivate infectivity of free virus particles. IUDR did not prevent adsorption and penetration of polyoma virus to cells. The events sensitive to IUDR treatment occurred at around 20 hours after infection. The cytopathic effects of polyoma virus, including emergence of DNA containing-inclusions in the nucleus, were observable in infected cells in which viral replication was completely arrested by IUDR. It was shown by fluorescent antibody technique in infected mouse embryo cells and by complement fixation test in infected mouse kidney cells that IUDR inhibited completely the synthesis of viral antigen. No virus-like particles were demonstrated in the IUDR-treated infected-mouse kidney cells by electron microscope examinations.     

Bovine Adenovirus

Two adenovirus strains were isolated in calf testicle cell cultures from blood specimens of cattle in Japan. This is the first isolation of bovine adenovirus reported in Japan. The isolates were antigenically similar to each other and distinct from the hitherto described serotypes 1, 2 and 3 of bovine adenovirus. Unfortunately, bovine adenovirus types 4 and 5 were not available for comparison, and hence, until the matter is settled, the virus will be called “Bovine adenovirus type Nagano”. Nagano virus was identified as adenovirus on the bases of the inhibitory effect of 5-iodo-2′-deoxyuridine on virus replication, ether-resistance, effect of temperature and pH on infectivity, and fine structure of the virus particle. The virus grew and formed intranuclear inclusion bodies, a characteristic of adenovirus, in bovine testicle cells but not in bovine kidney cells. The virus agglutinated rat erythrocytes very poorly, but not sheep, goat, cattle, horse, guinea pig, hamster, chicken, and mouse cells. The virus produced adenovirus group-specific antigen in cell cultures. Sero-negative calves were readily infected with the virus by the intravenous, subcutaneous, oral or intranasal routes of inoculation. The infected animals produced antibodies and showed a mild clinical reaction comprised of rhinorrhea, diarrhea and a degree of pyrexia; low-titered viremia of short duration and leukopenia were also observed. A serologic survey indicated wide-spread dissemination of the virus among Japanese cattle, but further studies are needed to determine the etiologic significance of the virus in the natural disease in cattle.     

Adenovirus inhibition of cell translation facilitates release of virus particles and enhances degradation of the cytokeratin network.

Infection of animal cells by a number of viruses generally results in an array of metabolic defects, including inhibition of host DNA, RNA, and protein synthesis, and morphological alterations known as cytopathic effects. For adenovirus infection there is a profound loss of cell structural integrity and a marked inhibition of host protein synthesis, the latter generally assumed necessary to enhance virus production. We examined the purpose of viral inhibition of cell translation and found that it was related in part to cytopathic wasting of infected cells. We show that viral shutoff of host translation promotes destruction of the intermediate filament network, particularly cytokeratins which are proteolysed at keratins K7 and K18 by the adenovirus late-acting L3 23-kDa proteinase. We found that if adenovirus is prevented from inhibiting cell translation, the intermediate filament network remains relatively intact, keratin proteins are still synthesized, and cells possess an almost normal morphological appearance and lyse poorly, reducing the release of nascent virus particles by several hundredfold. Remarkably, in tissue culture cells the accumulation of late viral structural proteins is only marginally reduced if host translation shutoff does not occur. Thus, a surprising major function for adenovirus inhibition of cellular protein synthesis is to enhance impairment of cellular structural integrity, facilitating cell lysis and release of progeny adenovirus particles.     

Fluctuation of antiviral resistance in the intestinal tracts of nude mice infected with a mouse adenovirus

Oral administration of adenovirus strain K87 to BALB/c nude mice resulted in viral proliferation in the intestinal tract up to around week 6 at which point replication was suppressed. In other words, the host acquired resistance. However, this resistance was temporary and the viral infection persisted over a long period with repeated periods of proliferation and resistance. That the appearance of this resistance is the result of infecting mice with the virus and is not due to age difference per se was made clear through experimentation with nude mice of different age groups. However, it was indicated that increase in age is involved in the decreased rate of reproliferation following initial suppression. No evidence of the virus was obtained from any other organ throughout the infection. Furthermore, throughout the persistent infection, even during the aforementioned periods of resistance, no neutralizing antibody was detected from sera, intestinal wall or intestinal content. When spleen cells from BALB/c heterozygous littermate mice was transferred to the nude mice, an earlier onset of antiviral resistance was seen than in nude mice without the transfer, and this was accompanied by a rise in neutralizing antibody titer. From these results, it is believed that the resistance characteristic of nude mice infected by mouse adenovirus is dependent on some factor other than the neutralizing antibody invoked resistance exhibited by euthymic mice.     

SJL/J Mice Are Highly Susceptible to Infection by Mouse Adenovirus Type 1

Mouse adenovirus type 1 (MAV-1) targets endothelial and monocyte/macrophage cells throughout the mouse. Depending on the strain of mouse and dose or strain of virus, infected mice may survive, become persistently infected, or die. We surveyed inbred mouse strains and found that for the majority tested the 50% lethal doses (LD(50)s) were >10(4.4) PFU. However, SJL/J mice were highly susceptible to MAV-1, with a mean LD(50) of 10(-0.32) PFU. Infected C3H/HeJ (resistant) and SJL/J (susceptible) mice showed only modest differences in histopathology. Susceptible mice had significantly higher viral loads in the brain and spleen at 8 days postinfection than resistant mice. Infection of primary macrophages or mouse embryo fibroblasts from SJL/J and C3H/HeJ mice gave equivalent yields of virus, suggesting that a receptor difference between strains is not responsible for the susceptibility difference. When C3H/HeJ mice were subjected to sublethal doses of gamma irradiation, they became susceptible to MAV-1, with an LD(50) like that of SJL/J mice. Antiviral immunoglobulin G (IgG) levels were measured in susceptible and resistant mice infected by an early region 1A null mutant virus that is less virulent that wild-type virus. The antiviral IgG levels were high and similar in the two strains of mice. Taken together, these results suggest that immune response differences may in part account for differences in susceptibility to MAV-1 infection.