Infected Suckling Mice Research Articles

Cross-reacting protein in sera of rabbits and hamsters hyperimmunized with phlebotomus fever viruses.

SummaryHyperimmunization of rabbits or hamsters with infected suckling mouse brain of various Phlebotomus fever viruses led to the development of viral-specific and nonspecific cross-reacting antibodies which can be detected by immunodiffusion techniques. The cross-reacting antibody(s) was shown also to react with other nonrelated arbovirus antigens. Elucidation of the phenomenon showed that the reaction was not due to C-reactive protein, heterophile, or normal mouse brain antibody; rather it appears to be the result of a modified brain antigen present in the central nervous system following infection with arboviruses.

Dengue Virions and Antigens in Brain and Serum of Infected Mice

The temporal relationships of the production of infectious dengue-2 virus and its antigens were investigated in intracerebrally infected suckling mice. Infectious virus, a slowly sedimenting noninfectious hemagglutinin (SHA), and a noninfectious soluble complement-fixing antigen (SCF) were found in the brain. Serum contained high concentrations of SCF antigen relative to infectivity when compared to SCF to infectivity ratios in the brain. Degradation of virions by Tween-80 and ether produced two antigens with sedimentation characteristics similar to the noninfectious antigens occurring naturally in infected tissues. However, the virionderived SHA differed from native SHA when examined by electron microscopy and by equilibrium centrifugation in cesium chloride. Virion-derived SCF (as well as the virions and both SHA antigens) was denatured by sodium lauryl sulfate (SLS) and 2-mercaptoethanol (2-ME), whereas native SCF retained its complement-fixing activity. SLS and 2-ME treatment of dengue-1 and dengue-2 sucrose-acetone antigens increased their serotypic specificity. The hemagglutinin present in sucrose-acetone antigens was predominantly native SHA.

Preparation of noninfectious arbovirus antigens.

Noninfectious arbovirus antigens were prepared from borate-saline suspensions of infected suckling mouse brain buffered with tris(hydroxmethyl)aminomethane and treated with beta-propiolactone (BPL). The activity and stability of these antigens were enhanced by altering the buffering system, by passing the virus seed through a series of four or more continuous passages in the brains of suckling mice, or by a combination of these procedures. The titers of group A and B arbovirus antigens were comparable to titers of antigens extracted by the conventional sucroseacetone-BPL (SA-BPL) method. Antigens prepared from some ungrouped and Bunyamwera arboviruses by either the borate-saline-BPL or SA-BPL method produced inconsistent results and will require the development of more unique procedures to obtain suitable hemagglutinating antigens.

Protective effect on infections with Vibrio cholerae in suckling mice caused by the passive immunization with milk of immune mothers.

The oral infection in suckling mice, strain ICR, with strains of Vibrio cholerae has been reported, in which the multiplication of vibrios, accumulation of fluid in the intestine, diarrhea, and death were observed [1]. In the study, it was observed that experimental cholera was produced only in sucklings younger than about 10 days of age. The infant rabbit [2, 3] and canine [4, 5] models of experimental cholera had already been studied and developed by several authors. Compared with these models, suckling mice offer the following advantages, (i) The experimental cholera is easily produced by oral infection with a small inoculum of vibrios without any particular treatment except starvation for 1 day. (2) Experiments with a large number of mice are easy in contrast to those with infant rabbits or dogs. The ICR strain in particular has beneficial characteristics for use in experimental cholera: mothers have more than 10 babies in each litter and do not refuse to raise sucklings born of other mothers.

Preparation of Noninfectious Arbovirus Antigens

Noninfectious arbovirus antigens were prepared from borate-saline suspensions of infected suckling mouse brain buffered with tris(hydroxmethyl)aminomethane and treated with beta-propiolactone (BPL). The activity and stability of these antigens were enhanced by altering the buffering system, by passing the virus seed through a series of four or more continuous passages in the brains of suckling mice, or by a combination of these procedures. The titers of group A and B arbovirus antigens were comparable to titers of antigens extracted by the conventional sucroseacetone-BPL (SA-BPL) method. Antigens prepared from some ungrouped and Bunyamwera arboviruses by either the borate-saline-BPL or SA-BPL method produced inconsistent results and will require the development of more unique procedures to obtain suitable hemagglutinating antigens.

PROTECTIVE ACTION OF BENZIMIDAZOLE DERIVATIVES AGAINST VIRUS INFECTIONS IN TISSUE CULTURE AND IN VIVO

2-(α-hydroxybenzyl)benzimidazole (H.B.B.), several of its monosubstituted derivatives, and 1-propyl-2-(α-methoxybenzyl)benzimidazole (1-propyl-O-methyl-H.B.B.; P.M.B.B.) have been examined for their effects on the onset of virus-induced cytopathic change in tissue cultures. All the compounds tested exerted high protective action towards cells infected with Coxsackie viruses B5, A9, and A21, echoviruses 6 and 11, and rhinovirus serotype 5 (Norman). Generally, lower activity was demonstrated against echovirus 28 and a selection of other rhinoviruses, but H.B.B. itself was inactive against echovirus 28. The compounds are known to be active against polioviruses. In most cases, activities against larger R.N.A. and D.N.A. viruses were small or nil. P.H.B.B. was tested in vivo for its effect on Coxsackie A9 infection in suckling mice. P.H.B.B. delayed the time of death, but did not reduce the mortality when the infecting dose of virus was large; with a smaller virus dose, there was delay and a reduction in mortality. A strain of Coxsackie A9 virus partially resistant to P.H.B.B. can be selected in tissue culture, but the results suggest that resistant strains may not be formed readily in young mice.

Ultrastructural lesions of the myocardial cell in Coxsackie B4 virus infected mice.

This study describes aspects of the specific changes which occur in the myocardial cells of Coxsackie B4 virus infected suckling mice. These changes consisted of cellular shedding, membrane-vesicle complex formation, disappearance of Golgi body, nuclear shrinkage and densification of chromatin and an increase in ribosomal population. These changes are in addition to the nonspecific changes which probably result as a nonspecific cellular response to injury.

Variable results of sodium deoxycholate tests of Nodamura virus, an ether and chloroform resistant arbovirus.

SummaryNodamura virus, an ether and chloroform resistant arbovirus, was likewise resistant to sodium deoxycholate by either of two SDC test methods when virus was tested in suspensions of infected suckling mouse brain, but by only one of two methods when tested in suspensions of infected suckling mouse muscle. By the method in which muscle suspensions of virus were incubated with 0.5% final concentration of SDC at 37° for 1 hr, and then diluted and tested in mice, inactivation occurred irregularly and unpredictably. These variable results were unexplained, but were not due to brand of SDC, duration of incubation, individual muscle suspensions, concentration of muscle tissue in virus-SDC mixtures, passage history of virus in mice or arthropods, dates of experiments or methods of sterilizing SDC. However, the results indicated that tissue source of virus and method of performing SDC tests can influence results.

Suckling Mouse Cataract Agent (SMCA) in Mice

Summary SMCA virus induces cataracts in a high percentage of infected suckling mice. Maximal SMCA-induced cataract rates were obtained only when virus doses of 104.0 ELD50 or greater were inoculated intracerebrally into suckling mice 4 days of age or younger. Virus inoculated by the intravenous or intraperitoneal routes did not produce cataracts. The SMCA-induced cataract rate was mouse strain-dependent. Cataract rates, based on eye examination at age 30 days, varied from 85% in strain C57B1/6Ha mice to 3% in strain A mice. After 30 days, rates decreased in most mouse strains, but in strain A mice the cataract rate gradually increased to a rate of 44% at 1 year (late cataract strain). Comparative virus assays were performed of the brains, liver-spleen tissues and eyes of SMCA-inoculated strain CFW(A) (30-day cataract rate 46%) and strain A (30-day cataract rate 3%) mice. In each mouse strain virus reached peak titers in all tissues studied in 7 to 15 days. Virus titers then slowly decreased, disappearing from liver-spleen tissues and most eyes by 60 days, but persisting indefinitely (up to 827 days) in the brain. Eye infection rates were similar (88% in A mice, 91% in CFW(A) mice) in both strains of mice. However, in the late cataract strain A mice, virus clearing from eyes and viscera occurred earlier, and peak titers of virus in eyes were lower than in CFW(A) mice.

The largest rabies-specific antigen in extracts of infected suckling mouse brains.

SUMMARY Extracts of rabies-infected suckling mouse brains purified by precipitation at pH 4·5, freed from smaller antigens by sedimentation at 161, 180g and digested with RNase, DNase and trypsin show in the ultracentrifuge a component of S 20 ≈ 16 to 18 which is lacking in extracts of normal suckling mouse brains similarly treated. The largest rabies soluble antigen (‘outer antigen’: Mead, 1962b) has a sedimentation constant S 20 ≈ 16 estimated by the ‘biological’ method of Polson & van Regenmortel (1961). The purified antigen appears to consist of rings or possibly single-turn helices about 100 Å in diameter containing about 0·57 μ g. pentose (as ribose) per μg. total nitrogen. The antigen also appears to contain deoxypentose. It is resistant to pancreatic RNase, DNase, trypsin and chymotrypsin, has a density of about 1·34 g./cm3. in CsCl and an electrophoretic mobility about 7/8 that of rabbit serum albumin at pH 8·5. Preparative density-gradient centrifugation in the analytical rotor of the Model E Spinco centrifuge is described. This allows the method to be applied to smaller particles than can be treated in the S.W. 39 rotor.

Purification and structure of Semliki Forest virus isolated from mouse brain.

Semliki Forest virus from brains of infected suckling mouse was separated by cesium chloride equilibrium density gradient centrifugation into two fractions, both able to agglutinate red blood cells of goose. The heavy fraction (HF density 1.24) had more infectivity, had a sedimentation coefficient of 285 S, and contained intact virus particles, diameter about 75 mμ. The light fraction (LF density 1.205) of equivalent HA (hemagglutinating activity), had less than 1% of the infectivity of the HF and did not contain intact virus particles. It consisted of "empty" virus particles and fragments of the fragile envelope material. The complete virus particle consists of an envelope of complex character surrounding a well-defined core. Shadow-cast preparations of HF show pointed and truncated shadows which suggest cubical symmetry, and the core of full particles, in thin sections, shows an angular, often pentagonal or hexagonal configuration. This suggests that the cubical symmetry of the full particle depends upon the fundamental symmetry of the core, which appears to be icosahedral.

ブタからのA群アルボウイルスの分離について

During June and September, 1965, 14 agents were recovered from blood samples of swine collected in Gumma Prefecture.Thirteen of the isolates were Japanese encephalitis virus, and one strain (JaGAn 18565) was identified as group A arbovirus.This virus was referred to as Haruna virus because it was isolated from swine at Haruna area.Haruna virus shared a common antigen with group A arbovirus and it was closely related antigenically to Getah virus recovered from mosquitoes in Malaya.Sucrose-acetone extraction of infected suckling mouse brains with Haruna virus yielded hemagglutinins and antigens additionally treated with protamine sulfate were more potent than untreated antigens. Haruna virus produced the most potent hemagglutinin than any Getah complex viruses.

Aino virus, a new a new member of simbu group of arbovirus from mosquitoes in Japan.

Three strains of non-Japanese encephalitis virus were isolated from mosquitoes caught in mid-September, 1964, in Aino town, Nagasaki prefecture, Japan. Two of them were recovered from pools of Culex tritaeniorhynchus and one from a mixed pool of Culex pipiens and Culex pseudovishnui. All of these viruses were shown to be fatal to the adult mouse by intracerebral inoculation, ether sensitive, inactivated by desoxycholate and not precipitated with protamine sulfate. These characteristics of the viruses, therefore, are in good accord with those of arbovirus.Since viral hemagglutinin could not be detected by sucrose-aceton extraction of the infected suckling mouse brain, immunological identification of the viruses was achieved by the complement fixation test. As a result, these viruses were antigenically alike and furthermore turned out to be a new member of Simbu group of arbovirus. It is proposed to name this new virus Aino.Later, in 1965 and 1966 attempts to reisolate the virus from mosquitoes collected in Nagasaki area were unsuccessful.

A Simplified Method for Preparing Eastern and Western Equine Encephalomyelitis Complement Fixing Antigens

1. Western equine encephalomyelitis and Eastern equine encephalomyelitis CF antigens were prepared by grinding the brains of infected suckling mice in a borate stock buffer (BSB), allowing the 20% suspension to stand for 24 hours at 4°C, centrifuging, and harvesting the supernate. The “BSB Antigens” prepared in this manner were shown to be of greater potency and sensitivity than commercial products. Inactivation of viral antigens with ethylene oxide gas affected neither the potency nor the sensitivity of the antigen when compared to untreated BSB virus suspension. Ethylene oxide treated BSB mouse brain EEE and WEE CF antigen-antiserum systems did not cross react. 2. Reduction of the incubation time for primary reagents in CF tests from 18 hours at 4°C to 2 hours at 4°C gave little if any change in antiserum or BSB antigen titer. Elimination of the incubation period altogether vitiated the activities of commercial antigens or normal mouse brain, but did not appreciably affect that of the BSB antigen.

Reovirus Infection in Suckling Mice: Immunofluorescent and Infectivity Studies

Summary When reovirus type 1 was inoculated subcutaneously into suckling mice a generally nonfatal pantropic infection ensued. Infectious virus was recovered from most of the tissues tested from 1 hr to at least 20 days later. The greatest amounts of virus were recovered from 3 to 12 days after inoculation. Similarly, viral antigens were observed by fluorescent antibody staining in numerous tissues from 3 to 12 days after inoculation, with maximal antigenic distribution occurring from the 6th to the 9th days.

Rapid identification of Venezuelan and eastern equine encephalitis viruses using infected suckling mouse serum as the hemagglutinating antigen.

"Rapid Identification of Venezuelan and Eastern Equine Encephalitis Viruses Using Infected Suckling Mouse Serum as the Hemagglutinating Antigen" published on May 1966 by The American Society of Tropical Medicine and Hygiene.

Pathogenesis of Venezuelan Equine Encephalomyelitis Virus

Summary The pathogenesis of an attenuated and an unattenuated strain of Venezuelan equine encephalomyelitis virus infection in suckling mice as studied by infectivity titrations and direct fluorescent antibody-staining technique was described. Both strains of VEE virus caused a pantropic infection in suckling mice regardless of the concentration of inoculum used or the route of inoculation given. Viral antigen was detected in many tissues, notably in the central nervous system and in the pancreas. However the intensity of antigenic fluorescence was rather weak, making it difficult to discern sequential development of the viral antigen in infected tissues.

PURIFICATION OF JAPANESE ENCEPHALITIS VIRUS AND ITS ANTIGENIC PARTICLES FROM INFECTED SUCKLING MOUSE BRAINS.

Effort has been made to purify Japanese encephalitis (JE) virus from suckling mouse brains infected with the Nakayama strain. The brain contains so many extractable contaminating materials that purification of the virus from brain suspension by mere repetition of fractional centrifugation has been difficult (Kitaoka, 1955) . Recently, Nojima et al. (1964) reported a purification procedure of JE virus from infected suckling mouse brains by adsorption technique on hydroxyapatite column. The recent progress of sedimentation techniques has opend a simple and efficient way for purification of many animal viruses from infected tissues and tissue culture cells. The present paper will describe in detail the purification procedure of JE virus from infected suckling mouse brains by the protamine treatment, fractional centrifugation and sucrose gradient centrifugation. Also the structure of virus particles will be discussed in regard to hemagglutinating (HA) and complement fixing (CF) substances and infectious principle.

Purification, size, and morphology of a mosquito-borne animal virus, Semliki Forest virus

A plaque assay procedure for Semliki Forest virus (SFV) with an efficiency close to 100% has been described. A concentrated SFV preparation of about 97% purity has been obtained from infected suckling mouse brains by a combination of centrifugations and protamine sulfate treatment. The purification procedure results in an increase of more than 1.5 × 10 5-fold in ability to agglutinate goose erythrocytes, with no significant loss of infectivity. The concentration of plaque-forming units of the preparation is 0.9 times that of the virus-size particles, as determined by a count of the particles in microdrops. Sedimentation velocity and electron microscope studies on the particles of the SFV preparation, combined with measurement of infectivity, lead to the conclusion that the virus particles have a sedimentation constant of 320 S and are nonrigid spheres, with a diameter of 50 mμ, or disks. Some of the morphological aspects of SFV particles have been examined in electron micrographs of the suspension and pellet of the virus preparation.

Complement fixation with antigens prepared from bluetongue virus-infected mouse brains.

Six strains of bluetongue virus were compared by cross complement-fixation tests performed on Perspex sheets according to the method of Fulton & Dumbell using antigens derived from crude saline extracts, and acetone and ether extracts, of infected suckling mouse brains. Only minor differences were encountered with the former and no significant differences with the latter. The reasons for this are discussed.In the neutralization tests important differences were demonstrated between strains.There is evidence that the soluble antigen is composed of particles of varying size, that the smaller particles are responsible for the marked overlapping in the complement-fixation tests, and that increasing complexity associated with increasing particle size may be concerned with increasing serological specificity.