Kokernot, R.H.

MacIntosh, B. M.

English

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21 November 1959 S.A. TYDSKRIF VIR GENEESKUNDE 987ISOLATION OF WEST NILE VIRUS FROM A NATURALLY INFECTED HUMABEING AND FROM A BIRD, SYLVIETTA RUFESCENS (VIEILLOT)*R. H. KOKER.NOT, D.V.M., M.D., M.P.H., Rockefel/er Foundation, andB. M. MclNTosH, D.V.Sc. (PRET.), Poliomyelitis Research Foundation, South African Institute for Medical ResearchFrom the Arthropod-borne Virus Research UnittIn a previous paper in this Journal Weinbren1 presented areview of the literature concerning the clinical aspects ofinfection with West Nile virus. The same publication reportedthe results of a serological survey for the presence of WestNile neutralizing antibodies in sera collected from variousvertebrate hosts, including man, which indicated that thevirus was active in the Union of South Africa. The presentpaper describes the isolation of West Nile virus from theserum of a naturally infected human being and from thetissues of a wild-eaught bird, Sylvietta rufescens (Vieillot)commonly called a crombec warblp.f. Both specimens werecollected in Tongaland, Union of South Africa, in April-May1958, as a part of the regular field activities of this UnitA brief account of the field programme since its initiationin 1954 is given in the introduction of a previous paper(Kokemot et al. 2).MATERIALS AND METHODSThe methods used in the documentation and processing ofbuma sera for attempted virus isolation have been reportedelsewhere.2 Briefly it may be stated that the acute and con-valescent sera from the case cited here were proved to havebeen derived from the same donor by a comparison of twosets of fingerprints! taken at the different times when theblood specimens were obtained.Experience has shown that a significant number of indi-viduals engaged in various aspects of the field programmehave acquired clinical illness during the work periods. Theaetiology of such cases has been confirmed by virus isolationand demonstrated by serological studies to have been dueto infection with some one of the arthropod-borne viruses.2• 4Careful surveillance of field personnel is now maintained andincludes initial fingerprinting for identification, obtainingof pre- and post-employment blood specimens (the latter todetect possible inapparent infections), and taking of tempera-tures orally, morning and afternoon.The crombec warbler§ was captured in a Japanese mistnet and taken to the field laboratory, where it wasanaesthetized. The spleen and a sample of liver were removedaseptically and stored together in a sterile glass container onCO2 ice. At the Johannesburg laboratory the tissues weresuspended in a volume of bovine plasma-albumin estimatedto give a 10% suspension and then centrifuged. After treat-* The studies and observations on which this paper is basedwere financed jointly by the South African Institute for MedicalResearch, the Poliomyelitis Research Foundation, the SouthAfrican Council for Scientific and Industrial Research and theRockefeller Foundation, and were conducted with the collabora-tion of the Union Health Department.t P.O. Box 1038, Johannesburg.t We acknowledge with thanks the examination of and opinionon these prints by a member of the South African Police force,Lt. G. Retief, Officer-in-eharge, Local Finger Print Office, Johan-nesburg.§ The authors acknowledge with thanks the identification ofthis bird by Mr. R. Liversidge, Ornithologist on the staff of thePort Elizabeth Museum.ment with penicillin and streptomycin the supernate wasinoculated intracerebrally into newborn and adult mice.The method used in identifying the virus strains isolatedfrom the human serum and the tissues of the bird was similarto that described by Smithburn et al. 3 All neutralizationtests, however, were done with sera previously heat-inactivatedin a 6O"C water bath for 30 minutes. An equal volume offresh normal rhesus monkey serum (obtained 1-2 hourspreviously) was added to the inactivated sera just before thesetting up of the test proper. This step was taken to assurethe presence of the heat-labile accessory substance presentin normal serums,s which augments the activity of neutralizingantibodies to West Nile virus. Casals et aP have describedthe methods utilized in doing complement-fixation (CF)tests. Haemagglutination-inhibition (Ill) tests were doneacc.ording to methods described by Clarke and Casals.8The strain of West Nile virus used in these studies was theoriginal isolate described by Smithbum et af.9 in its 33rdmouse-brain passage.RESULTSIsolation and identification of virus strain from the human beingAn African male (J.M.), aged 26, was employed duringthe period 23 April- I May 1958, by the field team -atNdumu to assist in the capture of wild birds with the aid ofJapanese mist nets. Each morning he arrived at the netsshortly after sunrise, and remained there until sunset. Histask was to remove from the nets birds that were caught,and in the intervening periods he stayed near by in the grass,obscured from birds under clumps of bush. On 28 April,after 4 days of such activity, he reported at about 18.00hours to the doctor present at the station with vague com-plaints of feeling ill and having a headache. Although in noobvious distress, he appeared ill and had an oral temperatureof 10l·2°P. A venipuncture was made and about 15·0 rnI.of blood taken. The specimen was designated H 442. Theserum was separated the following morning and stored in alusteroid tube on CO2 ice. The day after onset the individualwas afebrile and able to resume his normal duties.On 28 May, after it had been stored for 3 days on CO2ice and 27 days in a frozen state in a mechanical deep-freeze(- 20°C), the specimen H 442 was inoculated intracerebrallyinto 2 litters of one-day-old mice. After a 4-day incubationperiod, 3 infants from one litter were missing (presumablyeaten by their mother) and all others of both litters weresick. Brains of the sick mice were harvested for passage,pathological examination and storage. Passage was successfuland a serially transmissible agent was established. The2nd-passage infected mouse-brain material was pathogenicfor adult mice inoculated intracerebrally. The infective agentwas easily filterable through a Seitz pad.In the attempt to re-isolate the agent from H 442 serum,made on 2 June 1958, 3 different dilutions were made andeach was inoculated intracerebrally into 2 litters of newborn988 S.A. MEDICAL JOURNAL 21 Nove~ber 1959TABLE I. RESULTS OF A- Il'.'TRACEREBRJ\L ADULT-MOUSE NEUTRALIZA-TIO_ TEST WITH H 442 VIRUS AGAINST 3 SERA OBTAINED FROM THEPERSON FROM WHOM THE vrRUS WAS ISOLATEDTABLE IT. RESULTS OF HI TEST WITH H 442 ANTIGEN SHOWING ORIGINFROM A HUMAN BEING- AND RELATIONSHIP TO WEST NILE Ai'm AN 2842VIRUSESA rise in HI antibodies to the homologous virus as well asto West ile virus and AN 2842 (designation given virusstrain isolated from the crombec) was also demonstrated ina test with the donor's one acute- and two convalescent-phasesera. In this test, one of the later serum specimens was takenover 7 months after onset of illness and a significant drop mHI antibodies was noted. These results are shown in Table IT.Fate of mice CalculatedSerum with dare Dilution titre ofofbleeding of virus Died Survived virus 1 to:10-0 6 0Routine pre- 10-' 5 0employment 10-8 3 3 130,000,00024.4.58 10-' 1 510.1• 0 610-8 6 0Acute-phase 10-7 6 028.4.58 10-8 4 2 178,000,00010-' 0 610-1• 0 6Convalescent- 10-< 5 1phase 10- 5 1 5 31,0004.6.58 10-8 0 6AN 28422·63·02·5H4425·04·74·0West Nile3·34·04·2West ileH442A 2842SerumDISCUSSIONThe isolation of West ile virus from a naturally infectedhuman being in the Union of South Africa should serve toalert the medical profession to a consideration of this agentm the differential diagnosis of cases of pyrexia. The factthat it has been isolated from a bird as well tends to confirmthe results of the South African serological survey presentedby Weinbren,l which indicated that neutralizirrg antibodiesdue to West ile virus occur in these two vertebrate hosts.The aetiology of West ile virus infection m man can bedetermined with certainty only if the virus is isolated or ifthe convalescent serum shows a significanJ rise in antibodytitre over the acute-phase serum. In attempts at virusTABLE I1J. RESULTS OF CROSS-NEUTRALIZATION TESTS WITH WEST!'.'1LE, H 442 AND AN 2842 VIRUS STRAINS AGAINST THEIR RESPECTIYEPRE- AND POST-INOCULATION GUINEA-PIG SERAVirus strain, logs neutralizedIsolation and identification of virus strain from tissues of aerombee warbler-Sylvietta rufescens (Vieillot)This virus strain was isolated from a suspension of pooledspleen and liver tissue taken from a crombec caught m oneof the Japanese mist nets on 7 May 1958. It is significantthat one week before the capture of the crombec the donorof H 442 virus had been assigned to the net detail for an8-day period and that in the mterval the position of the netsremained unchanged.The pooled tissues from the crombec were kept on CO 2ice until processed in Johannesburg on 12 May. In the litterof 6 newborn mice inoculated intracerebrally with 0·03 ml.of the suspension, one was moribund on the 7th post-inoculation day and the remaining 5 remained well for 21days. The group of 6 adult mice similarly inoculated remainedwell for the same period. A brain suspension prepared fromthe moribund infant mouse and moculated intracerebrallyinto newborn and adult mice caused illness in both after a4-day incubation period. The infective agent in 2nd-passagebrain material was readily filterable through a Seitz pad.The agent was designated AN 2842 virus strain to correspondwith the accession number given to the tissues obtained fromthe crombec.A crude HA antigen was prepared from AN 2842 virus-infected infant-mouse brains. The results of a screeningtest with this antigen indicated that AN 2842 was a group-Bagent related more closely to West Nile virus than to anyof the other agents of the group represented ID the test.In a screening CF test with AN 2842 antigen and 5 group-B-virus mouse hyperirnmune sera, complement was fixed onlym the presence of West Nile immune serum.The results of cross-neutralization test with the humanisolate (H 442), the bird isolate (AN 2842), and the prototypestram of West Nile virus, against their respective pre- andpost-inoculation guinea-pig sera are presented in Table ITLThe results of these tests indicate the relationship of thevirus strain isolated from the human being to the strainisolated from the crombec. Furthermore, they show thatboth strains are identical with or closely related to Westile Virus.Antigen*H442 AN 284210 10640 64040 80of serum dilution givingmice. Eleven of the 12 infants inoculated with a 10-0.3serum dilution were either missing, dead or sick betweenthe 5th and 7th days following inoculation. In 2 littersinoculated with a 10-1 • 3 serum dilution, 2 mice were missingon days 4 and 7, while the rest remained well for 21 days.In the litters inoculated with a 10-2• 3 dilution, only one mousedied, on day 9.An intracerebral adult-mouse neutralization test withH 442 virus was done against a serum obtained from J.M.4 days before onset of illness as well as his acute- and con-valescent-phase sera. The test showed a significant rise inneutralizing antibodies during convalesce~ce of the donorand thus confirmed the fact that the virus had originatedwith him. The results of this test are shown in Table I.SerumtWest NileAcute28.4.58 10Convalescent4.6.58 6409.12.58 80* Eight units antigen used.t Results expressed as reciprocalcomplete HI.An intracerebral adult-mouse cross-neutralization testwith West Nile and H 442 viruses against their respectivepre- and post-moculation guirrea-pig sera indicated a signifi-cant reciprocal cross-relationship between the two viral strains.These results, incorporated m Table ITI, thus identify H 442virus as a strain identical with or closely related to WestNile virus.21 November 1959 S.A. TYDSKRIF VIR GENEESKU_TDE 989isolation, the blood specimen should be obtained in the earlystage of illness and during the period of pyrexia, since theperiod of viraemia in arthropod-borne virus diseases isshort. If the diagnosis is to be made by serological study,it is necessary to have paired sera, the first taken as earlyas possible in the course of the illness and the second duringthe 2nd or 3rd week of convalescence. Both specimensshould be refrigerated without actual freezing and handledin such manner as to prevent bacterial contamination andminimize haemolysis.SUMMARYI. The isolation of a strain of West Nile virus from theblood of a naturally infected human being is described.2. Following infection there was a significant increase intitre of both neutralizing and haemagglutination-inhibitingantibodies in tests controlled by the inactivated acute serumfrom which the virus was isolated.3. The isolation of a strain of West ile virus from pooledtissues (liver and spleen) of a wild-caught crombec warbleris also described.REFERENCESI. Weinbren, M. P. (1955): S. Arr. Med. l., 29, 1092.2. Kokernot, R. H., Smithburn, K. c.. de Meillon, B. and Paterson, H. E.(1958): Amer. J. Trap. Med. Hyg., 7, 579.3. Smithbum, K. c., Kokernot, R. H., Weinbren, M. P. and de MeiUon, B.(1957): S. Afr. J. Med. Sci., 22, 113.4. Heymann, C. S., Kokemot, R. H. and de Meillon, B. (1958): S. Afr. Med. l.,32, 543.S. Morgan, I. M. (1945): J. Immuno!., 50, 359.6. Whitman, L. (1947): Ibid., 56, 97.7. Casals, J., Olitsky,P. K. and AnsIow, R. O. (l95t): J. Exp. Med., 94, 123.8. Clarke, D. H. and Casals, J. (1958): Amer. J. Trop. Med. Hyg., 7, 561.9. Smithburn, K. C., Hughes. T. P., Burke, A. \ . and Paul, J. H. (1940):Amer. J. Trap. 1ed., 20,471.A HAZARD ASSOCIATED WITH THE USE OF A FACE MASK: CASE REPORTs. V. POTGIETER, M.B., CH.B., and J. W. MOSTERT, M.B., CH.B., D.A. (LOND.), F.F.A.R.eS. (ENG.),*Karl Bremer Hospital, University of StellenboschCASE REPORTJ.M.S., a European female patient aged 35 years, underwentan appendectomy, the anaesthetic time being 1t hours. Pre-medication consisted of 50 mg. of pethidine, 25 mg. of phenergan,and 0·65 mg. of atropine. For induction of anaesthesia thepatient received 400 mg. of thiopentone, and anaesthesia wasmaintained with halothane vaporized from a RowbOlham bottleHazards directly associated with the clinical application offace masks for purposes of anaesthesia, of resuscitation, orboth, are not generally known. A sequela is here reported,with a brief discussion of the care needed in the use of rubberface masks, and the results of an investigation prompted byour suspicion (which pro.ved to be unfounded) that thenew chemically-active anaesthetic halothane (fiuothane)might be incriminated.At the Karl Bremer Hospital it is standard practice for theanaesthetic face masks to be washed with hexachlorophenesoap and water only after each use and powdered with talcwhen dry.inserted into the inspiratory limb of a semi-closed Boyle mark-I Icircle absorption system. A dose of 60 mg. of gallamine ('flaxedil')was given fractionally to aid relaxation. Oxygen only, at a flowrate of 1 litre per minute, was led into the absorption system.A size 3 M. & LE. 'Everseal' rubber mask (Figs. 1 and 2) wasfixed tightly in position by a harness, over a rubber Water'soropharyngeal airway, in such a way that the chin was held upby the mask, so that manual support of the jaw was unnecessary.On the following morning a chemical burn exhibiting some darkpigmentation (Fig. 3) withstood the attempts of one of the nursesto remove it; the lesion resembled an impression of the face maskso closely that the nurse tried to rub it 011' believing it to be astain.DISCUSSIONWhile it is obvious that the eyes need protection from a mask,neither of us had seen or heard of such a burn on the face.Trivial lacerations of the lips are known to result from im-properly applied masks, but there was nothing to suggestsignificant pressure or allergy in the present case. Dr. J.Marshal!, consultant dermatologist at the hospital, confinnedthe diagnosis of primary chemical bum followed by increasedpigmentation which in time would disappear entirely. Thelesion faded and was, in fact, hardly visible 14 days later.ow of King Edward VIII Hospital, Durban.*

Isolation of West Nile virus from a naturally infected human being and from a bird, Sylvietta rufescens(Vieillot)

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Isolation of West Nile virus from a naturally infected human being and from a bird, Sylvietta rufescens(Vieillot)

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