THE ULTRACENTRIFUGE AS AN AID IN THE DETECTION OF POLIOMYELITIS VIRUS

1. By means of differential ultracentrifugation, a purified and concentrated macromolecular fraction has been regularly obtained from infected human, monkey, and chimpanzee stools. This fraction was isolated from sixteen stools in which virus was thought to be present, and inoculated intracerebrally into sixteen monkeys, of which fifteen developed poliomyelitis. 2. Eleven stool specimens in which virus was suspected, when tested separately in eleven monkeys by the intra-abdominal-intranasal method, produced poliomyelitis in two of these animals. When the same specimens were tested separately by the ultracentrifugal-intracerebral method, poliomyelitis developed in ten monkeys out of eleven inoculated. 3. With the intra-abdominal-intranasal method, it has been customary to inoculate the virus present in 1.6 gm. of stool. With the ultracentrifugal-intracerebral method, the virus present in as much as 30 gm. of stool has been inoculated. 4. From one titration experiment it would appear that the ultracentrifugal-intracerebral method is at least 100 times more sensitive than the intra-abdominal-intranasal method

COMPLEMENT-FIXING ANTIBODIES TO TYPE 2 (LANSING) POLIOMYELITIS VIRUS IN A NORMAL POPULATION OF A SUBTROPICAL AREA

Sera collected in 1950 from the native population in the vicinity of Cairo, Egypt, have been tested for complement-fixing antibodies to Type 2 (Lansing) poliomyelitis virus. Complement-fixing antibodies are confined to the age of 1 to 9 years if a serum dilution of 1:4 is used in the test, or to the age of 1 to 4 years with a serum dilution of 1:16. A comparison has been made of the findings obtained in this study with the results for neutralizing antibodies previously reported. Complement-fixing antibodies were found to be temporary in nature while neutralizing antibodies were maintained for long periods of time. On this basis, criteria for "recent," "old," or "no infection" in poliomyelitis have been established. Sixteen of the children were bled again in 1952, 18 months after the first bleeding and both series of sera were compared in complement fixation and neutralization tests. All children who were negative in 1950 and who developed c-f antibodies by 1952, also developed neutralizing antibodies to a titer of more than 1:250 during the same period. Some children who failed to develop c-f antibodies, did develop neutralizing antibodies although of lower titer than found in children in whom c-f antibodies made their appearance. These findings are discussed in the light of theories regarding the mode of acquisition of antibodies to poliomyelitis virus

INTERFERENCE BETWEEN POLIOMYELITIS VIRUSES IN TISSUE CULTURE

The inhibition of multiplication of one poliomyelitis virus by a poliomyelitis virus of another immunologic type has been established by using tissue cultures of monkey testes. The degree of interference varied from none, to partial, to complete, depending upon the time between inoculation of the interfering and the challenge viruses, and the amount of each virus inoculated. Reciprocal interference was demonstrated between Types 1, 2, and 3 poliomyelitis viruses. Under conditions which resulted in complete suppression of the growth of one poliomyelitis virus by another, interference by poliomyelitis virus with the multiplication of four antigenically distinct "orphan" viruses and of three antigenically related strains of Coxsackie virus could not be demonstrated. Poliomyelitis virus rendered non-infective by formalin or by irradiation with high energy electrons or with ultraviolet light, or treated so that only traces of residual active virus remained, failed to interfere with the propagation of active homologous virus

IMMUNOLOGICAL REACTIONS OF THE COXSACKIE VIRUSES : I. THE NEUTRALIZATION TEST: TECHNIC AND APPLICATION

The neutralization test is a reliable and useful procedure for following immunological reactions of the Coxsackie viruses (C virus). The standard procedure has been an incubation period of 1 hour at room temperature followed by subcutaneous inoculation into newborn mice. However, this time and temperature are not critical, for the virus in neutralized within 10 minutes of mixing with immune serum and remains neutralized for long periods. During the variable incubation periods used, the control virus remained active, even in dilute suspensions. The neutralization test is not affected by the presence or absence of complement. Neutralizing antibody is stable at 65°C. for 30 minutes, and immune serum has to be heated to 80°C. for 30 minutes before the antibody is no longer detectable. As the quantity of virus is increased, the quantity of serum required for neutralization likewise increases, but not in a regular or predictable fashion. Neutralized mixtures of the virus can be made infective again by simple dilution before inoculation. The neutralization test is a satisfactory means for typing Coxsackie viruses. At least seven antigenic types have been identified. Similar antigenic types have been found to be scattered over wide areas. Thus the Conn.-5 type was present in 1948 in Massachusetts, Connecticut, New York, and North Carolina. The Texas-1 type was present in 1943 in Connecticut and in 1948 in North Carolina and Texas. Further information on the specificity of the neutralizing antibody response has been obtained from a study of the occurrence and development of antibodies in 6 patients who contracted infections with one or another of the C viruses while working with them in the laboratory. From each patient a virus was isolated during the illness. No patient had detectable antibodies to his strain before his illness, but each soon thereafter developed antibodies to his own strain and to the prototype strain to which it was related. By means of the neutralization test, it has been shown that a family epidemic may include two different immunological types of virus. Neutralizing antibodies appear at the time of or soon after onset of illness, increase rapidly to titers of about 1:1000 which are maintained during the period of 1 to 3 months following infection, and are still present 2 years later, although at lower levels. Neutralizing antibodies are present in the normal population. In North Carolina, over 80 per cent of the children have antibodies at birth. The level falls rapidly to a minimum of 14 per cent at the age of 1, and then it quickly rises to reach the adult level at the age of 7. Gamma globulin collected in various parts of the United States between 1944 and 1949 and in Denmark in 1949 neutralizes at least four antigenically different Coxsackie viruses

IN VITRO DIFFERENTIATION OF VIRULENT AND ATTENUATED POLIOVIRUSES BY THEIR GROWTH CHARACTERISTICS ON MS CELLS

The MS character of polio-virus particles is described. Strains that produced relatively large plaques, up to 6 mm. in diameter, on a monkey stable (MS) cell line, were classified as MS+. Such strains were found to include the highly virulent poliovirus strains. Attenuated strains appeared deficient in this gene, for they produced tiny plaques less than 1 mm. in size, or no visible plaques at all; they were considered as MS mutants. Strains of maximum neurovirulence for the monkey possessed the MS+ and d+ characters, while those of greatest attenuation were MS d. Strains possessing the MS d+ character showed high or intermediate attenuation. Virulent strains grew equally well in MS or primary monkey kidney (MK) cells. Attenuated strains gave lower titers in MS cultures than in MK cultures. While MS cells after infection with a virulent virus yielded about 100 to 200 PFU per cell, only one PFU per MS cell was detected after infection with an attenuated virus. A study of newly isolated Type 3 strains showed them to consist of the MS+d+ or MS d+ type. The MS+d+ virus proved to be highly paralytogenic for monkeys even by the intramuscular route, while the MS d+ virus was of the partially attenuated type. The study of in vitro characters of viruses from children fed attenuated poliovirus offers a possibility for following genetic changes of the viruses after multiplication in the human enteric tract

THE INFECTION OF CHIMPANZEES WITH ECHO VIRUSES

The oral and parenteral infections of chimpanzees receiving echo Types 6 and 4 viruses successively are described. The two infections, spaced 2½ months apart, and given by the same route in each animal, failed to induce overt disease. The inapparent infections were demonstrated by virus excretion in the throat and the stools and the development of neutralizing antibodies. Complement-fixing antibodies also appeared after Type 6 infection, but fell more rapidly than the neutralizing antibodies. After oral infection, echo-6 virus was found for equal periods in both the throat and feces, but echo-4 persisted in the throat for much longer periods than in the lower bowel. Almost no virus carriage occurred after parenteral inoculation. No true viremia was exhibited in any of the animals. One of the chimpanzees had neutralizing antibodies against Type 6 virus in its pre-inoculation serum. It responded extraordinarily to the Type 6 exposure, developing antibody levels of 1:50,000 to echo-6, of 1:1024 against the echo-6' variant, and of 1:64 against the echo 6'' variant. Although Type 4 antibodies developed after the exposure, they proved difficult to measure by ordinary methods. However, they could be satisfactorily assayed by the plaque reduction method. Three other chimpanzees fed echo-2, echo-3, and an untypable echo virus, respectively, yielded results confirming those established with Types 4 and 6

THE CELLULAR CHANGES PRODUCED IN TISSUE CULTURES BY HERPES B VIRUS CORRELATED WITH THE CONCURRENT MULTIPLICATION OF THE VIRUS

A sequential study is reported of the morphological changes occurring after herpes B virus infection of cells as revealed in ultrathin sections under the electron microscope. Monolayer cultures of renal epithelial cells prepared from the natural host of the virus, the monkey, were infected, and the cellular alterations were correlated with the appearance of infective virus in the culture fluids. The morphological changes consisted in swelling of the cells and disappearance of the nucleolus, followed by margination and gradual decrease of the nuclear chromatin. The inclusion material corresponded to the clear central areas of the nucleus, where the chromatin had disappeared. In the late stages of infection this inclusion material filled the nucleus and formed a classical type A inclusion body. Characteristic particles appeared in the nucleus and cytoplasm of the infected cells a few hours after inoculation. They had a dense center surrounded by one or two membranes. Those with one membrane ranged in size from 60 to 100 mµ and those with two from 120 to 180 mµ. Particles showing the same wide variation in size and structure were seen both in the nucleus and in the cytoplasm. They were first visible on the external surface of the swollen but intact cells at about the same time new infective virus became detectable in the culture fluid. A small number of the extracellular, and cytoplasmic, virus particles appeared "binucleated," containing two central bodies, each having its own membrane, both being surrounded by a single external coat. About 180 mµ in diameter, they were randomly distributed among the "mononucleated" particles

DOUBLE INFECTIONS OF SINGLE CELLS WITH ECHO 7 AND COXSACKIE A9 VIRUSES

Virus particles derived from single cells infected with two enteroviruses have been studied. Evidence was obtained to indicate that phenotypic, but not genotypic, mixing occurs between Coxsackie A9 (CAP) and ECHO 7 (E7) viruses. Monkey kidney cultures in monolayer were doubly infected with high multiplicities of CA9 and E7 viruses. During the latent period, the infected cells were suspended, diluted, and distributed under oil into droplets each containing a single cell, as checked by microscopic observation. The virus particles released by individual cells into the microdrop were characterized in differential plaque neutralization tests. Fifteen per cent of the microdrops contained doubly neutralizable particles, 53 per cent yielded either CA9 or E7 particles, and 34 yielded particles of an intermediate character (deficits between 37 and 75 per cent). On passage, the doubly neutralizable particles yielded progeny of both parental types. All passage strains behaved like the corresponding parent strain as regards pathogenicity for newborn mice, which is to say that this property was limited to virus particles with CA9 antigenicity. Coxsackie A9 has a more rapid growth cycle than ECHO 7 in rhesus monkey kidney cell cultures, and a slower one in patas cultures. In rhesus, when E7 virus was added first, CA9 could be added up to 2 hours later, and still a significant number of cells yielded either CA9 or doubly neutralizable virus. The converse was observed in patas cells