Am. I. Trop. Med. Hyg., 30(3), 1981, pp. 509—515 Copyright ©1981 by The American Society of Tropical Medicine and Hygiene

OPPORTUNITIES AND RESPONSIBILITIES OF THE REFERENCE CENTER*

ROBERT E. SHOPE Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale School of Medicine, Box 3333, New Haven, Connecticut 06510

The title of this talk must seem forbidding to Let me sketch the background of the Arbovirus many of you. A reference center is a service or Reference Center at Yale, then I shall share with ganization which on the surface, and especially to you my personal biases about the responsibilities the uninitiated younger scientists, connotes a mu and opportunities which a reference center a.f seum with doddering curators. I hope today to fords. counter that image and to impart a philosophy Reference centers do not begin as reference cen which has been successful in the hands of those ters. They begin as collections of scientists with with whom I have been fortunate enough to col a common research interest, and recognition that laborate over the past 15 years, and which will be protoplasm in the form of type species must be essential in the future if reference centers are to conserved. They require a physical and financial continue to be effective. resource sufficient to characterize and maintain As you may guess, I shall draw heavily from type species, and a spirit to cooperate and share my own experience with the World Reference freely with others. I cannot emphasize too much Center for Arboviruses at the Yale School of Med that a reference center cannot succeed without icine. The same philosophy with principles and cooperation and sharing. corollaries extends to a wide variety of similar The center at Yale was an outgrowth of The programs which most of you either operate or in Rockefeller Foundation's program on arthropod teract with. I have in mind for instance the World borne viruses. The Foundation in 1953 set up a Health Organization Centres for Research and world-wide network of laboratories' to study the Reference, the American Type Culture Collection, distribution, epidemiology, and disease potential the Pan American Health Organization designat of viruses biologically transmitted by mosquitoes, ed dengue reference center at the Walter Reed ticks, and other biting arthropods. A base labo Army Institute of Research, the Research Refer ratory was also established in New York City, ence Reagents Program of the Research Resources directed by the late Max Theiler. The New York Branch of the National Institute of Allergy and City laboratory trained personnel in serologic Infectious Diseases, the developing National In identification techniques, many of which were ac stitutes of Health programs for cryopreservation tually developed there. It also produced reference of filarids such as Wucheraria bancrofti, the re reagents and received viruses for identification. A pository for Aedes mosquitoes at Notre Dame concept was adhered to in the Rockefeller Foun University, the Center for Disease Control region dation field laboratories—no exotic viruses were al center for arboviruses at Fort Collins, Colo to be introduced. This dictum insured that the rado, the National Institute of Allergy and Infec agents sent to the reference center were isolated tious Diseases tick collection at the Rocky in the field and also originated there; the new vi Mountain Laboratory, and the U.S. Department ruses could not be laboratory contaminants. of Agriculture facilities in Ames, Denver, and At about the same time as The Rockefeller Plum Island. In fact, each of you who maintains Foundation program started, other organizations a cell culture, an animal, an insect, or a micro including the U.S. Army, the U.S. Navy, the U.S. organism in your research program is in reality a Public Health Service, and several universities reference center. and foreign governments embarked on similar field programs. In many cases, viruses were re ferred from these programs to the New York Lab

* Presidential Address given before the 29th Annual oratory for identification, and a serological clas Meeting of the American Society of Tropical Medicine sification of arboviruses was refined. and Hygiene, Atlanta, Georgia, 6 November 1980. In 1965 the New York Laboratory was moved 509 510 ROBERT E. SHOPE

to the Yale School of Medicine under the direction Fort Detrick; Allen had supplied 40 ml of Rift of Wilbur Downs and was designated formally by Valley fever sheep antiserum which had passed the World Health Organization as the Interna safety tests and was received with U.S. Depart tional Arbovirus Reference Centre. ment of Agriculture permission. It had been vir The Yale laboratory today maintains a collec tually unused (but not forgotten) stored in the tion of over 400 distinct serotypes of arboviruses freezer for 10 years. When I asked Wil Downs and other zoonotic viruses including the arenavi why he requested it, he answered “Iknew we ruses. Virtually all described arboviruses and would need it,―and indeed we did. many other zoonotic viruses are maintained ex Working in vertical laminar flow biosafety cab cept for those prohibited by the U.S. Department inets, we inoculated the virus into baby mice and of Agriculture—Rift Valley fever, African swine BHK-2 1 cells. The complement-fixation test of fever, African horsesickness, exotic bluetongue Jim Meegan and Jordi Casals, using mouse anti types, and Nairobi sheep disease—and those gen, was positive for Rift Valley fever almost si zoonotic viruses so hazardous that they can be multaneously with the hemagglutination-inhibi worked with only in maximum security facili tion test using mouse serum antigen and with ties—Machupo, Lassa, Ebola, and Marburg. electron microscopy by Owen Wood which I would like, now, to give you several examples showed bunyavirus particles in the BHK-2 1 cells. of the opportunities presented by reference center The diagnosis was transmitted simultaneously to material, each of which illustrates a principle or the World Health Organization, Geneva; through a significance factor which enriched the reference the U.S. Navy to Egyptian authorities; and to the center experience. foreign quarantine authorities of the U.S. De The first principle is: interaction among many partment of Agriculture and the U.S. Public scientific organizations is essential to exploit the Health Service. The agriculture inspector was at full wealth of a reference center. In October 1977 Yale by 8 o'clock the next morning to supervise I received a phone call from Jack Schmidt of the disinfection of the hoods in which the work was Navy Research and Development Command. done. The Rift Valley fever strain was shipped to Jack said that one of their virologists at NAM Plum Island and to Fort Detrick; the remaining RU-3 in Cairo, James Meegan, working with materials at Yale were autoclaved. No fewer than acute phase sera supplied by Imam Zaghloul seven organizations helped in the identification. Imam of the Egyptian Ministry of Health, had Within 3 days the diagnosis was secure, but it isolated a virus from fever cases from Sharquia could not have been made without a tremendous and Qalyubia Governorates northeast of Cairo. reference resource built on cooperation of many Could Yale help identify the virus? We receive organizations and years of preparation. dozens of such requests each year but what made The second principle is: cooperation between this different was the magnitude and seriousness veterinary and medical professions is essential to of the problem. As we were to find out, about progress. This is not an original concept, but one 200,000 persons were ill and by official count, 600 which we tend to forget. I shall continue the Rift had died or were to die.2 My notes of the phone Valley fever story. The Egyptian health authori conversation indicate that we discussed the dif ties rapidly determined that huge numbers of cat ferential diagnoses: dengue and yellow fever, un tie, sheep, and camels had aborted or died of the likely in the absence of Aedes aegypti in lower disease, and its spread to Egypt stimulated work Egypt; Lassa and Marburg viruses, unlikely to ers at Plum Island to test U.S. Army-produced kill mice in the pattern observed; Bunyamwera vaccines in sheep and cattle both for altruistic rea and Semliki Forest viruses, do not usually kill sons vis a vis world needs, and for U.S. readiness persons; and Rift Valley fever, fits most of the should the disease be introduced to our soil. We observations, but what would it be doing in Egypt at Yale retained a vicarious interest in Rift Valley where it had never before occurred? fever. While waiting for Jim Meegan's arrival from But opportunities arise under the strangest cir Egypt, I tried to locate a Rift Valley fever anti cumstances. Jerry Callis and Jerry Walker of the serum. The virus and its antiserum are not readily U.S.D.A. Plum Island Animal Disease Center available in the U.S. Fortunately, with tremen phoned during late July last year. There had been dous foresight, Wilbur Downs in 1967 had con an airline strike affecting J. F. Kennedy airport. tacted W. P. Allen, then with the U.S. Army at Some Nile rats from the Sudan had come through PRESIDENTIAL ADDRESS 511

U.S.D.A. quarantine inspection there, but were microbiologist starts an experiment with an infec delayed, then finally shipped on to Denver where tious agent. It may seem simplistic to state that they were to be used in rat control experiments. the agent should be identified before the experi On arrival in Denver, some rats were dead and ment and even during and after the experiment. others were sick. I was told by Dr. Walker that This may be easy with parasites but is not so easy an animal caretaker in Denver had also taken ill with viruses. We have discovered instances of with fever and malaise. Could this be an impor mistaken identity or contamination in our own tation of Rift Valley fever virus in the Nile rats? laboratory, and in a surprisingly large percentage Two of the sick rats had recovered and their of materials tested from other virus laboratories. blood, along with some inactivated Rift Valley This does not mean they are poor laboratories; on fever hemagglutinating antigen produced under the contrary, the best laboratories request inde auspices of the Army Research and Development pendent certification. Probably the best docu Command, were available for testing. I agreed to mented series originated in the program of the do the tests. Research Resources Branch of the National Insti This request again was similar to dozens we tute of Allergy and Infectious Diseases.4 The Ref receive every year for reference service. The re erence Center at Yale was commissioned to find sults are often negative and reside forever in our out if the arboviruses used as starting seed prep notebooks with a brief negative report to the or arations for reference reagents were correctly la ganization which requested the test. In this case beled and were pure, i.e., without contaminants. there was some urgency since an introduction of The wisdom of the NIAID was shown when we Rift Valley fever constitutes both a veterinary and uncovered over 10% of the reagent sets with one a medical emergency. or another complication. The tests involved: 1) I collected the specimens at the airport on neutralizing the virus with an immune serum from Thursday afternoon and performed the hemagglu another isolate of the same virus to find a contam tination-inhibition tests the next day. I included inating agent which would break through the im antigens to Wesselsbron, Germiston, chikungun mune barrier; 2) testing the immune reagent for ya, and the phlebotomus fever group virus, Saint contaminating homologous or heterologous virus Floris, since these agents could logically have been es; 3) testing both the seed virus and the immune encountered in rats from Sudan and might make reagent from the seed virus for reaction with ref an animal caretaker sick. A totally unexpected erence antigen and antibody of viruses known to result was observed. The phlebotomus fever be in the producer's laboratory; and 4) testing by grouping antibody, included to control the reac complement-fixation the resulting immune re tivity of the Saint-Floris antigen, inhibited the agent with a battery of 181 antigens of mouse Rift Valley fever antigen. As so often happens the pathogenic viruses. The results included such sur rodent sera were negative, but here was Rift Val prises as finding: a seed virus which was a mixture ley fever, thought by me and everyone else to be of two unrelated arboviruses; the inadvertent in unique and unrelated, revealing its secrets. It was troduction of an extraneous arbovirus into the im a member of the phlebotomus fever serogroup. By munizing antigen; a contaminating homologous Monday we had the confirmatory results which virus in the immune reagent; several contaminat have now been published.3 The finding stimulated ing murine viruses; and the mislabeling of bottles an entire new set of areas of research in vaccine by the packager of the immune reagent. development, epidemiology, genetics, and bio Reference centers have helped investigators chemistry, the results of which are not yet fully avert major errors of interpretation of experi evident. The finding was only possible because of ments. The most common happening in our ex 1) cooperation between the veterinary and medical perience with arborviruses is contamination with professions, 2) the availability of the resources of an alphavirus. Semliki Forest virus made major the reference center, and 3) the willingness to do headline news during 1980 when it was cloned tests without expectation of great rewards, a prin using recombinant DNA technology following a ciple which is sometimes the most difficult to in presumed cross-contamination of Sindbis virus. culcate. Had the starting material been submitted to a ref There is another important concept: the Ref erence center, the mix-up could have been rec erence Center can and should be used regularly to ognized early. Drs. Casals and Buckley at Yale certify the identity of infectious agents. Every have detected eastern encephalitis in a putative 512 ROBERT E. SHOPE

Sindbis preparation, Kunjin in a putative Murray to: 1) complete prohibition on the import of live Valley preparation, and Semliki Forest and chi ruminants by U.K. and New Zealand; 2) a ban kungunya viruses on several occasions in suppos on imports of meat by U.S.S.R. in spite of the edly normal Aedes cell lines. In each case the in fact that large shipments were already in Soviet vestigator who submitted their material to the waters in 1977 when the announcement was reference center for testing was saved the embar made; 3) partial embargoes on shipment of live assment or time and effort entailed by a mix-up, sheep to the Middle East which imported nearly and none of these cases have become headline 5,000,000 head from Australia in 1978; and 4) a news. temporary ban by the People's Republic of China Reference center findings can have sudden and on the import of wool and hides.5 unexpected repercussions on world veterinary The Australian authorities, I am happy to re trade. Australia exports three bfflion dollars' worth port, were understanding of the reference center's of livestock and livestock products annually.5 responsibilities, the loss of $3,000,000,000 notwith During the summer of 1977 Christina Frazier, standing. In fact since 1978 they have made an working with me as a post-doctoral fellow, called annual grant to support the Yale Reference Cen me over to see the results of a complement-fixation ter. In addition, the Australian scientific com test. I had suggested that she try to identify some munity has responded to its plight by making unknown viruses from Australia. I had second major scientific advances in bluetongue research thoughts about saddling her with rather routine since the identification of bluetongue virus in Aus identification of viruses, but she was enthusiastic tralia in 1977. and insisted that she would help out in addition Let me now relate another Australian experi to her regular research program. The comple ence which illustrates that the reference center ment-fixation test showed that specimen CSIRO findings can foretell the future. In 1956, Dr. S. G. 19 was bluetongue virus.6 After working the Anderson of the Walter and Eliza Hall Institute week-end to repeat the finding, we cabled Ralph of Medical Research, Melbourne, collected 16 Doherty of the WHO Regional Centre for Arbo pairs of acute and convalescent sera from patients virus Reference in Brisbane and Toby St. George suffering from epidemic polyarthritis and rash. of the Commonwealth Scientific and Industrial The patients became ill during an epidemic of one Organization Veterinary Laboratory. The signif to two thousand cases which occurred in Mildura icance of this is awesome, when one realizes that in the Murray Valley.TMNot only were these people bluetongue was not known in Australia and the quite sick with debilitating and painful arthritis, inevitable result would be embargo of the three but also the disease occurred during the vacation billion dollar livestock export trade by Australia's season and the area suffered financially. Dr. An friends. derson sent the sera to Max Theiler and indepen Now I wish there had been some better way to dently to the late Kenneth Smithburn in South let our Australian colleagues know because, as Africa. Dr. Theiler asked me to examine the sera expected, the impact was sudden and terrible. We to see if antibody to chikungunya virus were pres could not just whisper the news though, because ent. We both knew that chikungunya virus caused we also had a sacred responsibility to APHIS, the in Africa a disease somewhat like the Australian U.S.D.A. quarantine authority, to let them know epidemic polyarthritis and rash. Theiler, in his we had been studying in New Haven, albeit un wisdom, failed to tell me that Smithburn had al wittingly, a class 5 exotic animal pathogen. ready tested the sera by neutralization test and Predictably, the cost to Australia was high had found them negative with chikungunya virus. about $3,000,000,000.@ Ironically, CSIRO-19 virus Influenced by Jordi Casals and Loring Whit when it was tested for its ability to cause disease man, I designed the tests differently from Ken in sheep and cattle, was nearly benign.7 Yet in Smithburn in two ways-I used the hemaggluti fection and disease are not differentiated in the nation-inhibition test which cross-reacts broadly reporting requirements of OlE, which mandates within the alphavirus genus, and I used two al that the 16 diseases on their List A be reported phaviruses from the reference center collection, immediately by member countries for internation Bebaru and Getah from Malaya, viruses which I al notification.5 had previously studied there in 1957 when I was In the case of Australia, the identification of a member of the U.S. Army Medical Research bluetongue virus by the Yale Reference Center led Unit. The test results showed serologic rises in PRESIDENTIAL ADDRESS 513 titer in six of the pairs to Bebaru and Getah vi virus is related to, but different from a virus from ruses. We stated: “Thefact that six of 16 patients a shrew―—Kemp's virus which was later to be developed antibodies to group A arthropod-borne named ‘Mokola'—―andrabies does not have any viruses during attacks of polyarthritis and rash in serorelatives.― I had also just found that Mokola Mildura in 1956 is presumptive evidence that the virus was related by complement-fixation test to epidemic was caused by an arthropod-borne virus the virus from Sudanese mosquitoes (subsequently related to those used in serological testing. The named ‘Obodhiang') and how could rabies virus pattern of HI response, however, is not one that come from mosquitoes? would be expected from any given single virus The whole set of relationships was so beset with among those used in testing. Accordingly, it seems coincidences and so anti-dogma that I shared the probable that the virus isolated will be found to findings only with Wil Downs and a few other differ from the existing identified members of close friends (and I am sure Fred Murphy also group A.―8 was silent) until later when we established that In 1963, the isolation of Ross River virus from these viruses were indeed related to rabies both Aedes vigilax mosquitoes was reported by Doh serologically and ultrastructurally.'6 Dorothy erty et al. ,°who also showed the Ross River an Moore subsequently placed the Culicoides isolate, tigen to be excellent for diagnosing epidemic poly kotonkan, in the rabies serogroup,'5 and Tignor arthritis and rash. Ralph Doherty suggested that et al. identified another rabies-related virus, Du Ross River virus was itself the cause of the dis venhage, which was isolated from a rabid South eases. Only last year,'° 20 years after the original African man bitten on the lip by a bat. 17.18 prophecy did Rosen, Tesh, Gubler, and investi It was only through efforts to maintain a wide gators from New Zealand and Australia isolate ranging network of collaborators that it was pos Ross River virus strains in large numbers from sible to bring together this unlikely meld of viruses blood of patients in Fiji, Samoa, and other South into what now appears to be an evolutionarily west Pacific islands. The prophecy was fulfilled. sound scheme, the rabies serogroup, recently dig A reference center must receive material from nified by a genus name, Lyssavirus. as wide a variety of sources as possible. Imagine A corollary to “Areference center must receive if you will, a virus isolated in 1964 by Jack material from as wide a variety of sources as pos Schmidt from Mansonia uniformis mosquitoes sible―is “neverneglect uninteresting material.― near Malakal, Sudan;― a virus isolated in 1956 by The agent with the lowest priority for character Drs. Boulger and Porterfield from Eidolon hel ization may be the missing piece in the taxonomic vum fruit bats in Lagos, Nigeria;'2 a virus isolated puzzle. The finding of the rabies serogroup stim in 1968 by Graham Kemp from Crocidura spp. ulated research on four continents and has led to shrews originating near Mokola Circle in Ibadan, the once revolutionary, but now accepted, concept Nigeria,'3 and subsequently from human cerebro of antigenic diversity of rabies virus and the hy spinal fluid;'4 and a virus isolated in 1968 by Ver pothesis that perhaps different vaccines are need non Lee from Culicoides midges captured from ed for different parts of the world.'9 cattle at the University of Ibadan farm in A principle which cannot be overemphasized is Nigeria.'5 At the same time imagine that Fred the reference center collection must be ready as Murphy of CDC, Atlanta, and I are jointly study an invaluable resource when new technology ap ing systematically, and I confess in a rather uni pears. Possibly the most important opportunity maginative fashion, the known rhabdoviruses of offered by a reference center, especially world ref animals including rabies virus. Fred examined the erence center, is application of new technology to ultrastructure and I the serological reactions. We a large and varied collection. were both independently doing similar procedures It is little known that Max Theiler had grouped with unidentified viruses received for reference the orbiviruses more than 20 years ago. He was study. fascinated with the effect of bile salts on viral in One day the pieces of a puzzle suddenly fell fectivity,20 and for years tested each new virus together. Fred telephoned to say that Lagos bat that was referred to the arbovirus center. He virus was a rhabdovirus by electron microscopy. thought arboviruses should be sensitive to bile He said, “Itlooks like rabies; could a mistake have salts and could not understand a virus set which been made?― I said, “No,because I have just was obviously arthropod-borne but showed only shown by complement-fixation test that Lagos bat slight sensitivity. The exceptional viruses were 514 ROBERT E. SHOPE recorded one by one in a separate section of his antibodies. Many of these are of primarily scien notebook. One of these was Colorado tick fever tific rather than commercial value. It is time now virus. to refine and to implement inexpensive methods I do not recall the exact sequence of events, but of preservation and to prepare repositories. I know Fred Murphy and Ernie Borden at CDC, Existing type culture collections and reference Atlanta, were busily applying thin-section electron centers are technically equipped to maintain these microscopy during the 1960s; maybe it was not new collections. There is no question about the strictly speaking a new technology, but certainly need. The challenge is firstly in the willingness of it was not widely applied before this to arbovi investigators to share freely their new-found prod ruses. They showed that Colorado tick fever virus ucts and secondly in the ability of reference cen had the characteristic morphology we now attrib ters to handle what promises to be massive num ute to orbiviruses—cytoplasmic, icosahedral par bers of new acquisitions. I believe we are up to ticles with accompanying matrix and tubular the challenge. forms. The special section in Max Theiler's note book suddenly assumed significance. Could these ACKNOWLEDGMENTS bile-resistant viruses also be like Colorado tick fe ver virus? The studies reported herein were supported in Max Theiler carefully located each of these part by The Rockefeller Foundation, the World agents, gave them to me for serologic comparison, Health Organization, the CSIRO of the Austra and I in turn shipped each to Atlanta for electron lian Government, U.S. Army contract DADA-17- microscopy. Every one was an orbivirus. Within 72-C-2170, U.S. Navy contract N00014-78-C- 6 months the entire genus was characterized and 0104, and U.S. PHS grant Al 10984 and contract named. Although Max Theiler steadfastly refused NO1-Al 42517. recognition through authorship on the publica tions,2―22 it was his systematic bile sensitivity REFERENCES testing of viruses from the collection that estab lished the basis for the genus. The collection had 1. Theiler, M., and Downs, W. G., 1973. The Ar been ready and waiting when the electron mi thropod-Borne Viruses of Vertebrates. Yale Uni croscopy technology applied by Fred Murphy was versity Press, New Haven and London, pp. xiii xxiv. ripe. 2. Meegan, J. M., Hoogstraal, H., and Moussa, M. The arbovirus collection continues to be used I., 1979. An epizootic of Rift Valley fever in effectively, a recent example being the extensive Egypt in 1977. Vet. Rec., 105: 124—125. genetic and molecular studies of the family Bun 3. Shope, R. E., Peters, C. J., and Walker, J. S., 1980. Serological relation between Rift Valley fever vi yaviridae being done at the University of Ala rus and viruses of phlebotomus fever serogroup. bama and at Fort Detrick.@24 Lancet, 1: 886—887. Some future responsibilities and opportunities. 4. Cunningham, S., ed., 1978. NIAID Catalog of Re We are going through a period of rapid techno search Reagents 1978—1980.DHEW Publication logical advance. The reference centers of the No. (NIH) 78-899, U.S. Department of Health, Education and Welfare, PHS, National Institutes world must keep up. The storage and classifica of Health, Bethesda. tion of type species, whether they be insects, vi 5. Gee, R. W., 1979. Bluetongue—effects on trade. ruses, protozoa, or other parasites, will remain as Pages 35—37 in T. D. St. George and E. L. a basic function. There are two major technolog French, eds., Arbovirus Research in Australia. Commonwealth Scientific and Industrial Re ical advances which promise to generate enor search Organization and Queensland Institute of mous numbers of new collections: recombinant Medical Research, Brisbane. DNA and monoclonal antibodies. Certain prod 6. St. George, T. D., Standfast, H. A., Cybinski, D. ucts of these technologies are commercially at H., Duce, A. L., Muller, M. J., Doherty, R. L., tractive, as reflected by the rapid advance in the Carley, J. G., Filippich, C., and Frazier, C. L., 1978. The isolation of a bluetongue virus from price of Genentech stock during 1980. You can Culicoides collected in the Northern Territory of bet these commercial products will be preserved Australia. Aust. Vet. J., 54: 153—154. well by industry as long as profits are to be had. 7. St. George, T. D., and McCaughan, C. I., 1979. There will be many other products useful to trop The transmission of the CSIRO- 19 strain of blue tongue type 20 to sheep and cattle. Aust. Vet. J., ical medicine—cloned viral and parasitic prod 55: 198—199. ucts, and hybridomas which produce monoclonal 8. Shope, R. E., and Anderson, S. G., 1960. The vi PRESIDENTIAL ADDRESS 515

rus etiology of epidemic exanthem and polyar Koch, H., 1971. An unusual case of human ra thritis. Med. J. Aust., 47: 156—158. bies thought to be of chiropteran origin. Afr. 9. Doherty, R. L., Gorman, B. M., Whitehead, R. Med. J., 45: 767—769. H., and Carley, J. G., 1964. Studies of epidemic 18. Tignor, G. H., Murphy, F. A., Clark, H. F., polyarthritis. The significance of three group A Shope, R. E., Madore, P., Bauer, S. P., Buckley, arboviruses isolated from mosquitoes in Queens S. M., and Meredith, C. D., 1977. Duvenhage land. Aust. Ann. Med., 13: 322—327. virus: Morphological, biochemical, histopatho 10. World Health Organization, 1980. Ross River virus logical and antigenic relationships to the rabies surveillance. Weekly Epidemiol. Rec., 12: 87. serogroup. J. Gen. Virol., 37: 595—611. 11. Schmidt, J. R., Williams, M. C., Lule, M., Mivule, 19. Shope, R. E., 1975. Rabies virus antigenic rela A., and Mujomba, E., 1965. Viruses isolated tionships. Page 151 in G. M. Baer, ed., The Nat from mosquitoes collected in the southern Sudan ural History of Rabies, Volume 1. Academic and western Ethiopia. East Afr. Virus Res. Inst. Press, New York. Rep., 15: 24—26. 20. Theiler, M., 1957. Action of sodium deoxycholate 12. Boulger, L. H., and Porterfield, J. S., 1958. Iso on arthropod-borne viruses. Proc. Soc. Exp. lation of a virus from Nigerian fruit bats. Trans. Biol. Med., 96: 380—382. R. Soc. Trop. Med. Hyg., 52: 421—424. 21. Borden, E. C., Shope, R. E., and Murphy, F. A., 13. Kemp, G. E., Causey, 0. R., Moore, D. L., Ode 1971. Physicochemical and morphological rela lola, A., and Fabiyi, A., 1972. Mokola virus. tionships of some arthropod-borne viruses to Further studies on IbAn 27377, a new rabies-re bluetongue virus-a new tazonomic group. Phys lated etiologic agent of zoonosis in Nigeria. Am. icochemical and serological studies. J. Gen. Vi J. Trop. Med. Hyg., 21: 356—359. rol., 13: 261—272. 14. Familusi, J. B., and Moore, D. L., 1972. Isolation 22. Murphy, F. A., Borden, E. C., Shope, R. E., and of a rabies-related virus from the cerebro-spinal Harrison, A. K., 1971. Physicochemical and fluid of a child with aseptic meningitis. Afr. J. morphological relationships of some arthropod Med. Sci., 3: 93—96. borne viruses to bluetongue virus-a new taxo 15. Kemp, G. E., Lee, W. H., Moore, D. L., Shope, nomic group. Electron microscope studies. J. R. E., Causey, 0. R., and Murphy, F. A., 1973. Gen. Virol., 13: 273—288. Kotonkan, a new rhabdovirus related to Mokola 23. Bishop, D. H. L., and Shope, R. E., 1979. Bun virus of the rabies serogroup. Am. J. Epidemiol., yaviridae. Pages 1—156in H. Fraenkel-Conrat 98: 43—49. and R. R. Wagner, eds., Comprehensive Virol 16. Shope, R. E., Murphy, F. A., Harrison, A. K., ogy, Volume 14. Plenum Press, New York. Causey, 0. R., Kemp, G. E., Simpson, D. I. H., 24. Robeson, G. K., El Said, L. H., Brandt, W., Dal and Moore, D. L., 1970. Two African viruses rymple, J., and Bishop, D. H. L., 1979. Bio serologically and morphologically related to ra chemical studies on the phlebotomus fever group bies virus. I. Virol., 6: 690—692. viruses (Bunyaviridae family). J. Virol., 30: 339— 17. Meredith, C. D., Rossouw, A. P., and Van Praag 350.