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CRIMEAN - CONGO HEMORRHAGIC FEVER

JORDI CASALS
Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, New Haven,Conn.06510,USA.

INTRODUCTION

Crimean hemorrhagic fever was first described as it occurred in Crimea in 1944 by a team of Soviet investigators led by M.P. Chumakov. The disease reappeared in the same area in the course of the following year and was shown by work in human volunteers to be caused by a filterable agent present in larvae and adult forms of a tick, Hyaloma marginatum marginatum, as well as in the blood of patients during the febrile stage; for various reasons the virus was not maintained serially in laboratory animals and was lost. Subsequent events in the characterization of -he disease were: the use of newborn mice as experimental animal which led to the isolation and continuous maintenance of the virus in the laboratory (1967); the demonstration that CHF and Congo viruses, the latter isolated in 1956, were serologically indistinguishable (1969); and the demonstration that viral strains from Central Asia, European USSR and Bulgaria were similar (1970), thus establishing a single disease entity instead of two or more.

The virus. CCHF virus is placed in the family Bunyaviridae even though few of the properties required for inclusion in the family are known for the virus. CCHF virus is morphologically and in size, 90100nm, like a bunyavirus; it contains RNA, possibly singlestranded; and it is inactivated by lipid solvents and detergents. Additional properties required for inclusion in the Bunyaviridae have not been reported for the virus: RNA in 3 or 4 segments with a total molecular weight of 6 x 10^6 daltons; helical symmetry of the nucleocapsid; presence of at least 3 polypeptides of which 2 are glycopeptides and located in the envelope. Since, in addition, CCHF virus is not part of the Bunyamwera supergroup its position in the Bunyaviridae is that of "other possible members", not in the Orthobunyavirus genus.

The antigenic characterization of the virus is, on the other hand, far better established than its position in the International Committee on Taxonomy of Viruses (ICTV) scheme. Exhaustive and continued efforts by hemagglutination inhibition (HI), complementfixation (CF) and agar gel diffusion and precipitation (AGDP) tests have shown the virus to be antigenically related to no other viruses except: to Hazara with which it constitutes the CCHF group, Table 1; and possibly to Nairobi sheep disease (NSD), Tables 2 and 3. The latter relationships is thoroughly intriguing, as it involves also Ganjam and Dugbe viruses, and should be carefully investigated as it might result in the creation of a second or third genus in the Bunyaviridae; unfortunately restrictions by the Department of Agriculture, U.S., preclude such study in our laboratory.

TABLE I
Results of HI and CF Tests with CHF-C Group Viruses

   

Antigen, 8 units

   

CHF-C

 
   

IbAr 1200


Ug 3010


Hazara


Test no

Serum


HI


CF


HI


CF


HI


CF


1


Man, convalescent CHF


80 (a)


-


-


-


40


-

 

Man, convalescent Congo fever


20


-


-


-


0


-

 

Mouse, immunized,CHF-C, JD 206


160


-


-


-


20


-

 

Mouse, immunized, Hazara (serum A)


640


-


-


-


1280


-

 

Sheep, presumed natural exposure to CHF-C or a related virus


640


-


-


-


80


-


2


Mouse, immunized, CHF-C, IbAr 10200


640


256


640


256


80


16

 

Mouse, immunized, CHF-C, Ug 3010


160


256


640


256


20


16

 

Mouse, immunized, Hazara (serum B)


160


8


160


8


320


128


a) Reciprocal of serum titer; 0, no inhibition at serum dilution 1:10, lowest used.

TABLE 2
SEROLOGICAL RELATIONSHIP BETWEEN CCHF AND NAIROBI SHEEP DISEASE (NSD) VIRUSES (From : Dr. F.G. Davies, Kabete, Kenya)


-


Antigen NSD


Mouse ascitic fluid


CF


IF


IHA (a)


NSD


128 (b)


640


10240


NSD (Gangam)


64


320


2560


Dugbe


4


20


< 20


Hazara


trace


20


320


CCHF (IbAr 10200)


0


10


320


Bhanja


0


0


<20


T-176 (c)


4


20


320


Palyam


0


0


<20


Ephemeral Fever


0


0


<20


a) Indirect hemagglutination with tannic acid treated sheep erythrocytes.

b) Reciprocal of serum titer.

c) Virus isolated from Amblyoma cohaerens.

TABLE 3
SEROLOGICAL RELATIONSHIP BETWEEN CCHF AND NAIROBI SHEEP DISEASE (NSD) VIRUSES BY COMPLEMENT-FIXATION TEST


Antigen



Mouse serum or ascitic fluid


NSD


Ganjam I 3159


Ganjam I 619


Dengue 1


CCHF


Drozdov


0


0


0

 
 

U3010


4/2


16/4 - 4/2


4/2


0



IbAr 10200


0


0 - 0


0


0



JD 206


0


0

 

0


Hazara


 

trace


0

 

DGK

   

0


0

 

Uukuniemi

   

0


0

 

Nyamanini

   

0


0

 

Lanjam

 

0


0


0


0


WEE

 

0


0


0


0


Normal

   

0


0

 

Reciprocal of serum titer/reciprocal of antigen titer;

0, no fixation at dilutions 1:2 of serum and 1:2 of antigen.

Antigenic comparison of strains. CCHF virus has a wide geographic and ecologically varied distribution, as will be seen later. By analogy with some other arboviruses it could be anticipated that such distribution might be reflected in antigenic differences; thus far this has not been the case.

Investigation of antigenic differences among strains of this virus has been limited by technical reasons; the only methods generally applicable until now have been CF and AGDP. Serological methods that could detect small but reproducible strain differences are the plaque reduction (PR) neutralization and the kinetic HI tests, neither of which is generally applicable to all strains of the virus. CCHF virus replicates poorly or not at all in most cell lines tried with one exception, CER cells, and in all instances with no visible CPE under fluid medium. Plaque formation by some but not all strains has been reported in LLC-MK2, CV-1 monkey cell line and African green monkey kidney primary cultures; such plaques are usually very small, delayed in appearing and require skilled technical handling. HA antigens are not easily prepared with most strains, require special conditions in carrying out the test and the titers at best are in the order of 1:50 to 1:100.

Replication of the virus in CER cells, a hamster kidney cell line of somewhat dubious parentage (Smith et al.(1) , is excellent but again with no CPE or plaque formation; the use of foci of infection detected by immunofluorescence (IF) and their neutralization by immune sera may lend itself to quantitative serological comparisons, as may also be done by titration of sera by indirect IF; no results are yet available.

An illustration of the type of cross-reactions among isolates of the virus observed by CF is shown in Table 4, in which isolates from widely separated areas were compared; they are nearly identical.

TABLE 4

COMPLEMENT-FIXATION TEST


Cross reactions among six strains of Congo-Crimean hemorrhagic fever virus


Antigen, strain


serum



3010


10200


10248


7620


206


Drozdov


Congo Ug 3010


128/512


256/1024


256/512


256/1024


128/512


128/512


Congo IbAr 10200


128/256


256/256


256/256


256/256


128/256


128/256


Congo IbAn 10248


64/512


256/512


256/1024


128/512


128/512


128/512


Congo IbAn 7620


128/256


512/256


256/256


512/512


256/128


256/256


Congo Pak JD 206


128/512


256/512


128/512


256/512


128/512


256/512

Crimean HF, Drozdov


128/1024


256/1024


128/1024


256/1024


128/1024


256/1024


Reciprocal of serum titer/reciprocal of antigen titer.

World distribution. A notable feature of this virus and of the disease it causes is its wide geographic distribution which encompasses the three continents of the Old World and three faunal regions: Palearctic, Oriental and Ethiopian.

As far as is known natural infection with the virus causes disease in man only; infection of lower animals has been abundantly documented by serological surveys that have included domestic animals--cattle, sheep, goats, camels--but no disease has been reported in them.

In the accompanying map, Figure 1, are shown the distribution of the virus' activities :



Fig. 1. World distribution of Crimean-Congo Hemorrhagic Fever Virus

DV - disease, virus

D - disease, no virus

V - virus, no disease

A - antibodies, man

Aa - a., lower animals

1) Disease confirmed by virus isolation and development of antibodies in survivors has occurred in Bulgaria, USSR (Crimea, Rostov-on-Don, Astrakhan, Armenia, Azerbaijan, and the Central Asian Repubiics of Kazahk, Uzbek, Turkmen, Kirgiz and Tadzhik), Pakistan, Uganda and Zaire. Laboratory infections are not included.

2) Disease, clinically diagnosed, unconfirmed by virus isolation has been reported in Yugoslavia.

3) Virus in the absence of naturally acquired disease has been isolated in Senegal, Nigeria, Central African Empire, Kenya and Ethiopia.

4) Antibodies in man, not associated with disease, have been reported in Yugoslavia, Turkey, Iran, India, Nigeria and Egypt.

The antibodies in Yugoslavia were, in our estimate, questionable; about 15 sera presumably containing CF antibodies when tested in that country were sent to our laboratory where they were found to be negative or non-specific.

Antibodies in Turkey were detected in the course of a seroepidemiological survey conducted at YARU (Serter, Casals, and Buckley, 1975, unpublished observations). About 1100 sera were tested by HI of which 26 were positive: 18 at dilution 1:20, 6 at 1:40, and one each at 1:80 and 1:160. By PR test, 8 of these 26 sera were positive of which 7 had a titer of 1:10 and one a titer of 1:40 against 80 PFU; see Table 5.

TABLE 5
ANTIBODIES AGAINST CCHF VIRUS DETECTED IN SEROLOGICAL SURVEYS CONDUCTED AT YARU


Origin


Year collected


Species


Number


All sera


Test Selected sera

 

   

AGDP


AGDP


HI


CF


N


PRN


Iran


1972-73


Man


351


48


30/55


5/55


0/38


0/15

 

 

Sheep


728


280


77/105


68/105

     

 

Goat


135


48

         

 

Cattle


130


23

         

 

Camel


157


0

         

 

Small mammal


274


8

         

Turkey


1974


Man


1074

   

26

   

8


West Africa


1973-75


Man


636

     

1

   

Antibodies in Iran, were first reported by Chumakov and Smirnova (2) in sheep and cattle. In a survey conducted in our laboratory (3) the results shown in Table 5 were noted; antibodies were found in sheep, goat, cattle and a few small mammals. The antibodies in man, detected by AGDP open to question by ourselves as being to a great extent nonspecific; but there is little doubt that at least 5 persons had significant and specific titers by HI.

Antibodies in man were reported in India (4) by AGDP test; 9 of 633 human sera were positive, originating in Pondichery and Kerala States (Southern India). The same authors also reported antibodies in lower animals.

Neutralizing antibodies in man have been reported in Nigeria 5 by means of a mouse intracerebral test; 24 of about 250 persons had a log neutralization index of 1.5 or greater. This seems to be a very high proportion of positives by this test, particularly in view of the fact that no reports are available of natural disease.

Antibodies in lower animals but not in man, have been reported from other parts of the world, mainly by AGDP test, which test in our estimate is not without problems of specificity. The data have been assembled by Hoogstraal (6). Two bats, species not identified, of 19 were positive in an area of the French Pyrenees near the Catalan border; 6 of 687 cattle and 15 of 48 sheep in Hungary had antibodies; about 10% of 233 cattle were positive in Afghanistan (7,see 6) ; a number of sheep, goat and camel as well as 1 human sera were found positive by CF in Egypt (Darwish et al., 1977 unpublished). Antibodies in lower animals, particularly in sheep and cattle, have also been detected in areas in which the virus has been isolated.

The Disease. From descriptions by USSR and Bulgarian authors, it is evident that CHF is a truly hemorrhagic fever in which acute loss of blood is often a life threat. This conclusion has been borne out in a recent nosocomial episode in Pakistan, March 1976, in which there were 6 secondary cases; the index and 5 secondary cases had severe hemorrhages which may have been the cause of death in 3 of the 4 fatal cases.

The clinician with undoubtedly the most first hand experience with this disease as it occurs in the Soviet Union, based on observation of more than 150 cases in the Rostov-on Don area, is Dr. E.V. Leshchinskaya; she has on two occasions given the members of the US Hemorrhagic Fevers Delegation to the USSR the benefit of her knowledge. The following description is based on her reports.

The disease is generally acquired as the result of infection by a tick-bite or, in fewer instances, by contagion. The incubation period is between 2 and 7 days; the onset is sudden, acute, with fever, chills, severe muscular pains,

Intense headache, vomiting and pain in the epigastric and lumbar regions. Hemorrhages develop from the 3rd to the 5th day of onset and appear in the skin-from petechiae to purpura--and from mucous membranes, nasal, gingival, gastric, intestinal, urinary and uterine; the patients are in critical condition at this time. Physical examination reveals marked lassitude and somnolence, at other times altered consciousness and irritability; there is dry tongue with blood crusts, injected conjunctiva and numerous skin hemorrhages, many at the sites of injections. The pulse is slow to begin with but with blood loss soon becomes fast and feeble, along with lowered blood pressure and dull heart sounds, all indicating impending shock and vascular collapse. Palpation of epigastric region is very painful; liver and spleen are often enlarged. Leucopenia and thrombocytopenia are usual. Death occurs in from 5 to 30% of cases, usually on days 7 to 9 from onset. Autopsy reveals numerous hemorrhages in many organs and often large amounts of blood in stomach and intestines. In favorable cases, the temperature falls between the 10th and 20th day, hemorrhages diminish and the patient improves; the convalescence is long, from 2 to 4 weeks, with a tendency to fatigue and weakness. Loss of hair may occur.

In addition to the severe cases, there occur also according to Leshchinskaya, mild forms with a 2 or 3 days febrile course and no hemorrhages.

The disease in Africa has been reported in 12 cases occurring between 1956 and 1965 (8); one of the patients died 8 or 10 days after he left the hospital with gastric hemorrhages, caused by a gastric ulcer. No association with tick bites was reported in any of the patients; some had contracted the disease as a result of laboratory exposures.

The existence of inapparent infections with this virus has been questioned or denied by some Soviet investigators, on the grounds that they had found antibodies only in persons who had had the disease. On the other hand, Bulgarian investigators (9) in a survey by CF and AGDP of 3012 persons found antibodies in 156 who came from endemic areas and none in 586 originating in non-endemic areas; since not all the positives had a recollection of having had the disease, this was taken as evidence for subclinical infection. As mentioned above, antibodies were detected in man in Nigeria, Turkey, Iran and Egypt; however, in most of the positives there was no way of ascertaining whether they had had overt infections, severe or mild, in the past.

There is need for improvement in the tests used for seroepidemiological surveys with this virus. Some of the neutralization test results reported are open to question due to the known non-specific neutralizing activity of normal sera from many anwmal species, including man, on the virus (10). The majority of surveys

reported have been done by CF and AGDP tests; even in persons who had an overt infection there is a marked loss of positives within 3 or 4 years from onset, particularly among those who had a mild disease

The use of IF for antibody surveys must be considered. Twenty-nine sera from as many persons who had been diagnosed as cases of CCHF in Bulgaria, were kindly supplied to us by Dr. V.E. Vasilenko; they were tested by indirect IF in our laboratory using as antigen CER cells infected with CCHF virus, strain IbAr10200 with the result shown in Table 6. The sera were tested only at dilution 1:4, except 2 that were titrated and had titers, respectively, 1:32 and 1:64. Assuming that the patients resided in the endemic areas, the possibility of boosting exposures after their initial disease cannot be excluded as an explanation for the long-lasting antibodies.

TABLE 6
ANTIBODIES IN PERSONS RECOVERED FROM CCHF IN BULGARIA DETECTED BY IMMUNOFLUORESCENCE. SERA COLLECTED IN 1973 SUPPLIED BY DR. S.M. VASILENKO


Years after onset


Number


Positive


Negative


18-19


8


5


3


6-9


11


9


2


1-5


9


8


1


?


1


1

 

Sera tested at dilution 1:4 only.

Epidemiological Considerations. The association of CHF with ticks has been established since the 1944-45 outbreaks in Crimea. After 1967, due in great part to the use of the newborn mouse as experimental host, great progress has been made not only on the characterization of the virus but on the epidemiology of the disease.

1. Biological properties. Certain biological properties of the virus have epidemiological significance. As far as it is known, the virus in nature causes disease in man only; there are indications, however, that infection of cows and sheep may be accompanied by a febrile reaction. The virus has been isolated from naturally infected cattle, goat and hedgehogs; and hares (7). Experimental inoculation of the virus by peripheral routes to 2 month old calves and 2-to 2 1/2 month old lambs results in viremia from 1 to 10 days after injection, in the absence of disease .12). Hare (Lepus europaeus) after intracardiac or intramuscular inoculation also develop viremia for a similar period with titers up to 10^- 4 / 0.02 cc, again with no disease (13).

The recognition of CHF foci with very large numbers of birds, rooks, heavily parasitized by ticks including species closely connected with the disease in man, led first to the assumption that these birds may be a reservoir of the virus. However, in spite of numerous attempts CCHF virus has never been isolated from birds, some heavily infested with ticks from which virus was isolated; nor have antibodies been found in birds with one questionable exception. Furthermore, experimental inoculation of rooks and other birds did not result in viremia nor antibody development.

Naturally acquired antibodies have been reported on numerous occasions in domestic animals, chief among them cattle, sheep, goat and camel; also in a number of wild animals including halre, tolai hare (L. capensis) and jird.

2. Isolations from arthropods. Attempts to isolate CCHF virus from mosquitoes in the Soviet Union and elsewhere have been uniformly negative. Although Causey et al. 14 isolated a strain from 1 to 377 pools of Culicoides species, the evidence is that insects have no part in the cycle of maintenance of the virus in nature.

Ticks. A remarkable feature of CCHF virus is the large number of tick species from which it has been isolated; in this respect, the virus is unique among tick-borne arboviruses, none approaching it, as stated by Hoogstraal (6) who compiled the data, in the "diversity of the reservoir-vector species linked with it and the numerous ecological environments in which it circulates". As shown in Table 7, CCHF virus has been isolated from 25 different species or subspecies including one from which no actual isolation was made but which was found to contain virus antigen by IF.

TABLE 7
TICKS FROM WHICH CCHF VIRUS HAS BEEN ISOLATED


( From Dr. Harry Hoogstraal, 1977)


Argasidae


Argas persicus


Eurasia


Isodidae


Amblyomma variegatum


Africa

 

Boophilus annulatus


Eurasia



Boophilus decoloratus


Africa



Boophilus microplus


Eurasia



Dermacentor daghestanicus (x)


Eurasia



Dermacentor marginatus


Eurasia



Haemaphysalis punctata


Eurasia



Hyalomna anatolicum


Eurasia



Hyalomna asiaticum


Eurasia



Hyalomna dedritum


Eurasia



Hyalomna impeltatum


Africa



Hyalomna impressum


Africa



Hyalomna marginatum marginatum


Eurasia



Hyalomna marginatum rufipes


Africa



Hyalomna marginatum turanicum


Eurasia



Hyalomna nitidwn


Africa



Hyalomna truncatum


Africa



Ixodes ricinus


Eurasia



Rhipicephalus bursa


Eurasia



Rhipicephalus pulchellus


Africa



Rhipicephalus pumilio


Eurasia



Rhipicephalus rossicus


Eurasia



Rhipicephalus sanguineus


Eurasia



Rhipicephalus turanicus


Eurasia


(x) No isolation, detection in salivary glands by immunofluorescence.

An important epidemiological question concerns survival of the virus in ticks. Experimental studies by Lee and Kemp (15) with Hyalomma m. rufipes fed on calves; by Zgurskaya et al. (16) with H.m. marginatum fed on hare and hedgehogs; and by Kondratenko (17) with H.m. marginatum, R. rossicus and D. marginatus fed on suslik give evidence of transstadial survival and, even more important, transovarian transmission in these ticks. The fact supports the view that ticks are, not only the vector but a reservoir as well, perhaps not the only one but capable of maintaining the virus activity in certain foci for long periods of time. Once an individual tick is infected the virus seems to persist in it for the arthropod's life time, which given the life expectancy of the tick, is more than sufficient to carry the virus from an epidemic season to the next.

3. Natural cycle of the infection. Most ticks epidemiologically and virologically associated with CCHF virus infection in Eurasia are two-or three-host species (6). The greater the number of vertebrate hosts parasitized, the greater are the chances of maintaining and amplifying the virus and transmission to man. Immature forms of these ticks, larvae and nymphs, stay on the same host, usually birds or small mammals; on detaching they molt to adults on the ground. The adult attaches to larger animals, cattle, sheep or man; the females oviposit and the egg develops into larvae which will then attach to ground feeding birds, hare or other smaller mammals. Since birds are not susceptible to the virus, their role is to amplify the number of ticks and to disperse them.

The vector most commonly associated with disease transmission in Central Asia is H. anatolicum anatolicum; in the European part of the USSR, H. marginatum marginatum; and in Bulgaria, the latter and also Ixodes ricinus.. There has been little opportunity to investigate the question in Africa, for lack of disease.

H.m. marginatum is an efficient reservoir of the virus (Hoogstraal, 1977, in preparation). Among mammals, the hare (L. europaeus) is considered an important reservoir in the European USSR and Bulgaria; in Asia its role may be played by hedgehogs, gerbil, and the tulai hare (L. capensis).

4. Disease incidence. There seems to be no central epidemiological bureau in the USSR that reports the nation-wide annual incidence of the disease.

In Crimea the disease occurred in 1944 and 1945 with a total number of about 200 cases; in the subsequent 25 years cases occurred sporadically, not annually and were few in number. From 1970 to 1974 no cases were seen, in spite of the fact that the virus was repeatedly isolated from ticks (18). In the Strakhan area, about 150 cases in all were reported between 1955 and 1969; in the Rostovon Don district, 321 cases were registered between 1963 and 1969 but very few in later years (19). The incidence in the Central Asian Republics of the USSR is difficult to document with accuracy; there have been numerous anecdotal reports but little overall coverage. it appears that few, if any, cases of the disease have been observed in the USSR in recent years (Gaidamovich, 1977, personal communication).

In Bulgaria, 717 cases were observed from 1953 to 1965 (20); in the period between 1968 and 1973, about 130 cases were reported (21).

Twelve cases of Congo virus disease have been described in Africa, between 8 1956 and 1965, of which one was fatal and 5 were laboratory acquired (8).

The most recent outbreaks of the disease have occurred in Pakistan in 1976, one in January-March in the northern part of the country involving 14 persons; and the second in March and April involving 17 cases, in the south-west in the proximity of Quetta (Burney, 1977, personal communication).

5. Transmission to man. Man acquires the disease in two different ways through a tick bite or contact or by contagion.

The great majority of cases are due to infection after a tick bite; occasionally the tick may not have actually bitten but the patient squashed it between his fingers as a means of self protection. This type of infection occurs in persons who have outdoors occupations, farmers, dairy maids or woodsmen.

A substantial number of cases of CHF have been acquired by contagion, particularly through contact with the blood of patients with hemorrhages, either in the home or in hospitals. The disease appears to be more severe following this type of exposure than after tick bite, probably due to the large mass of virus penetrating the body of the victim either through mucosae, inhalation, cuts or abrasions. The Soviet literature has abundant descriptions involving hospital personnel or relatives and friends, particularly in Central Asia, in which 5, 6 or more secondary cases developed following close contact with a bleeding index case. In Bulgaria (20), in a period of 13 years, 42 cases with 17 deaths occurred in medical and nursing personnel. During the JanuaryMarch, 1976 episode in north Pakistan, 6 secondary cases resulted from exposure to the index case, with 3 deaths, including a surgeon who operated on the index case; 7 tertiary cases were either moderately severe or mild, with no deaths.

Another source of contagion is in the laboratory, by exposure to infected materials and either i nhalation or mucosae absorption; Badalov et al. (22) describe the infection of a laboratory assistant who died following heavy exposure to the contents of a broken centrifuge tube. Laboratory infections have also been reported in Africa.

Exposure to the blood of infected animals, particularly cattle and sheep, during slaughtering or wool shearing has led to severe, often fatal infections. Chumakov et al. 7 describe an occurence in 1973 in which three farmers who skinned a sick cow were taken ill, hospitalized and died 6 to 8 days later.

Specific Diagnosis. After Butenko, in 1967, introduced the use of newborn mice intracerebrally inoculated with blood from the patient, virus isolation is easily accomplished. In a series of cases, Butenko et al. (23) reported 40 isolations from 47 blood samples taken between days 1 and 7 from onset, and 4 isolations from 10 samples taken from day 8 to 12. The highest titers of virus in the blood with an LD50 of 10^-4.5 /0.02ml, were between days 1 and 5 from onset.

Antibodies have been routinely detected by CF and AGDP tests, and perhaps lately, by IF; the neutralization and plaque reduction tests are not satisfactory. CF and AGDP antibodies are observed (11) first between 11 and 15 days from onset and remain positive with diminishing titers for at least 3 to 5 years.

Unresolved Questions. As with other viral hemorrhagic fevers a high priority problems to resolve concerns rapid, early diagnosis. Even with current methods the time required for specific diagnosis might possibly be shortened; testing pressure smears of brain tissue from mice inoculated with suspect material or CER cells in chamberslides similarly inoculated by IF may well shorten the time required by from 1 to 3 days.

The fastest way to make a diagnosis would be by detecting the virus or its antigens in clinical specimens. Considering the high titer of virus in the blood between days 1 and 5, reportedly 10^6.2 LD50/ml, it may be possible to detect it by means of IF or radioimmunoassay.

A highly sensitive test for detection of neutralizing antibodies requires urgent development, not only for seroepidemiological surveys, but also for the determination of possible antigenic differences among strains of this virus. Conceivably studies of subvirionic particles might help in resolving the question of antigenic differences; however, the extent to which techniques for purification, fractionation, and polynucleotide analysis can be used may be curtailed by the risk involved in studies of the kind with this agent.

There are undoubtedly many other unresolved questions with CCHF virus, particularly in epidemiology and treatment; we have only pointed those in fields with which we are more familiar.

ACKNOWLEDGEMENT

I wish to express my deepest gratitude to Dr. Harry Hoogstraal for allowing me to see the manuscript of a "Review Article on Crimean Hemorrhagic Fever-Congo Virus" that he is in the process of publishing; and also for the supply of translations of numerous articles in Russian. Without his generous help, my task would have been considerably harder.

REFERENCES
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2. Chumakov, M.P., Smirnova, S.E. (1972) in "Actual Problems of Virology and Prophylaxis of Viral Diseases, M.P. Chumakov, ed., Academy of Medical Sciences USSR, Moscow, pp. 367-368 (Translation from the Russian, H. Hoogstraal).
3. Saidi, S., Casals, J., Faghih, M.A. (1975) Am. J. Trop. Med. Hyg., 24: 353-357.
4. Shanmugam, D., Smirnova, S.E., Chumakov, M.P. (1973) in "Medical Virology", M.P. Chumakov, ed. Works Institute Poliomyelitis and Virus Encephalitis, Moscow, pp. 149-152 (Translation from the Russian, H. Hoogstraal).
5. David-West, T.S., Cooke, A.R., David-West, A.S. (1974) Bull. World Health Organ., 51 : 543-546.
6. Hoogstraal, H. (1977) J. Med. Entomol., in preparation.
7. Chumakov, M.P., Vafakulov, B.Kh., Zavodova, T.I., Karmysheva, V.Ya., Maksumov, S.S., Martynova, L.I., Rodin, V.I., Sukharenko, S.N. (1974) in "Medical Virology", M.P. Chumakov, ed., Works Institute Poliomyelitis and Virus Encephalitis, Moscow, pp. 29-34 (Translation from the Russian, H. Hoogstraal).
8. Simpson, D.I.H., Knight, E.M., Courtois, G., Williams, M.C;, Weinbren, M.P Kikukamusoke, J.W. (1967) East African Med. J., 44 : 87-92.
9. Vasilenko, S., Katsarov, G., Levi, V., Minev, G., Kovacheva, 0;, Genov, I., Arnaudov, G., Pandurov, S., Arnaudov, Kh., Kutsarova, Yu. (1972) in "Actual Problems of Virology and Prophylaxis of Viral Diseases", M.P. Chumakov, ed., Academy of Medical Sciences USSR, Moscow, p. 338.
10. Casals, J., Tignor, G.H. (1974) Proc. Soc. Exp. Biol. Med., 145: 960-966.
11. Karinskaya , G.A., Chumakov, M.P., Butenko, A.M., Badalov, M.E. Rubin, S.G. (see Badalov et al.) 1974 Miscellaneous Publications, Entomological Society of America, 9: 142-144.
12. Zarubinski, V. Ya., Kondratenko, V.F., Blagoveshchenskaya, N.M., Zarubina, L.V., Kuchin, V.V. (1976) Tezisy Dokl. 9. Vses. Konf. Prirod. Ochag. Bolez. Chelov. Zhivot., Omsk, pp. 130-131.
13. Perelatov, V.D., Kuchin, V.V., Donets, M.A., Zarubina, L.V., Kondratenko,V.F., Blagoveshchenskaya, N.M., Vostokova, K.K., Novikova, L.D., Novikova, E.M. (1972) in "Actual Problems of Virology and Prophylaxis of Viral Diseases". M.P. Chumakov, ed., Academy Medical Sciences USSR, Moscow, pp. 354-355. Translation from the Russian, H. Hoogstraal).
14. Causey, O.R., kemp, G.E., Madbouly, M.H., David-West, T.S. (1970) Am. J. Trop. Med. Hyg., 19: 846-850.
15. Lee, V.H., Kemp, G.E. (1970) Bull. Entomol. Soc. Nigeria, 2: 133-135.
16. Zgurskaya, G.N.. Berezin, V.V., Smirnova, S.E., Chumakov, M.P. (1971) Works Institute of Poliomyelitis and Virus Encephalitis, Moscow, pp.217-220.
17. Kondratenko, V.F. (1976) Quoted by H. Hoogstraal, J. Med.Entomol., in preparation.
18. Chumakov, M.P., Andreeva, S.K., Zavodova, T.I., Zgurskaya, G.N., Kostetsky, N.V., Martyanova, L.I., Nikitin, A.M., Sinyak, K.M., Smirnova, S.E., Turta, L.I., Ustinova, E.O., Chunikin, S.P. (1974) in "Medical Virology", M.P. Chumakov, ed., Works Institute of Poliomyelitis and Virus Encephalitis, Moscow, pp. 19-24 (Translation from the Russian, H. Hoogstraal).
19. Perelatov, V.D. (see Badalov et al.) (1974) Miscellaneous Publications, Entomological Society of America, 9: 154-156.
20. Donchev, D., Kebedzhiev, G., Rusakiev, M. (see Badalov et al.) (1974) Miscellaneous Publications, Entomological Society of America, 9: 194-200.
21. Vasilenko, S., Ktsarov, G., Kirov, I., Radev, M., Arnaudov, G. (1972) in "Actual Problems of Virology and Prophylaxis of Viral Diseases", M.P. Chumakov, ed., Academy of Medical Sciences USSR, Moscow, p.337.
22. Badalov, M.E., Lazarev, V.N., Koimchidi, E.K., Karinskaya, G.A. (1974) in a translation of "Crimean Hemorrhagic Fever". Papers at the Third Regional Workshop at Rostov-on-Don, 1970. Miscellaneous Publications, Entomological Society of America, 9: 160-161.
23. Butenko, A.M., Chumakov, M.P., Smirnova, S.E., Vasilenko, S.M., Zavodova,T.I., Tkachenko, E.A., Zarubina, L.V., Bashkirtsev, V.N., Zgurskaya, G.N., Vishnivetskaya, L.K. (see Badalov et al.) (1974) Miscellaneous Publications, Entomological Society of America, 9: 128-134.
DISCUSSION
J. McCormick : Dr. Casals, if we are looking in an acutely ill patient for fluorescent antibodies against this organism, how early should we expect to see it ?
J. Casals : Antibodies seem to appear by the eighth, ninth, tenth day. The Soviet studies indicate that they rise for the first month and then gradually go down so that by the end of the third, fourth year they are still positive. Those are by complement fixation and by agar gel diffusion. I must say that following exposure, virus circulates in the blood until the tenth day at least.

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