618 TRANSACTIONS OFTHEROYALSOCIETY OF TROPICALMEDICINEANDHYGIENE,VOL. 73, No. 6, 1979
ROYAL
SOCIETY
OF TROPICAL
Ordinary Manson
House,
Thursday,
MEDICINE
AND
HYGIENE
Meeting 14th December,
1978
The President: DR. S. G. BROWNE, CMG, OBE, &ID, FRCP, DTM,
Symposium
on Rift Valley
FKC, in the Chair
Fever
The Rift Valley fever epizootic in Egypt 1977-78 1. Description of the epizootic and virological studies JAMES M. MEEGAN U.S. Naval Medical Research Unit No. 3 (NAMRU-3) U.S. Embassy, Cairo, Arab Republic of Egypt
Summary From October to December 1977, an extensive eDizootic occurred in Egypt resulting in abortions and increased mortality m domestic animals, and severe clinical disease with fatalities in man. Rift Valley Fever (RVF) virus was isolated and identified as the causative agent. In humans, acute febrile, encephalitic, ocular and fatal haemorrhagic diseases were documented as resulting from RVF virus infection. A retrospective serological survey indicated RVF was recently introduced into the area. The 1977 eoizootic extensively involved five Governorates.-In the summer and autumn of 1978, epizootic RVF reappeared in Egypt and spread to nreviouslv uninfected areas. Virological, serological and epidimiological studies, and factors related to the spread of RVF are discussed. Introduction et al. (1931) studied an epizootic disease in the Rift Valley of Kenya which primarily affected sheep and resulted in an increased incidence of abortion in ewes and of mortality in lambs. The investigation revealed: (a) the causative agent was a virus, most probably arthropod-borne; (b) the fatal form of the disease occurred predominantly among young sheep, goats and cattle, with the characteristic pathological lesion being focal liver necrosis; and (c) man could be infected by the virus, but exhibited only a mild febrile illness. The name Rift Valley fever (RVF) was suggested, and discussions with local inhabitants indicated RVF had most probably been present in the area for at least 20 years. In the 48 years since this initial study, comparatively little data have been accumulated about this viral disease. Most investigations have been concerned with the pathology of the animal disease. They revealed that a wide variety of wild, domestic and laboratory animals are susceptible to RVF virus (WEISS, 1957). Characteristically, a short incubation period was followed by a definite febrile phase, In
1930,
DAUBNEY
development of focal liver necrosis and death, frequently associated with haemorrhagic signs. The SMITHBURN et al. (1948) &tomological studies in Uganda established RVF as an arthrooodborne virus-by isolating six strains of virus irom species of two genera of mosquitoes. Since then, approximately 18 mosquito species have been implicated as possible RVF virus vectors (GEAR et al., 1955; STEYN & SCHULZ, 1955; KOKERNOT et al., 1957; WEINBREN et al., 1957; WILLIAMS, et al., 1960). Due to this wide vertebrate and invertebrate host range, the virus has circulated in a number of ecological settings. Major epizootics occurred in 1931 in Kenya, in 1951 and 1975 in South Africa (GEAR, 1951; VAN VELDEN et al., 1977), and.1975 in the Sudan (EISA et al., 1977). The virus was also isolated from various sources during smaller outbreaks in Nigeria, Uganda and Rhodesia, and serological surveys have detected antibodies to RVF virus in a number of southern African nations (EASTERDAY, 1965). However, until 1977 the virus was geographically limited to Sub-Saharan Africa. In most epizootics, small numbers of human infections were documented and no fatalities were directly ascribable to RVF. Retinitis was the only complication suggested to result from this otherwise benign human febrile illness (SCHRIRE, 1951; FREED, 1951; COHEN & LUNTZ, 1976). This ocular syndrome remained questionable since definitive diagnosis was not established by virus isolation or serological conversion in paired serum samples. Recently, however, VAN VELDEN et al. (1977) documented that RVF virus infection could result in acute clinical complications by confirming four human RVF fatalities and 12 cases of encephalitis among a small number of individuals infected during the 1975 South African epizootic. Considering this history, one understands why the sudden spread of RVF to Egypt was unexpected and why an extensive epidemic of an unprecedented disease with severe clinical manifestations and numerous fatalities represented an enigma.
619
J. M. MEEGAN
MEDITERRANEAN
SEA
.FAOUS ~* ‘.
/
‘,
..
NILE DELTA I- GIZA
,. ; ,I ,’ ,*’ 1 I: : : : I \ ,SNA’i“‘.. c
NILE VALLEY IOII 12, 12, 13, 14, IS16,
‘.,’
.:._ 1 ‘.
GOV.
BENI SUEF FAYUM YINYA ASYUT SOHAG OENA ASWAN
“I’--’
t t : : \ , ,: i i ,’ : ‘, : ASWAN 1;
GOV.
2-OALYUBIYA 3-SHAROIYA 4- MINUFIYA 3- GHARBIYA 6 - DAOAHLIYA 7, KAFR EL SHEIKH 6- BEHEIRA S-DAMIETTA
1 Fig. 1. Map outlining
major Governorates
in Egypt.
Description of the epizootic During the first week of October, 1977, an increased incidence of acute febrile disease and a fatal haemorrhagic-like illness were reported from the Inshas (also known as Basateen-el-Ismailiya) group of villages (population approximately 10,000) in Sharqiya Governorate, 70 km north-east of Cairo (Fig. 1). The villages are situated in a heavily irrigated agricultural area planted with peanuts, fruit trees and rice. The principal water supply is the Ismailiya canal running from the Nile near Cairo north-east along the south-eastern border of the Nile Delta. During the second and third weeks of October,
additional human cases were reported in many villages along the canal, both to the north near Zagazig, and to the south near El Khanka in Qalyubiya Governorate (Fig. 1). At this time, a focal outbreak of human cases occurred in the Imbaba area of Giza Governorate, which borders Cairo on the west. However, Cairo itself was not involved in the epidemic. During October, the Egyptian Ministry of Agriculture received a number of reports from the epidemic area documenting an increased incidence of mortality and abortion in sheep, cattle, domestic buffalo and camels. In retrospect, it appeared that animal disease had been occurring in the epidemic
620
EGYPTIAN RVF EPIZOOTIC 1977-78.
1 DESCRIPTION AND VIROLOGICAL STUDIES
area since September, and to the south in Aswan Governorate since late August (MEEGAN et al., 1979). The Egyptian Government responded with aerial insecticiding using varying concentrations of malathion. However, no study was made of the effectiveness of this effort. The predominant species of mosquito in the area is C&x pip&s (see HOOGSTRAAL et al., pp. 624-629), and ‘the unusually long summer of 1977 might have increased the mosquito population densities, although this cannot be documented. Reports of elevated rates of human febrile illness and death in these areas continued through November and the first two weeks of December. Clinics and hospitals also observed an increased incidence of encephalitic and ocular disease. With the onset of the colder winter weather in mid-December (temperatures range from 2°C to 24°C from December to March) and the concomitant decrease in mosquito population densities, reports of human and animal disease subsided.
antigens were loo/ mouse brain suspensions prepared in buffered physiological saline. Arthropodborne viral antigens from 12 serological groups were selected for testing, and represented viruses present in Egypt and others which could cause fatal human disease. No pattern of serological conversion could be detected with these antigens. Concurrently, virus isolation was attempted by intracerebral inoculation (i.c.) of undiluted sera into two-day-old suckling mice. The original serum samples yielded 39 viral isolates which were lethal in one and a half to two days in suckling mice (Table I). Five isolates were passaged twice in suckling mice and a virus stock was prepared from a 10% brain suspension. These isolates were lethal in two to four days for adult mice, adult hamsters and rats when inoculated by the i.c., subcutaneous or intraperitoneal routes. Histopathological examination of the experimental animals revealed focal liver necrosis. The isolates were sensitive to sodium deoxycholate and ether, and a sucrose-acetone virus preparation (CASALS, 1967) haemagglutinated goose erythrocytes at a pH of 6.3 at 37°C. One isolate (NAMRU-Z41) was diluted 1:4 and tested by CF against mouse hyperimmune antisera from 12 serological groups of arthropod-borne viruses (cross reactions within groups allowed screening of approximately 300 viruses). All tests were negative (antisera to RVF virus were not available in Egypt at that time). Isolate 241 was then tested at the Yale Arbovirus Research Unit (World Health Organization Arbovirus Reference Center) against a larger number of reference antisera. In collaboration with Drs. J. Casals, R. Shope, J. Converse and 0. Wood, the isolate was identified as Rift Valley fever virus by the CF and haemagglutination-inhibition (HI) tests (MEEGAN et al., in preparation). Electron microscopic studies of infected Vero cell cultures showed a spherical viral particle, 100 nm in diameter and similar in morphology to Bunyamwera-like viruses. An additional 40 acute sera were collected in November from patients examined in detail at the
Virological and serological studies On 14th October, Dr. Imam Z. Imam of the Egyptian Ministry of Public Health provided NAMRU-3 with 66 acute and five convalescent serum samples from patients seen at hospitals in the Zagazig area (Sharqiya Governorate) and displaying both the acute febrile and fatal haemorrhagic-like disease (the condition and amounts of the samples permitted virological studies on only 51 sera; the sample numbering system to identify corresponding clinical disease states was lost during transport). Extensive bacteriological tests disclosed no pathogen. The clinical symptoms of the acute febrile disease (including fever, headache, joint pain and severe lower back pain) suggested a viral aetiology. Consequently, convalescent samples were analysed with the complement fixation (CF) test to determine serological conversion to a number of reference viral antigens. Sera were heat inactivated (56°C for 30 min), diluted (starting at 1:4), and tested in the CF test as outlined by CASALS (1967). Reference
Table I-Isolation November, 1977
of RVF
virus
from
human
and sheep serum
Type of disease
No. of isolates/ No. of samples
Location
Date collected
Zagazig Sharqiya
l-7 October
Acute febrile and/or fatal haemorrhagic
39/51
Sharqiya Qalyubiya Giza
l-22 November
Acute febrile
11/33
samples
Titre of virus* in sera 4-2
Sharqiya
J l-7 November
Fatal haemorrhagic
417
Aborting
214
sheep
* Titre = Suckling mouse intracerebral LD 50/ml ** Range based on 4 samples titred for each location-only
collected
10
No.
in OctoberRe-isolation/ of Attempts
8.1** -10
6.1 10 -10 4.1 10 -10
15/15 8.6 7.2
8.9 10.0 10 +10
two sheep sera were titred
313 414 l/l
621
J. M. MEEGAN Zagazig fever hospital, the Abbassia fever hospital and NAMRU-3, and 15 isolates were derived from the samples (Table I). Two virus isolates were selected as prototypes: one (Zagazig 548) from a 52-year-old male patient with the acute febrile illness and a second (Zagazig 501) from a 12-yearold Zagazig female who died of the haemorrhagiclike disease. These isolates reacted positively in the HI and CF tests onlv with antisera to RVF virus. Mouse neutralization (N) tests were performed according to the procedures of CASALS (1967), and log neutralization indices of greater than three were obtained in cross-neutralization tests between Z-501, Z-548 and RVF virus Entebbe strain (provided by the U.S. Army). Convalescent serum samples from six patients with the acute febrile disease showed greater than four-fold rises in CF and HI antibodies to Z-501 antigen. Paired acute and convalescent samples collected from 10 patients displaying an encephalitic disease (see LAUGHLIN et al.,-pp. 630-633) revealed greater than four-fold rises in CF and HI antibodv &res to Z-501. Paired samples from two patients displaying an ocular disease- also exhibited-a fourfold or ereater rise in HI and CF antibodv titres to Z-~O~-(SIAM & MEEGAN, 1980). Since RVF virus appears to be similar to the Bunyaviridae, and because Bunyaviruses have a segmented genome (OBIJESKI & MURPHY, 1977) which can reassort to produce genetic recombinants (GENTSCH & BISHOP, 1976), the increased incidence of human complications observed during the epidemic possibly resulted from a recombined strain of RVF virus. Preliminary comparative investigations are in progress, and initial results from this laboratory and from the laboratory of Dr. C. J. Peters (U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland), indicate that the Zagazig strain (Z-501) is significantly more pathogenic in certain laboratory animals than previous isolates of RVF virus from Sub-Saharan Africa. A retrospective serological survey is now in progress on stored human sera collected from 1971 to 1976 from the Qalyubiya Governorate and from the greater Cairo area. Preliminary results from 500 samples tested for HI antibodies to RVF virus have revealed no evidence of RVF endemicity in Eevot before the 1977 eoidemic. A similar survey of over 5,000 domestic animal sera from throughout Egypt also has revealed no evidence of antibodies to RVF virus before 1977 (Dr. Medhat Darwish, personal communication). Thus> it is most probable that the virus was introduced during 1977, although the source of the introduction remains in question (see HOOGSTRAAL et al., pp. 624-629). In an attempt to determine the extent of the epizootic during 1977, the NAMRU-3 Medical Zoology and Veterinary Medicine departments in collaboration with the Egyptian Ministry of Agriculture collected over 13,000 domestic animal sera for serological analysis from every major Governorate in Egypt during the winter months of 1977-78. The final HI test results have not been completed and will be reported in detail when finalized. However, Tables II and III show initial results from representative sheep and cattle samples selected for
Table II-Summary of domestic animals seropositive for HI antibodies to RVF virus in Nile Delta Governorates 1977-78 Percentage positive (Total Sample)* Governorate
Sheep 23.2 37.0 52.0 0.0 2.8 0.0 1.1
Giza Qalyubiya Sharqiva Gharbiya Daqahliya Minufiya Beheira * Positive = Titre
(181) (540) (275) (252) (761) (295) (549)
Cattle 19.1 14-9 36.0 3.6 0.4 0.8 1 .o
(277) (377) (125) (307j (244) (259) (419)
3 l/40
Table III-Summary seropositive for HI Valley Governorates
of domestic animals antibodies to RVF Nile 1977-78 Percentage positive (Total Sample)*
Governorate Aswan Qena** Asyut Minya Beni Suef Faiyum
Sheep 32.0 l-5 40.5 9 *5 o-0 9-4
(301) (261) (178) (243) (179) (93)
Cattle 57.5 0.0 45.5 5.2 0.0 0.0
(134 (164 (11 (133 (133 (52
* Positive = Titre Z l/40 ** Excluding Isna which borders Aswan and was heavily infected testing from the larger collection, and indicate that five Governorates (Sharqiya, Qalyubiya, Giza, Asyut and Aswan) were heavily involved in the epizootic (Fig. 1). Other Governorates have evidence of local incursions but most of the animal populations lacked protective antibodies after the 1977 outbreak. Definitive demographic data on these areas of Egypt is not available but estimates are that the area involved in the epizootic has a human population of one to three million. Extensive epidemiological data could not be collected. Official Egyptian Government figures for the 1977 enidemic indicate morbiditv of about 18,000 and mortality of 598 (howe;er, other estimates of clinical cases range to greater than 200.000). About 150 blood samnles were able to be collected from two villages within the centre of the epidemic area in Sharqiya. A serological conversion rate of 359; was measured in the first 75 sera analysed by the HI test. No incidence rates for ocular and encephalitic disease were determined (anecdotal reports from local ophthalmologists indicate that about 800 ocular cases appeared to result from RVF virus infection). There-were few reoorts of clinical disease or death in Upper _* Egypt -_(southern Nile Valley).
622 Table
EGYPTIAN RVF EPIZOOTIC 1977-78. 1. DESCRIPTION AND VIROLOGICAL STUDIES
IV-Summary
Date
2 3~1~ 2 July 28 August 13 October
2 -Tulv_ 7 Tulv 26 September 11 November
of isolations
of RVF virus
Area
from
Egypt
during
1978
Area involved in 1977 Epizootic
Material
El Sawamil Sharqiya Asyut Aswan Sharqiya
+
Fakoos Sharqiya Minva Gharbiya Gharbiya
-
Human sera
-
Sheep sera Sheep sera Human sera
Recurrence and spread of RVF in 1978 The historv of RVF in Sub-Saharan Africa includes a series of epizootics interspread between enzootic periods of little or no virus circulation (DAVIES, -1975). The possible overwintering of RVF virus in Eevut. with the subseauent establishment of endemiziy,-was a viable concern considering that most animals and humans lacked antibodies. In early July, 1978, an increased incidence of human and animal disease was reported from villages near Belbeis (Fig. 1). The Upper Egypt Governorates of Minya and Asyut reported increases in abortions among sheep and cattle. Table IV records the history of our isolations and identifications of RVF virus from various areas of Egypt in 1978. The virus reappeared in villages which were involved in the 1977 outbreak and also spread to new villages and Governorates which were not involved in 1977. Isolations of virus continued until early December, although a restricted number of samples were able to be collected for virological studies. Virtually no accurate epidemiological data is available on the 1978 outbreak. In 1944, African horsesickness virus, which is transmitted by Culicaides spread from Sudan north to Egypt, and subsequently across the Suez canal on to the Asian continent (ALEXANDER, 1948). Considering this historical precedent, we obtained serum samples from United Nations (U.N.) troops stationed on the Sinai peninsula. A, recent collaborative study with Dr. B. Nichlasson of the Swedish U.N. Forces indicated that about 1”; of the Swedish battalion were exposed to RVF virus during their tour of duty in 1978. Although numerous implications can be drawn from these preliminarv data (see Discussion), the additional fact that a -local inhabitant (who had never travelled outside the Sinai) also had serological evidence of RVF makes it highly probable tha
ROYAL
SOCIETY
OF TROPICAL
Ordinary Manson
House,
Thursday,
MEDICINE
AND
HYGIENE
Meeting 14th December,
1978
The President: DR. S. G. BROWNE, CMG, OBE, &ID, FRCP, DTM,
Symposium
on Rift Valley
FKC, in the Chair
Fever
The Rift Valley fever epizootic in Egypt 1977-78 1. Description of the epizootic and virological studies JAMES M. MEEGAN U.S. Naval Medical Research Unit No. 3 (NAMRU-3) U.S. Embassy, Cairo, Arab Republic of Egypt
Summary From October to December 1977, an extensive eDizootic occurred in Egypt resulting in abortions and increased mortality m domestic animals, and severe clinical disease with fatalities in man. Rift Valley Fever (RVF) virus was isolated and identified as the causative agent. In humans, acute febrile, encephalitic, ocular and fatal haemorrhagic diseases were documented as resulting from RVF virus infection. A retrospective serological survey indicated RVF was recently introduced into the area. The 1977 eoizootic extensively involved five Governorates.-In the summer and autumn of 1978, epizootic RVF reappeared in Egypt and spread to nreviouslv uninfected areas. Virological, serological and epidimiological studies, and factors related to the spread of RVF are discussed. Introduction et al. (1931) studied an epizootic disease in the Rift Valley of Kenya which primarily affected sheep and resulted in an increased incidence of abortion in ewes and of mortality in lambs. The investigation revealed: (a) the causative agent was a virus, most probably arthropod-borne; (b) the fatal form of the disease occurred predominantly among young sheep, goats and cattle, with the characteristic pathological lesion being focal liver necrosis; and (c) man could be infected by the virus, but exhibited only a mild febrile illness. The name Rift Valley fever (RVF) was suggested, and discussions with local inhabitants indicated RVF had most probably been present in the area for at least 20 years. In the 48 years since this initial study, comparatively little data have been accumulated about this viral disease. Most investigations have been concerned with the pathology of the animal disease. They revealed that a wide variety of wild, domestic and laboratory animals are susceptible to RVF virus (WEISS, 1957). Characteristically, a short incubation period was followed by a definite febrile phase, In
1930,
DAUBNEY
development of focal liver necrosis and death, frequently associated with haemorrhagic signs. The SMITHBURN et al. (1948) &tomological studies in Uganda established RVF as an arthrooodborne virus-by isolating six strains of virus irom species of two genera of mosquitoes. Since then, approximately 18 mosquito species have been implicated as possible RVF virus vectors (GEAR et al., 1955; STEYN & SCHULZ, 1955; KOKERNOT et al., 1957; WEINBREN et al., 1957; WILLIAMS, et al., 1960). Due to this wide vertebrate and invertebrate host range, the virus has circulated in a number of ecological settings. Major epizootics occurred in 1931 in Kenya, in 1951 and 1975 in South Africa (GEAR, 1951; VAN VELDEN et al., 1977), and.1975 in the Sudan (EISA et al., 1977). The virus was also isolated from various sources during smaller outbreaks in Nigeria, Uganda and Rhodesia, and serological surveys have detected antibodies to RVF virus in a number of southern African nations (EASTERDAY, 1965). However, until 1977 the virus was geographically limited to Sub-Saharan Africa. In most epizootics, small numbers of human infections were documented and no fatalities were directly ascribable to RVF. Retinitis was the only complication suggested to result from this otherwise benign human febrile illness (SCHRIRE, 1951; FREED, 1951; COHEN & LUNTZ, 1976). This ocular syndrome remained questionable since definitive diagnosis was not established by virus isolation or serological conversion in paired serum samples. Recently, however, VAN VELDEN et al. (1977) documented that RVF virus infection could result in acute clinical complications by confirming four human RVF fatalities and 12 cases of encephalitis among a small number of individuals infected during the 1975 South African epizootic. Considering this history, one understands why the sudden spread of RVF to Egypt was unexpected and why an extensive epidemic of an unprecedented disease with severe clinical manifestations and numerous fatalities represented an enigma.
619
J. M. MEEGAN
MEDITERRANEAN
SEA
.FAOUS ~* ‘.
/
‘,
..
NILE DELTA I- GIZA
,. ; ,I ,’ ,*’ 1 I: : : : I \ ,SNA’i“‘.. c
NILE VALLEY IOII 12, 12, 13, 14, IS16,
‘.,’
.:._ 1 ‘.
GOV.
BENI SUEF FAYUM YINYA ASYUT SOHAG OENA ASWAN
“I’--’
t t : : \ , ,: i i ,’ : ‘, : ASWAN 1;
GOV.
2-OALYUBIYA 3-SHAROIYA 4- MINUFIYA 3- GHARBIYA 6 - DAOAHLIYA 7, KAFR EL SHEIKH 6- BEHEIRA S-DAMIETTA
1 Fig. 1. Map outlining
major Governorates
in Egypt.
Description of the epizootic During the first week of October, 1977, an increased incidence of acute febrile disease and a fatal haemorrhagic-like illness were reported from the Inshas (also known as Basateen-el-Ismailiya) group of villages (population approximately 10,000) in Sharqiya Governorate, 70 km north-east of Cairo (Fig. 1). The villages are situated in a heavily irrigated agricultural area planted with peanuts, fruit trees and rice. The principal water supply is the Ismailiya canal running from the Nile near Cairo north-east along the south-eastern border of the Nile Delta. During the second and third weeks of October,
additional human cases were reported in many villages along the canal, both to the north near Zagazig, and to the south near El Khanka in Qalyubiya Governorate (Fig. 1). At this time, a focal outbreak of human cases occurred in the Imbaba area of Giza Governorate, which borders Cairo on the west. However, Cairo itself was not involved in the epidemic. During October, the Egyptian Ministry of Agriculture received a number of reports from the epidemic area documenting an increased incidence of mortality and abortion in sheep, cattle, domestic buffalo and camels. In retrospect, it appeared that animal disease had been occurring in the epidemic
620
EGYPTIAN RVF EPIZOOTIC 1977-78.
1 DESCRIPTION AND VIROLOGICAL STUDIES
area since September, and to the south in Aswan Governorate since late August (MEEGAN et al., 1979). The Egyptian Government responded with aerial insecticiding using varying concentrations of malathion. However, no study was made of the effectiveness of this effort. The predominant species of mosquito in the area is C&x pip&s (see HOOGSTRAAL et al., pp. 624-629), and ‘the unusually long summer of 1977 might have increased the mosquito population densities, although this cannot be documented. Reports of elevated rates of human febrile illness and death in these areas continued through November and the first two weeks of December. Clinics and hospitals also observed an increased incidence of encephalitic and ocular disease. With the onset of the colder winter weather in mid-December (temperatures range from 2°C to 24°C from December to March) and the concomitant decrease in mosquito population densities, reports of human and animal disease subsided.
antigens were loo/ mouse brain suspensions prepared in buffered physiological saline. Arthropodborne viral antigens from 12 serological groups were selected for testing, and represented viruses present in Egypt and others which could cause fatal human disease. No pattern of serological conversion could be detected with these antigens. Concurrently, virus isolation was attempted by intracerebral inoculation (i.c.) of undiluted sera into two-day-old suckling mice. The original serum samples yielded 39 viral isolates which were lethal in one and a half to two days in suckling mice (Table I). Five isolates were passaged twice in suckling mice and a virus stock was prepared from a 10% brain suspension. These isolates were lethal in two to four days for adult mice, adult hamsters and rats when inoculated by the i.c., subcutaneous or intraperitoneal routes. Histopathological examination of the experimental animals revealed focal liver necrosis. The isolates were sensitive to sodium deoxycholate and ether, and a sucrose-acetone virus preparation (CASALS, 1967) haemagglutinated goose erythrocytes at a pH of 6.3 at 37°C. One isolate (NAMRU-Z41) was diluted 1:4 and tested by CF against mouse hyperimmune antisera from 12 serological groups of arthropod-borne viruses (cross reactions within groups allowed screening of approximately 300 viruses). All tests were negative (antisera to RVF virus were not available in Egypt at that time). Isolate 241 was then tested at the Yale Arbovirus Research Unit (World Health Organization Arbovirus Reference Center) against a larger number of reference antisera. In collaboration with Drs. J. Casals, R. Shope, J. Converse and 0. Wood, the isolate was identified as Rift Valley fever virus by the CF and haemagglutination-inhibition (HI) tests (MEEGAN et al., in preparation). Electron microscopic studies of infected Vero cell cultures showed a spherical viral particle, 100 nm in diameter and similar in morphology to Bunyamwera-like viruses. An additional 40 acute sera were collected in November from patients examined in detail at the
Virological and serological studies On 14th October, Dr. Imam Z. Imam of the Egyptian Ministry of Public Health provided NAMRU-3 with 66 acute and five convalescent serum samples from patients seen at hospitals in the Zagazig area (Sharqiya Governorate) and displaying both the acute febrile and fatal haemorrhagic-like disease (the condition and amounts of the samples permitted virological studies on only 51 sera; the sample numbering system to identify corresponding clinical disease states was lost during transport). Extensive bacteriological tests disclosed no pathogen. The clinical symptoms of the acute febrile disease (including fever, headache, joint pain and severe lower back pain) suggested a viral aetiology. Consequently, convalescent samples were analysed with the complement fixation (CF) test to determine serological conversion to a number of reference viral antigens. Sera were heat inactivated (56°C for 30 min), diluted (starting at 1:4), and tested in the CF test as outlined by CASALS (1967). Reference
Table I-Isolation November, 1977
of RVF
virus
from
human
and sheep serum
Type of disease
No. of isolates/ No. of samples
Location
Date collected
Zagazig Sharqiya
l-7 October
Acute febrile and/or fatal haemorrhagic
39/51
Sharqiya Qalyubiya Giza
l-22 November
Acute febrile
11/33
samples
Titre of virus* in sera 4-2
Sharqiya
J l-7 November
Fatal haemorrhagic
417
Aborting
214
sheep
* Titre = Suckling mouse intracerebral LD 50/ml ** Range based on 4 samples titred for each location-only
collected
10
No.
in OctoberRe-isolation/ of Attempts
8.1** -10
6.1 10 -10 4.1 10 -10
15/15 8.6 7.2
8.9 10.0 10 +10
two sheep sera were titred
313 414 l/l
621
J. M. MEEGAN Zagazig fever hospital, the Abbassia fever hospital and NAMRU-3, and 15 isolates were derived from the samples (Table I). Two virus isolates were selected as prototypes: one (Zagazig 548) from a 52-year-old male patient with the acute febrile illness and a second (Zagazig 501) from a 12-yearold Zagazig female who died of the haemorrhagiclike disease. These isolates reacted positively in the HI and CF tests onlv with antisera to RVF virus. Mouse neutralization (N) tests were performed according to the procedures of CASALS (1967), and log neutralization indices of greater than three were obtained in cross-neutralization tests between Z-501, Z-548 and RVF virus Entebbe strain (provided by the U.S. Army). Convalescent serum samples from six patients with the acute febrile disease showed greater than four-fold rises in CF and HI antibodies to Z-501 antigen. Paired acute and convalescent samples collected from 10 patients displaying an encephalitic disease (see LAUGHLIN et al.,-pp. 630-633) revealed greater than four-fold rises in CF and HI antibodv &res to Z-501. Paired samples from two patients displaying an ocular disease- also exhibited-a fourfold or ereater rise in HI and CF antibodv titres to Z-~O~-(SIAM & MEEGAN, 1980). Since RVF virus appears to be similar to the Bunyaviridae, and because Bunyaviruses have a segmented genome (OBIJESKI & MURPHY, 1977) which can reassort to produce genetic recombinants (GENTSCH & BISHOP, 1976), the increased incidence of human complications observed during the epidemic possibly resulted from a recombined strain of RVF virus. Preliminary comparative investigations are in progress, and initial results from this laboratory and from the laboratory of Dr. C. J. Peters (U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland), indicate that the Zagazig strain (Z-501) is significantly more pathogenic in certain laboratory animals than previous isolates of RVF virus from Sub-Saharan Africa. A retrospective serological survey is now in progress on stored human sera collected from 1971 to 1976 from the Qalyubiya Governorate and from the greater Cairo area. Preliminary results from 500 samples tested for HI antibodies to RVF virus have revealed no evidence of RVF endemicity in Eevot before the 1977 eoidemic. A similar survey of over 5,000 domestic animal sera from throughout Egypt also has revealed no evidence of antibodies to RVF virus before 1977 (Dr. Medhat Darwish, personal communication). Thus> it is most probable that the virus was introduced during 1977, although the source of the introduction remains in question (see HOOGSTRAAL et al., pp. 624-629). In an attempt to determine the extent of the epizootic during 1977, the NAMRU-3 Medical Zoology and Veterinary Medicine departments in collaboration with the Egyptian Ministry of Agriculture collected over 13,000 domestic animal sera for serological analysis from every major Governorate in Egypt during the winter months of 1977-78. The final HI test results have not been completed and will be reported in detail when finalized. However, Tables II and III show initial results from representative sheep and cattle samples selected for
Table II-Summary of domestic animals seropositive for HI antibodies to RVF virus in Nile Delta Governorates 1977-78 Percentage positive (Total Sample)* Governorate
Sheep 23.2 37.0 52.0 0.0 2.8 0.0 1.1
Giza Qalyubiya Sharqiva Gharbiya Daqahliya Minufiya Beheira * Positive = Titre
(181) (540) (275) (252) (761) (295) (549)
Cattle 19.1 14-9 36.0 3.6 0.4 0.8 1 .o
(277) (377) (125) (307j (244) (259) (419)
3 l/40
Table III-Summary seropositive for HI Valley Governorates
of domestic animals antibodies to RVF Nile 1977-78 Percentage positive (Total Sample)*
Governorate Aswan Qena** Asyut Minya Beni Suef Faiyum
Sheep 32.0 l-5 40.5 9 *5 o-0 9-4
(301) (261) (178) (243) (179) (93)
Cattle 57.5 0.0 45.5 5.2 0.0 0.0
(134 (164 (11 (133 (133 (52
* Positive = Titre Z l/40 ** Excluding Isna which borders Aswan and was heavily infected testing from the larger collection, and indicate that five Governorates (Sharqiya, Qalyubiya, Giza, Asyut and Aswan) were heavily involved in the epizootic (Fig. 1). Other Governorates have evidence of local incursions but most of the animal populations lacked protective antibodies after the 1977 outbreak. Definitive demographic data on these areas of Egypt is not available but estimates are that the area involved in the epizootic has a human population of one to three million. Extensive epidemiological data could not be collected. Official Egyptian Government figures for the 1977 enidemic indicate morbiditv of about 18,000 and mortality of 598 (howe;er, other estimates of clinical cases range to greater than 200.000). About 150 blood samnles were able to be collected from two villages within the centre of the epidemic area in Sharqiya. A serological conversion rate of 359; was measured in the first 75 sera analysed by the HI test. No incidence rates for ocular and encephalitic disease were determined (anecdotal reports from local ophthalmologists indicate that about 800 ocular cases appeared to result from RVF virus infection). There-were few reoorts of clinical disease or death in Upper _* Egypt -_(southern Nile Valley).
622 Table
EGYPTIAN RVF EPIZOOTIC 1977-78. 1. DESCRIPTION AND VIROLOGICAL STUDIES
IV-Summary
Date
2 3~1~ 2 July 28 August 13 October
2 -Tulv_ 7 Tulv 26 September 11 November
of isolations
of RVF virus
Area
from
Egypt
during
1978
Area involved in 1977 Epizootic
Material
El Sawamil Sharqiya Asyut Aswan Sharqiya
+
Fakoos Sharqiya Minva Gharbiya Gharbiya
-
Human sera
-
Sheep sera Sheep sera Human sera
Recurrence and spread of RVF in 1978 The historv of RVF in Sub-Saharan Africa includes a series of epizootics interspread between enzootic periods of little or no virus circulation (DAVIES, -1975). The possible overwintering of RVF virus in Eevut. with the subseauent establishment of endemiziy,-was a viable concern considering that most animals and humans lacked antibodies. In early July, 1978, an increased incidence of human and animal disease was reported from villages near Belbeis (Fig. 1). The Upper Egypt Governorates of Minya and Asyut reported increases in abortions among sheep and cattle. Table IV records the history of our isolations and identifications of RVF virus from various areas of Egypt in 1978. The virus reappeared in villages which were involved in the 1977 outbreak and also spread to new villages and Governorates which were not involved in 1977. Isolations of virus continued until early December, although a restricted number of samples were able to be collected for virological studies. Virtually no accurate epidemiological data is available on the 1978 outbreak. In 1944, African horsesickness virus, which is transmitted by Culicaides spread from Sudan north to Egypt, and subsequently across the Suez canal on to the Asian continent (ALEXANDER, 1948). Considering this historical precedent, we obtained serum samples from United Nations (U.N.) troops stationed on the Sinai peninsula. A, recent collaborative study with Dr. B. Nichlasson of the Swedish U.N. Forces indicated that about 1”; of the Swedish battalion were exposed to RVF virus during their tour of duty in 1978. Although numerous implications can be drawn from these preliminarv data (see Discussion), the additional fact that a -local inhabitant (who had never travelled outside the Sinai) also had serological evidence of RVF makes it highly probable tha
