21. Rueckert, R.R., Studies on the structure of viruses of the Columbia SK group. 11. The protein subunits of ME-virus and other members of the Columbia SK group, Virology, 26, 345, 1965.

22. LaPorte, J., The structure of foot-and-mouth disease virus protein, J. Gen. Virology, 4, 63 1, 1969.

23. Maize], J.V., Jr., Phillips, B.A., and Summers, D.F., Composition of artificially produced and naturally occurring empty capsids of poliovirus Type I, Virology, 32,692, 1967.

24. Jacobson, M.F., and Baltimore, D., Morphogenesis ofpoliovirus. I. Association of the viral RNA with coat protein, J. Mol. Biol., 33, 369, 1968.

25. Jacobson, M.F., and Baltimore, D., Polypeptide cleavages in the formation ofpoliovirusproteins, Proc. Natl. Acad. Sci. U.S., 61, 77, 1968.

26. Hinuma, Y., Katagiri, S., Fukuda, M., Fukushi, K., and Watanabe, Y., Kinetic studies on the thermal degradation of purified poliovirus, Biken’s J., 8, 143, 1965.

27. McGregor, S., and Mayor, H.D., Biophysical studies on rhinovirus and poliovirus. I. Morphology ofviral ribonucleo- protein, J. Virology, 2, 149, 1968.

ions on the morphology and infectivity of rhinovirions. In manuscript. 28. Reeves, J.D. and Mayor, H.D., Biophysical and biochemical studies on picornaviruses. 111. The effects of hydrogen

29. Van Elsen, A., BoeyC, A., and Teuchy, H., Formation of fibrillar structures from poliovirus by alkaline disruption

30. Anderer, F.A., and Restle, H., Untersuchungen uber ein atteniuertes poliomyelitis-virus type 111, reindarstellung

31. Bachrach, H.L., Foot-and-mouth disease, Ann. Rev. Microb., 22,201, 1968.

and other treatments, Virology, 36, 51 1, 1968.

und physikalischechemische eigenschaften des Virus, Z. Naturforsch., 9b, 1026, 1964.

IMMUNOFLUORESCENT STAINING OF VIRAL ANTIGENS IN LEUCOCYTES AND MACROPHAGES:

A MEANS OF RAPID DIAGNOSIS OF VIRAL INFECTIONS

Immunofluorescence methodology offers rel- atively new techniques which, in some in- stances, may be advantageous for the investiga- tion of virus infections both in the diagnostic laboratory and in the research laboratory. In in vivo studies, the virus can be identified within exfoliated cells obtained from the site of pri- mary or secondary multiplication and often contained in pathological fluids and exudates. Virus-infected cells can be identified most readily after treatment by the indirect fluo- rescent antibody technique, in which the fluo- rescent label is attached to anti-species globu- lin. By means of such a technique, diagnoses are theoretically possible in any infection in which the virus is at an easily accessible site, even in the earliest stages of infection, by ex-

amination of material from the portal of entry or from the site of first multiplication. Recently it has been realized that the immunofluores- cence technique also holds possibilities for di- agnosis during the phase of spread from the primary multiplication site to the target organ, while the virus is actually present in the blood stream, carried by both polymorphonuclear leucocytes and mononuclear macrophages.

Sommerville has been among the most active investigators in development and utilization of immunofluorescence techniques, with particu- lar emphasis on early and rapid diagnosis of human viral diseases. In a recent article (Som- merville, 1968) he has reviewed knowledge on the detection of viruses within leucocytes and macrophages by means of immunofluorescence,

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and has correlated information gained from studies in experimental animals with that ob- tained from studies of human infections.

Polymorphonuclear leucocytes and macrophages in virus infections

Upon invasion of animals or man by a virus, the predominant cellular response is generally a mononuclear one, chiefly production of lym- phocytes and plasma cells. Mononuclear mac- rophages (which may be either fixed within the reticuloendothelial system or free in circu- lating blood) actively and indiscriminately pha- gocytose foreign particles with which they come in contact, rapidly and efficiently remov- ing them from circulation. A considerable body of evidence indicates that viruses are included among the materials so phagocytosed.’

Polymorphonuclear leucocytes, although not playing a large part in the direct response to invasion of the organism by viruses, undoubt- edly come into contact with invading virus particles. Immunofluorescence studies have in- dicated that these cells, also, become involved in phagocytosis of virus particles; and they have been clearly implicated in the carriage of virus antigens in experimental studies with lab- oratory animak2 3 In his article, Sommerville reviews in considerable detail the observations obtained from studies in experimentally infected animals and in cultures of polymorphonu- clear leucocytes and mononuclear macrophages infected with a variety of viruses. Animal studies have been conducted with poxviruses (vaccinia and ectromelia), herpesviruses, influenza,6 lymphocytic choriomeningitis, ra- b i e ~ , ~ canine distemper virus,8 infectious canine he pa ti ti^,^ l o poliovirus,2 and a number of other agents.

Liver macrophages have been particularly useful for immunofluorescence studies. In in- vestigations with mice, Mims’ found that larger viruses, such as vaccinia and vesicular stomatitis, were cleared from the bloodstream more quickly by liver macrophages than were smaller viruses. Poliovirus types 2 and 3 were cleared to some extent by these cells, but polio- virus type 1 was not cleared at all.

Observations in viral infections of human beings

As yet, only a small amount of work has

been done in this aspect of immunofluores- cence investigation. Human polymorphonu- clear leucocytes are obtained from patients for immunofluorescence tests by methods de- scribed in detail by Sommerville (1968).

Although with clinical materials it is difficult to study completely the course of viral infec- tion, the following facts have emerged:

1. Carriage of viral antigen by cells in “buffy coat” smears, from naturally infected human beings studied by immunofluorescence, has been detected chiefly in polymorphonuclear leucocytes and only rarely in mononuclear mac- rophages.

2. The number of immunofluorescent leu- cocytes varies widely-between 5 % and 85%-in any one preparation at one time.

3. The numbers of fluorescent cells appear to be maximal at the time that viremia is pres- ent.

4. Over a period of five to seven days, the number of immunofluorescent leucocytes grad- ually declines. Their disappearance often coin- cides with the appearance of serum antibody.

5 . With some virus infections, the pres- ence of immunofluorescent leucocytes may be of very short duration; with influenza, they rarely are observed later than 72 hours after onset of acute symptoms.

6. In some instances in which immunoflu- orescent leucocytes have been observed, the in- fecting virus has also been recovered from the same “buffy coat” concentrates of leucocytes that have been found positive by immunofluo- rescence.

Studies of human materials in vitro Evidence confirming the potential of human

polymorphonuclear leucocytes for phagocytos- ing viruses also has been obtained by Sommer- ville (1 968) in studies of cultured leucocytes from normal, healthy persons. The cultures were infected with poliovirus type 1, coxsackie- virus B 1, adenovirus type 6, vaccinia, influenza A and herpes simplex viruses, and the rate of virus incorporation into cells was studied both by immunofluorescence and by titration of the virus in suitably susceptible cell monolayer cul- tures.

In every instance, immunofluorescent evi- dence of virus phagocytosis by polymorphonu- clear leucocytes was seen within two hours

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after infection of the culture; by four hours, 50 to 85% of the cells were stained. At 24 hours after infection, most of the leucocytes were de- generating. In the early periods after infection, mononuclear macrophages included in the cul- tures contained no fluorescent inclusions, but by 24 hours most of the macrophages were stained.

With many of the viruses, titration revealed a rapid reduction in total viable virus; the re- duction was already very marked even at two hours after infection. Sommerville suggests that

this observation may explain why, in studies of natural infections in vivo, relatively few leuco- cyte concentrates yield virus when tested by methods based on viability of the virus, even though the virus can be identified by immu- nofluorescence. It appears that much of the virus antigen, which can be seen by immunoflu- orescence within leucocytes and at a later stage within macrophages, may be inactivated by cel- lular enzymes very soon after the virus enters the cell.

ARTICLES REVIEWED

Sommervile, R.G. Rapid diagnosis of viral infections by immunofluorescent staining of viral antigens in leucocytes and macrophages, Prog. Med. Virol., 10,398, 1968.

REFERENCES 1. Mims, C. A., Aspects of the pathogenesis of virus diseases, Bacteriol. Rev., 28, 30, 1964.

2. Kovacs, E., Baratawidjaja, R. K., Hamvas, J. J., Morrissey, L., and Labzoffsky, N. A., Direct visualization of fluores- cent labelled poliovirus in cells, Life Sci., 2,902, 1963.

3. Kovacs, E., Baratawidjaja, R. K., Walmsley-Hewson, A., and Labzoffsky, N. A., Demonstration of poliovirus in isolated leukocytes, blood and CSF smears of infected monkeys by immunofluorescence and biological means, Arch. ges. Virusforsch, 14, 143, 1963.

4. Sornmerville, R. G., Studies on the interaction between leucocytes and viruses. 11. Mousepolymorphonuclear leuco- cytes; vaccinia and herpes simplex viruses. (To be published.)

5. Baratawidjaja, R. K., Morrissey, L. P., and Labzoffsky, N. A., Demonstration ofvaccinia, lymphocyticchoriomenin- gitis and rabies viruses in leucocytes of experimentally infected animals, Arch. ges. Virusforsch, 17,273, 1965.

6. Boand, A. V., Jr., Kempf, J. E., and Hanson, R. J., Phagocytosis of influenza virus. I. In vitro observations, J. Immunol., 79,416, 1957.

7. Hanson, R. J., Kempf, J. E. and Boand, A. V., Phagocytosis of influenza virus. 11. Its occurrence in normal and im-

8. Liu, C. and Coffin, D. L., Studies on canine distemper infection by means of fluorescein-labelled antibody. I. The pathogenesis, pathology and diagnosis of the disease in experimentally infected ferrets, Virology, 3, 115, 1957.

mune mice, J. Immunol., 79,422, 1957.

9. Wright, N. G., Experimental infectious canine hepatitis. I. The pathology of the disease in guinea pigs, J. Comp. Pathol., 75,449, 1965.

10. Coffin, D. L., Coons, A. H., and Cabasso, V. J., A histological study of infectiouscanine hepatitis by meansoffluores- cent antibody, J. Erp. Med., 98, 13, 1953.

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