Emerg Infect DisEmerging Infect. DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention22172568331118911-103510.3201/eid1712.111035ResearchMolecular Epidemiology of Rift Valley Fever VirusMolecular Epidemiology of Rift Valley Fever VirusGrobbelaarAntoinette A.WeyerJacquelineLemanPatricia A.KempAlanPaweskaJanusz T.SwanepoelRobertNational Institute for Communicable Diseases of the National Health Service, Sandringham, South Africa (A.A. Grobbelaar, J. Weyer, P.A Leman, A. Kemp, J.T. Paweska, R. Swanepoel);University of Pretoria, Pretoria, South Africa (R. Swanepoel)Address for correspondence: Robert Swanepoel, Pathology Bldg, Rm 2-72, University of Pretoria, Prinshof Campus, PO Box 2034, Pretoria 0001, South Africa; email: bob.swanepoel@up.ac.za122011171222702276

Large-scale vaccination of animals might have influenced virus evolution.

Phylogenetic relationships were examined for 198 Rift Valley fever virus isolates and 5 derived strains obtained from various sources in Saudi Arabia and 16 countries in Africa during a 67-year period (1944–2010). A maximum-likelihood tree prepared with sequence data for a 490-nt section of the Gn glycoprotein gene showed that 95 unique sequences sorted into 15 lineages. A 2010 isolate from a patient in South Africa potentially exposed to co-infection with live animal vaccine and wild virus was a reassortant. The potential influence of large-scale use of live animal vaccine on evolution of Rift Valley fever virus is discussed.

Keywords: Rift Valley feverRift Valley fever virusvirusesmolecular epidemiologyphylogeneticsevolutionAfricaSaudi Arabia

Rift Valley fever (RVF) is an acute disease of domestic ruminants in Africa and the Arabian Peninsula. This disease is caused by a mosquito-borne virus of the family Bunyaviridae and genus Phlebovirus. Large outbreaks occur at irregular intervals when heavy rains favor breeding of mosquito vectors of the virus and are characterized by deaths of newborn animals and abortion in pregnant sheep, goats, and cattle. Humans become infected by contact with tissues of infected animals or from mosquito bites and usually show development of mildly to moderately severe febrile illness. However, severe complications, including ocular sequelae, encephalitis, and fatal hemorrhagic disease, occur in some patients (1).

Rift Valley fever virus (RVFV) has a negative-sense, single-stranded RNA genome comprising large (L), medium (M), and small (S) segments. The L segment encodes viral RNA polymerase. The M segment encodes envelope glycoproteins Gc and Gn, a nonstructural protein, and a 78-kDa fusion protein of nonstructural and Gn proteins. The S segment shows an ambisense strategy and encodes nucleocapsid protein N and a nonstructural protein (2).

Early genetic analysis involved nucleotide sequencing of M segment RNA fragments encoding glycoprotein that induced neutralizing antibody response in 22 isolates obtained over 34 years in 6 countries (3). This analysis showed remarkable stability of sections of the genome expected to be under greatest immune selection pressure. The diversity observed in isolates from Zimbabwe indicated that outbreaks do not invariably involve a single genotype of virus but can result from intensified transmission of multiple strains already circulating in RVF-endemic areas. Nevertheless, neutralization of isolates with monoclonal antibodies confirmed that a single vaccine should suffice to control the disease (3). Slightly greater variability was observed among isolates when nonstructural protein sequence data were analyzed (4). Partial sequences determined for all 3 RNA segments of the genome of 20 isolates sorted into 3 geographically linked lineages associated with western Africa, Egypt, and eastcentral Africa and showed evidence of reassortment of genome segments between some sub-Saharan isolates (5).

High-throughput technology facilitated whole-genome sequence analysis of 33 isolates and resulted in confirmation of low genetic diversity of the virus and separation of the isolates into 7 lineages. There was no mutually exclusive correlation between genotype and geographic origin; representatives of geographic areas tended to cluster, but isolates from distant locations occurred in each lineage, indicating continuous widespread dispersal of virus (6). The remarkable congruence of phylogenetic trees for the 3 genome segments suggested that reassortment was not common, but convergence of some lineages within genome segments implied that reassortment had played an evolutionary role in the history of RVFV. Bayesian analysis suggested that the time of divergence of RVFV isolates from a most recent common ancestor dated to 1880–1890, the colonial period when the introduction of large concentrations of susceptible sheep and cattle would have facilitated exploitation of a new niche by an unknown progenitor virus. The evolutionary rate of the virus was similar to that of other RNA viruses. Thus, low nucleotide diversity probably relates to recent derivation from a common ancestor rather than stability of the genome.

Whole-genome sequencing and Bayesian analysis of 31 isolates associated with the 2006–2007 outbreak of RVF in Kenya showed that 2 sublineages of virus had evolved separately before or during a large outbreak during 1997–1998, with continued expansion of 1 sublineage dating from 2–4 years before 2006, confirming that outbreaks in disease-endemic areas might be associated with multiple lineages of virus, and that virus activity and evolution can occur below the threshold of detection by public health or animal authorities during interepidemic periods (7). Other genetic studies have been more limited in scope and concerned with either locating and investigating mechanisms of pathogenicity or determining phylogenetic relationships of isolates involved in particular outbreaks (814).

Analysis of partial M segment sequence data for a large collection of isolates and derived strains obtained from various sources in Saudi Arabia and 16 countries in Africa during 1944–2010 showed phylogenetic relationships not apparent in studies involving a smaller range of isolates. A 2010 isolate from a patient in South Africa potentially exposed to co-infection with live animal vaccine and wild virus from a needle injury while vaccinating sheep plus selected other isolates were subjected to limited sequencing of all 3 segments of the genome to obtain evidence of reassortment. We present and discuss the epidemiologic implications of these findings.

Materials and MethodsViruses

Sequence data for 33 viruses were obtained from GenBank. The remaining 170 viruses for which we determined partial nucleotide sequences were obtained from the various institutions (Table A1).

RNA Extraction

Viral RNA was extracted directly from 140 μL of infected human or livestock serum, clarified 10% organ suspensions, reconstituted freeze-dried mouse brain suspensions, or Vero cell culture supernatant fluids. Extraction was performed by using a QIAamp Viral RNA Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions.

Reverse Transcription PCR and Nucleotide Sequencing of PCR Products

A 10-μL RNA aliquot was analyzed by using reverse transcription PCR with the Titan One Tube RT-PCR Kit (Roche Diagnostics, Penzburg, Germany) in a final volume of 50 μL as described (15). The forward primer FD1 (771/5′-CCAAATGACTACCAGTCAGC-3′/790) (3) and the reverse primer RVF E (1342/5′-CCTGACCCATTAGCATG-3′/1326) were selected to amplify a portion of the Gn glycoprotein gene of the virus; the primer positions correspond to the viral complementary DNA sequence of the M segment of the ZH501 human RVF virus isolate (16).

Amplicons were purified by using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Nucleotide sequences of PCR products were determined by using BigDye version 3.1 Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. For confirmatory purposes, sequences were obtained for both strands of PCR products by using primers FD1 and RVF E. Products were purified by using CentriSep spin columns (Princeton Separations Inc., Adelphia, NJ, USA) and analyzed by using a 377 GenAmp Sequencer and a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

On the basis of findings for M segment data, partial sequences were determined for S and L RNA segments of isolate SA184/10 from a patient potentially co-infected with live animal vaccine and wild virus; isolate SA54/10 from another patient in the same area; the batch of vaccine used (Smithburn neurotropic strain [SNS] 105/2010); SNS vaccine master seed virus; 95EG vaccine; and historical isolates 95EG Cow-2509, H1739, and H1825 (Table A1) to test for reassortment by using specifically designed primers. Primers F1 (1/5′-ACACAAAGACCCCCTAGTGC-3′/20) and R4 (1690/5′-ACACAAAGCTCCCTAGAGATAC-3′/1669) were used to amplify the S segment and the N gene (735 nt). Primers RVFL10 (4237/5′-GGTGTTGTGTCATCATTG-3′/4254) and L2 (4730/5′-GTGTGAGCTAGAGTTGCTTC-3′/4711) were used to amplify a 494-nt region of the L segment.

Sequence Analysis

Nucleotide and amino acid sequence alignments were generated by using ClustalW Multiple Alignment analysis software as implemented in BioEdit version 7.0.5.3 (17). Unique sequences generated in the study were submitted to GenBank and assigned accession numbers indicated in Table A1. Preliminary phylogenetic analysis was performed by using a neighbor-joining distance method in MEGA4 that applied a Jukes-Cantor model under 1,000 bootstrap iterations (18). Sequence divergence was determined by using MEGA4 to calculate mean pairwise distances within groups. A phylogenetic tree was constructed for 95 isolates exhibiting unique sequences and for 16 isolates that exhibited duplicate sequences but that were isolated in different years or countries, by using the maximum-likelihood method in PAUP* version 4.0b2 (Sinauer Associates, Inc., Sunderland, MA, USA).

Further analysis of partial M segment sequence data was performed by using the Bayesian software package, which included BEAST, BEAUTI, Tracer, TreeAnnotator, and FigTree (19), by using a Markov Chain Monte Carlo chain length of 3.0 × 107, a 3.0 × 106 burn in, a generalized time reversible plus gamma plus invariant nucleotide substitution model, a relaxed uncorrelated logarithmic normal molecular clock, and sampling every 1,000 states.

To check for recombination events within the RVFV alignment, the alignment was examined by using a set of 6 detection methods implemented in RDP3 (20). Partial nucleotide sequence data for S, M, and L RNA segments of 33 isolates for which whole-genome sequences have been reported (6) (all included in Table A1) were obtained from GenBank and used with sequences determined in the present study for 5 isolates plus the 3 animal vaccine viruses to perform analyses as described above to test whether genetic reassortment had occurred.

Results

Diversity of partial M segment sequences was low; pairwise differences ranged from 0% to 5.4% for nucleotides and from 0% to 2.8% for deduced amino acids. These values are similar to those identified for 33 whole-genome sequences (6). Neighbor-joining, maximum-likelihood, and Bayesian phylogenies were similar. Bayesian analysis indicated that divergence of isolates included in the study from a most recent common ancestor dated to 1892, which is similar to the estimate of 1880–1890 deduced earlier from 33 whole-genome sequences. However, posterior support values for the tree nodes were weak because a small nucleotide segment was analyzed. Despite low genetic diversity, the 5 derived strains and 198 isolates of RVFV circulating over the past 67 years produced 95 unique sequences that resolved into 15 lineages (A–O) (Figure A1; Table A1) with mean pairwise distances <0.017 within lineages and bootstrap values >70% (21).

Five lineages (B, D, F, J, and O) contained single isolates, and lineage I contained 2 isolates from South Africa. Six other lineages (A, C, E, G, H and N) contained clusters of isolates associated with outbreaks in individual countries or regions, but each also included isolates from distant locations or separate outbreaks. Thus, lineage A comprised isolates from outbreaks in Zimbabwe in 1978, Madagascar in 1979, and Egypt in 1977–1978 and 1993 and a 1974 isolate from an interepidemic period in Zimbabwe (Figure A1; Table A1).

Lineage C was most widely distributed and contained isolates from major outbreaks in Zimbabwe in 1978, Madagascar in 1991, eastern Africa (Kenya, Tanzania, and Somalia) during 1997–1998, Saudi Arabia during 2000–2001, Kenya in 2007, and South Africa during 2008–2009 and lesser outbreaks and interepidemic periods in the same countries and Mauritania during 1976–2009. It was also associated with an isolated fatal infection in Angola in 1985 in a visitor from South Africa ostensibly exposed to mosquito bites (22). Lineage E, which was found in the Central African Republic and Zimbabwe during 1973–1978, was isolated from 2 patients referred to South Africa for treatment in 1985 after a relative had died of a similar disease in Zambia during an outbreak of RVF in livestock confirmed serologically (22,23). Lineage G contained isolates from the Central African Republic, Zimbabwe, Guinea, and Senegal during 1969–1986, and lineage H contained isolates from the 2009–2010 outbreak in South Africa and an apparent antecedent from Namibia in 2004. Lineage N, originally designated West African (5), contained isolates from Senegal, Burkina Faso, and Mauritania during 1975–1993.

Lineages K, L, and M with a common root node contained the SNS neurotropic and the KCS hepatotropic strains derived from the same mosquito isolate (24) and isolates from Kenya, Zimbabwe, South Africa, and Egypt during 1951–2010, countries that used the SNS animal vaccine on a large scale during major outbreaks of RVF. Isolate KEN57 Rintoul, obtained from a cow in Kenya in 1951, when the first batch of SNS vaccine was sent from South Africa to Kenya, had a partial M segment sequence identical to that of the vaccine virus. The SNS 105/2010 batch of vaccine and Egyptian 95EG vaccine also had the same sequence as the SNS vaccine master seed stock produced in 1987 (Table A1).

Isolate SA184/10, from a patient potentially exposed to wild virus and SNS 105/2010 animal vaccine, grouped with the parent vaccine strain in lineage K, albeit with a divergence of 11 (2.2%) of 490 nt (2 aa), and distantly from 46 other human isolates from the 2008–2010 outbreaks in South Africa. Moreover, isolate 95EG Cow-2509 from the fetus of a cow that aborted after the administration of 95EG vaccine in Egypt grouped in lineage L distantly from all other isolates from Egypt in lineage A (12) (Figure A1; Table A1). Isolates H1739 and H1825, which also grouped in lineage L, came from the first human deaths caused by RVF during a major outbreak in South Africa during 1974–1976 during which animal vaccine was used on an unprecedented scale (25; National Institute for Communicable Diseases [NICD], unpub data) (Figure 1). These 5 isolates were investigated for genetic reassortment.

Annual sales of Smithburn neurotropic strain animal vaccine produced in South Africa in relation to cumulative viral lineages isolated and human deaths in major outbreaks of Rift Valley fever (RVF) in Africa and Saudi Arabia, 1944–2010. Broken arrows indicate RVF outbreaks without human deaths recorded, and solid arrows indicate RVF outbreaks with human deaths. RSA, Republic of South Africa; NAM, Namibia; ZIM, Zimbabwe; MOZ, Mozambique; KEN, Kenya; EGY, Egypt; SUD, Sudan; ZAM, Zambia; MAU, Mauritania; MAD, Madagascar; TAN, Tanzania; SOM, Somalia; SAU, Saudi Arabia; YEM, Yemen.

Partial nucleotide sequence data for S, M, and L RNA segments derived from whole-genome sequences of 33 isolates produced phylogenetic trees with the same topologies as those published for the complete sequences (6). Isolate SA184/10 from the patient who sustained a needle injury while vaccinating sheep sorted with SNS vaccine virus in the M segment tree but with isolate SA54/10 in the other segments, and was clearly a reassortant. SNS 105/2010 and 95EG batches of vaccine corresponded with the SNS master seed stock for all 3 segments of the genome. Analysis of isolates H1739 and H1825 obtained from the first recorded human deaths caused by RVF in 1975 and isolate 95EG Cow-2509 from Egypt did not show clear-cut evidence of reassortment but fell into a group that manifested convergence of lineages D and E in the M RNA segment tree in the 33 whole-genome study, corresponding to lineages L plus K, the vaccine lineage, in the present study. This phenomenon was interpreted as evidence of historical reassortment (6). Isolate SA75 also ostensibly came from a human infected in South Africa in 1975 (6), but we could not relate the designation to records at NICD. Analysis of partial M segment sequence data did not show evidence of recombination.

Discussion

Historical developments in RVF include spread beyond sub-Saharan Africa during 1977–2007 and fatal human infections during a large outbreak in South Africa during 1974–1976. Since that time, large outbreaks of the disease in livestock have invariably been associated with human deaths (1). Mechanisms for dispersal of RVFV fall beyond the scope of this report. However, refinements to phylogenetics, such as Bayesian-based and population-based genetic analysis, have shown that translocated virus does not necessarily arrive in receptive circumstances to trigger epidemics, but can initiate smoldering infection or seeding of the ground, which remains undetected until suitable climatic conditions precipitate outbreaks, as occurred ahead of the 2000–2001 and 2006–2007 outbreaks in the Arabian Peninsula and Kenya (6,7).

Recent outbreaks in South Africa showed an analogous pattern. After heavy rains in 2008, lineage C virus, which had been isolated during a limited disease outbreak in Kruger National Park in 1999, was associated with scattered outbreaks of RVF in adjacent parts of northeastern South Africa. In the first half of 2009, the same lineage caused limited outbreaks to the south in KwaZulu-Natal Province. In the second half of 2009, lineage H virus, which had been encountered in the Caprivi Strip of Namibia in 2004, caused focal outbreaks in Northern Cape Province and was progressively identified in coalescing outbreaks over much of interior South Africa in 2010 (Figure 2). Diversity of genotypes observed among recent isolates of the 2 lineages (Figure A1), and results of our limited Bayesian analysis imply that RVFV had progressively reinfiltrated the interior plateau of South Africa during a period of increasing rainfall, 3 decades after the major outbreak of 1974–1976. There were 26 deaths among 244 persons infected with lineage H virus, and while no deaths were recorded in areas where lineage C virus was active, only 22 cases were diagnosed (NICD, unpub. data).

Recent outbreaks of Rift Valley fever in South Africa. Lineage C virus (yellow areas), which caused a small outbreak in Kruger National Park in 1999, was associated with scattered outbreaks of disease in adjacent parts of northeastern South Africa in 2008 and limited outbreaks to the south in KwaZulu-Natal Province early in 2009. Lineage H virus (blue area), which was first encountered in the Caprivi Strip of Namibia in 2004, caused focal outbreaks in the Northern Cape Province late in 2009, and was associated with coalescing outbreaks over much of interior South Africa in 2010. Lines indicate province boundaries.

Reassortment between SNS vaccine virus and wild virus has implications for the safety of the vaccine and its possible role in evolution of RVFV. The SNS virus from Uganda (Table A1) was taken to mouse intracranial passage 102 and embryonated chicken egg passage 54 to produce avianized (decreased infectivity produced by repeated culture in chick embryo) animal vaccine in South Africa in 1951. After several adjustments, reversion was made in 1958 to SNS virus passed 103 times in mice only. Since 1971, the same virus has been propagated in BHK21 cells for preparation of freeze-dried vaccine (24,2629). In Kenya, animal vaccine was initially produced from avianized virus from South Africa, but since 1960, SNS virus has been used at mouse passage 106 (3032). SNS seed virus from South Africa was used to produce vaccine in Egypt in 1994, and South Africa and Egypt have used field isolates to produce inactivated animal vaccine (12,27,29). SNS vaccine is only partially attenuated, but accurate estimates for abortigenicity and teratogenicity were never determined because conflicting results were initially obtained in different sheep breeds and because pathogenicity varies with stage of pregnancy (26,33). However, there has been a tendency to regard risks associated with vaccine as acceptable in the face of an outbreak of RVF (34).

During 1951–1968, ≈1 million doses of SNS animal vaccine were sold in South Africa; sales were similar in Kenya (28,32). The vaccine was first used on a large scale in 1969–1970 when 6 million doses were issued, mainly to Zimbabwe, where a large outbreak occurred (29) (Figure 1). Sales decreased sharply by 1973 but increased to 22 million doses during the major outbreak in South Africa and Namibia during 1974–1976.

During the same period, 4.7 million doses of newly developed inactivated RVF vaccine and ≈14 million doses of Wesselsbron virus vaccine were sold (27,28,35). Wesselsbron is a mosquito-borne flavivirus initially thought to cause outbreaks analogous to RVF, and vaccine was produced by similar empirical attenuation of virus by intracranial passage in mice (33,35). Thus, >40 million doses of either vaccine were administered over 3 years in South Africa and Namibia, where the combined sheep, cattle, and goat population was <60 million. However, fewer than two thirds of the animals were in RVF-affected areas. Approximately 4.2 million doses of SNS vaccine were sold in 1977, much of it to South Africa, but also to Israel and Egypt. Approximately 3 million doses were sold in 1978–1979, mostly to Zimbabwe where an additional large outbreak of RVF had occurred. Subsequently sales remained <1 million doses per year until 1986, after which figures were no longer made public (28). However, substantial quantities of vaccine, mainly from South Africa, were used in eastern Africa and Saudi Arabia in recent years. In all locations except Israel and the Sinai Peninsula, where the threat of RVF was not proven, large numbers of animals were vaccinated only after onset of outbreaks (28,29).

Livestock vaccines are sold in multidose vials and commonly administered with automatic syringes and intermittent changes of needles. Vaccine is likely to be administered to some animals that are already infected. Thus, in view of intense viremia that occurs in RVF, serial transfer of wild virus by needle is a recognized hazard of vaccinating livestock during outbreaks (29). This vaccination likely resulted in co-infections with vaccine and wild virus, or even different wild genotypes, on multiple occasions over decades, particularly during 1969–1979, with an implied potential for the generation of recombinant and reassortant genotypes. Uptake and transmission of virus by vectors would have increased co-infections and potential for genetic interaction (36).

Although there is no corresponding evidence that reassortment or recombination occurred on a large scale, few isolates remain available from the 1969–1970 and 1974–1976 outbreaks in Zimbabwe and South Africa. Nevertheless, multiple new lineages were encountered during 1969–1981, particularly in Zimbabwe where intensive monitoring was instituted during 1971–1979 and where lineage A and C isolates were obtained (37) (Figure A1; Figure 1). Moreover, it can be deduced from reported Bayesian analysis (6) that emergence of new lineages and genotypes surged during this period. An alternative interpretation is that reassortment and recombination occur infrequently but that mutations result from as yet inadequately explored interactions during replication in co-infections or from the generation of neutralizing antibody–escape mutants (38).

Thus, the partial M segment nucleotide sequence of reassortant isolate SA184/10 differs from that of vaccine virus, and that of isolate 95EG Cow-2509 from the aborted fetus of a vaccinated cow differs even more markedly from that of vaccine virus and from all other known isolates from Egypt but has some relationship with isolates H1739 and H1825 in lineage L (Figure A1), which caused human deaths. The implication is that further investigation of evolutionary relationships in lineages K, L, and M are warranted, including whole-genome sequencing of additional isolates identified in the present study. It could be relevant that viruses attenuated through intracranial passage in mice may acquire new tissue tropisms and pathogenic properties (39,40).

New vaccines, some with natural or induced attenuating deletions or mutations in all 3 segments of the genome, are being developed (1). However, interactions of replication-competent strains with wild virus should be investigated, especially because some vaccines were deliberately modeled on viruses considered to be particularly virulent.

Suggested citation for this article: Grobbelaar AA, Weyer J, Leman PA, Kemp A, Paweska JT, Swanepoel R. Molecular epidemiology of Rift Valley fever virus. Emerg Infect Dis [serial on the Internet]. 2011 Dec [date cited]. http://dx.doi.org/10.3201/eid1712.111035

Acknowledgments

We thank J. Morvan, B.A. Botros, R.R. Arthur, F.G. Davies, H.C.W. Mbugua, G.H. Gerdes, C.A. Potgieter, C.J Peters, J.C. Morrill, and B.J. Erasmus for providing RVFV isolates and strains.

Virus isolates used for molecular epidemiologic analysis of Rift Valley fever virus*
IsolateYearCountrySourcePassageDonor†LineageGenBank accession no.
MgH8241979MadagascarHumanmic6, tc41AHM587040‡
MgAr8111979MadagascarAedes spp. mosquitoNA1AHM587041‡
ZH 5011977EgyptHumanmic2, tc3GenBankAM33072‡
94EG Tambul1994EgyptOvineNA2AHM587042‡
94EG Bahr1994EgyptBovineNA2ASeq as above
ZS63651979EgyptOvinemic2, tc2GenBankADQ 380205‡
ZH5481977EgyptHumanmic2, tc2GenBankAM33075‡
ZH17761978EgyptHumanmic2, tc2GenBankADQ 380203‡
ZM6571978EgyptCulex pipiens mosquitomic2, tc2GenBankADQ380204
ZC33491978EgyptBovinemic2, tc2GenBankADQ380206
93EG Abeer1993EgyptHumanmic1, tc22AHM587043‡
93EG Buffalo1993EgyptAsian buffalomic1, tc12ASeq as above
MP12§1977EgyptRVF isolate ZH548NAGenBankADQ380208‡
VRL2250/741974ZimbabweBovinemic3, tc2GenBankADQ 380209‡
VRL1516/781978ZimbabweOvinemic23AHM587044‡
VRL1750/781978ZimbabweOvinemic23ASeq as above
VRL1842/781978ZimbabweOvinemic23ASeq as above
S351972KenyaOvineNA4BHM587045‡
SPU52/99/11999South AfricaAfrican buffalomic25CHM587046‡
SPU52/99/51999South AfricaAfrican buffalomic25CHM587047‡
SPU52/99/21999South AfricaAfrican buffalomic25CHM587048‡
SPU52/99/31999South AfricaAfrican buffalomic25CHM587049‡
SPU52/99/41999South AfricaAfrican buffalomic25CSeq as above
SPU52/99/61999South AfricaAfrican buffalomic25CSeq as above
SPU50/99/11999South AfricaWaterbuckmic25CSeq as above
An2782000Saudi ArabiaOvine05CHM587050‡
Ar212182000Saudi ArabiaAe. vexans arabiensis mosquito05CSeq as above
Ar212192000Saudi ArabiaAe. vexans arabiensis mosquito05CSeq as above
Ar212202000Saudi ArabiaCx. tritaeniorhynus mosquito05CSeq as above
Ar212292000Saudi ArabiaAe. vexans arabiensis mosquito05CSeq as above
Ar212332000Saudi ArabiaAe. vexans arabiensis mosquito05CSeq as above
Ar212362000Saudi ArabiaAe. vexans arabiensis mosquito05CSeq as above
109112000Saudi ArabiaHumantc1GenBankCDQ 380197
SA01–13222001Saudi ArabiaAe. vexans arabiensis mosquitotc2GenBankCAF393745‡
SPU12/99/211998SomaliaGoat05CHM587051‡
SPU65/98/15981998TanzaniaHuman05CSeq as above
SPU65/98/13801998TanzaniaHuman05CSeq as above
SPU384/97/11997KenyaHuman05CHM587052‡
SPU384/97/51997KenyaHuman05CSeq as above
SPU384/97/201997KenyaHuman05CSeq as above
SPU384/97/241997KenyaHuman05CSeq as above
SPU2/98/11998KenyaHuman05CHM587053‡
SPU2/98/31998SomaliaHuman05CSeq as above
SPU12/98/21998SomaliaGoat05CSeq as above
VRL2241/981998ZimbabweBovinemic23CSeq as above
VRL2181/981998ZimbabweBovinemic23CSeq as above
VRL2104/981998ZimbabweOvinemic13CSeq as above
VRL2215/981998ZimbabweOvinemic23CSeq as above
VRL2413/981998ZimbabweBovinemic13CHM587054‡
SPU2/98/91998KenyaGoat05CHM587055‡
005231998KenyaHumantc2GenBankCDQ 380196‡
VRL1187/791979ZimbabweBovinemic33CHM587056‡
MgAn10021991MadagascarBovineNA1CHM587057‡
VRL1548/781978ZimbabweBovinemic23CHM587059‡
VRL2142/781978ZimbabweOvinemic23CSeq as above
VRL2354/781978ZimbabweBovinemic23CHM587058‡
MgAn9911991MadagascarBovineNA1CHM587060‡
MgAn9901991MadagascarBovineNA1CHM587061‡
SPU22/07/1182007KenyaHuman05CHM587062‡
SPU22/07/1252007KenyaHuman05CHM587063‡
SPU22/071292007KenyaHuman05CHM587064‡
SPU103/07/12007KenyaHuman05CSeq as above
SPU103/07/22007KenyaHuman05CSeq as above
SPU103/07/142007KenyaHuman05CSeq as above
SPU103/07/352007KenyaHuman05CSeq as above
H1MAU032003MauritaniaHuman0GenBankCEF160116‡
SPU86/092009South AfricaHuman05CHM587065‡
SPU89/092009South AfricaHuman05CSeq as above
SA72/092009South AfricaHuman05CSeq as above
SA77/092009South AfricaHuman05CSeq as above
SA152/082008South AfricaHuman05CHM587066‡
M84/082008South AfricaBovine05CSeq as above
M482008MadagascarBovineNAGenBankCHQ009512‡
H2MAU032003MauritaniaHuman0GenBankCEF160115‡
M37/082008South AfricaAfrican buffalo06CHM587067‡
SPU22/07/42007KenyaHuman05CHM587068‡
SPU22/07/1262007KenyaHuman05CSeq as above
SPU22/07/1202007KenyaHuman05CSeq as above
SPU22/07/121a2007KenyaHuman05CSeq as above
SPU22/07/121b2007KenyaHuman05CSeq as above
SA52/082008South AfricaHuman05CHM587069‡
J40/62008South AfricaAfrican buffalo06CSeq as above
J48/62008South AfricaAfrican buffalo06CSeq as above
N21312008South AfricaAfrican buffalo06CSeq as above
SPU204/851985AngolaHumanmic25CHM587076‡
B3091977KenyaBovineNA4CHM587070‡
VRL825/791979ZimbabweBovinemic33CHM587071‡
VRL1217/781978ZimbabweOvinemic23CHM587073‡
VRL845/781978ZimbabweBovinemic23CSeq as above
VRL2129/781978ZimbabweBovinemic23CSeq as above
VRL688/781978ZimbabweBovinemic13CSeq as above
VRL2051/761976ZimbabweHumanmic33CHM587072‡
214451983KenyaAe. mcintoshi mosquitoha1, tc17CHM587074‡
B11431977KenyaBovineNA4CHM587075‡
VRL1290/781978ZimbabweBovinemic23CHM587077‡
VRL1508/781978ZimbabweBovinemic23CSeq as above
73HB12301973CARHumanmic6, tc4GenBankDDQ 380221‡
VRL2140A/751975ZimbabweBovinemic23EHM587078‡
SPU44/851985ZambiaHumanmic1, tc35EHM587079‡
SPU45/851985ZambiaHumanmic1, tc35ESeq as above
VRL1887/781978ZimbabweBovinemic23EHM587080‡
VRL1905/781978ZimbabweBovinemic23ESeq as above
VRL3108/781978ZimbabweBovinemic23ESeq as above
VRL2230/781978ZimbabweBovinemic23ESeq as above
VRL1260/781978ZimbabweBovinemic23EHM587081‡
74HB591974CARHumanmic6, tc37EHM587082‡
73HB14491973CARHumanmic5, tc1GenBankEDQ 380211‡
Ar203641981South AfricaCx. zombaensis mosquitomic45FHM587101‡
Ar203681981South AfricaCx. zombaensis mosquitomic35FSeq as above
ArB1976 (Zinga)1969CARMansonia africana mosquitomic5, tc37GHM587083‡
AnK60871984GuineaMicropteropus pusillus batNAGenBankGAF134502‡
AnK38371981GuineaHipposideros caffer batmic7, tc17GHM587084‡
ArD1047691993SenegalAe. vexans arabiensis mosquitoNAGenBankGAF134499‡
VRL1032/781978ZimbabweBovinemic23GHM587085‡
VRL1853/781978ZimbabweBovinemic23G
HvB3751985CARHumanmic4, tc4GenBankGDQ 380218‡
CAR R16621985CARHumanmic4, tc37GHM587086‡
CAR R17521986CARHumanmic4, tc37GHM587087‡
SA71/102010South AfricaHuman05HHM587088‡
SA482/102010South AfricaHuman05HHM587089‡
SA1221/102010South AfricaHuman05HHM587090‡
SA59/102010South AfricaHuman05HHM587091‡
SA54/102010South AfricaHuman05HHM587092‡
SA79/102010South AfricaHuman05HSeq as above
SA122/102010South AfricaHuman05HSeq as above
SA197/102010South AfricaHuman05HSeq as above
SA226/102010South AfricaHuman05HSeq as above
SA227/102010South AfricaHuman05HSeq as above
SA257/102010South AfricaHuman05HSeq as above
SA360/102010South AfricaHuman05HSeq as above
SA377/102010South AfricaHuman05HSeq as above
SA420/102010South AfricaHuman05HSeq as above
SA486/102010South AfricaHuman05HSeq as above
SA509/102010South AfricaHuman05HSeq as above
SA533/102010South AfricaHuman05HSeq as above
SA578/102010South AfricaHuman05HSeq as above
SA591/102010South AfricaHuman05HSeq as above
SA611/102010South AfricaHuman05HSeq as above
SA663/102010South AfricaHuman05HSeq as above
SA1143/102010South AfricaHuman05HSeq as above
SA1147/102010South AfricaHuman05HSeq as above
SA1171/102010South AfricaHuman05HSeq as above
SA1325/102010South AfricaHuman05HSeq as above
SA276/102010South AfricaHuman05HHM587093‡
SA106/102010South AfricaHuman05HHM587094‡
SA100/102010South AfricaHuman05HSeq as above
SA195/102010South AfricaHuman05HSeq as above
SA225/102010South AfricaHuman05HSeq as above
SA325/102010South AfricaHuman05HSeq as above
SA328/102010South AfricaHuman05HSeq as above
SA492/102010South AfricaHuman05HSeq as above
SA423/102010South AfricaHuman05HHM587095‡
SA404/102009South AfricaHuman05HHM587096‡
SA373/102010South AfricaHuman05HHM587097‡
SA85/102010South AfricaHuman05HHM587098‡
SPU68/102010South AfricaHuman05HSeq as above
SA95/102010South AfricaHuman05HSeq as above
SA206/102010South AfricaHuman05HSeq as above
SA317/102010South AfricaHuman05HSeq as above
SA394/102010South AfricaHuman05HSeq as above
SA395/102010South AfricaHuman05HSeq as above
SA414/102010South AfricaHuman05HSeq as above
SA1108/102010South AfricaHuman05HSeq as above
SA1224/102010South AfricaHuman05HHM587099‡
SPU77/042004NamibiaHuman05HHM587100‡
An18301956South AfricaOvinemic55IHM587108‡
Ar741955South AfricaAe. circumluteolus mosquitomic75IHM587109‡
VRL2269/741974ZimbabweBovinemic2GenBankJDQ 380222‡
SNS¶1944UgandaEretmapodites spp. batmic103, tc28KHM587102‡
SNS 105/2010#1944UgandaEretmapodites spp. batmic103, tc58KSeq as above
95EG vaccine**1944UgandaRVF strain SNSNA2KHM587103‡
KEN57 Rintoul1951KenyaOvinemic31, tc17KHM587104‡
Entebbe/KCS††1944UgandaEretmapodites spp. batmip184, tc2GenBankKDQ380191‡
B3141963KenyaBovineNA4KHM587105‡
B6741962KenyaBovineNA4KHM587106‡
SA184/102010South AfricaHuman05KHM587107‡
VRL2373/741974ZimbabweBovinemic33KHM587121‡
H17391975South AfricaHumanNA5LHM587110‡
VRL763/701970ZimbabweBovinemic23LHM587111‡
Ar125681971South AfricaEr. quinquivittatus batmic35LHM587112‡
Ar111861969ZimbabweAe. lineatopennis mosquitomic55LHM587113‡
H18251975South AfricaHumanNA5LHM587114‡
35/741975South AfricaBovineNAGenBankLJF784387‡
95EG Cow-25091995EgyptBovineNA2LHM587115‡
KEN56/B2653/IB81963KenyaBovineha1, mic8, tc274LHM587117‡
Kitale 18401964KenyaBovineNA4LHM587116‡
SA751975South AfricaHumantc3GenBankLDQ380189‡
Lunyo1955UgandaAedes spp. mosquitomic35MHM587119‡
Ar1181955South AfricaAe. circumluteolus mosquitomic75MHM587120‡
OS-41988MauritaniaHumantc27NHM587122‡
OS-11987MauritaniaHumantc2GenBankNDQ380186
OS-31987MauritaniaHumantc2GenBankNDQ380184
OS-81987MauritaniaHumantc2GenBankNDQ380185
OS-91987MauritaniaHumantc2GenBankNDQ380183
HD475021987MauritaniaHumanNAGenBankNAF134494‡
HD473111987MauritaniaHumanNAGenBankNAF134495‡
ArD383881983Burkina FasoAe. cuminsi mosquitotc5GenBankNDQ380187‡
ArD384571984Burkina FasoAe. furcifer mosquitoNAGenBankNAF134497‡
HD474081987MauritaniaHumanNAGenBankNAF134496‡
HD219551975SenegalHumanmic57NHM587123‡
ArD386611983SenegalAe. dalzieli mosquitoNA7NHM587124‡
AnD1064171993SenegalBovineNAGenBankNAF134500‡
SA51 van Wyk1951South AfricaOvineov3, mic25OHM587125‡

*mic, mouse intracranial; tc, tissue culture; NA, not available; seq, sequence; RVF, Rift Valley fever; ha, hamster; CAR, Central African Republic; SNS, Smithburn neurotropic strain; mip, mouse intraperitoneal; ov, ovine. Isolates are listed in order of appearance in phylogenetic groups in the tree in Figure A1. Ninety-five isolates displaying unique sequences are indicated in boldface. Isolates in standard text have identical sequences to isolates indicated in boldface directly above.
†1, Institut Pasteur, Antananarivo, Madagascar; 2, US Naval Medical Research Unit 3, Cairo, Egypt; 3, Central Veterinary Research Laboratory, Harare, Zimbabwe; 4, Central Veterinary Research Laboratory, Nairobi, Kenya; 5, National Institute for Communicable Diseases, Johannesburg, South Africa; 6, Agricultural Research Council–Onderstepoort Veterinary Institute, Pretoria, South Africa; 7, US Army Medical Research Institute for Infectious Diseases, Frederick, MD, USA; 8, Onderstepoort Biologic Products, Pretoria, South Africa.
‡Sequences selected for analysis and inclusion in the tree (see Materials and Methods).
§Candidate vaccine strain derived by culturing isolate ZH548 from Egypt with the mutagen 5-fluorouracil.
¶SNS master seed stock derived from a 1944 mosquito isolate from Uganda by serial intracranial passage of infected brain suspension in mice and used to produce live animal vaccine in BHK cell cultures.
#Batch of live SNS animal vaccine from South Africa issued in 2010.
**Animal vaccine from Egypt derived from SNS vaccine from South Africa.
††Hepatotropic strain derived from the same 1944 mosquito isolate from Uganda used to produce the SNS strain, but produced by serial intraperitoneal passage of infected liver suspension in mice; referred to as the Entebbe strain in some publications.

Maximum-likelihood tree for a 490-nt section of the Gn glycoprotein gene of 111 isolates and derived strains of Rift Valley fever virus from Africa and Saudi Arabia, 1944–2010. The 95 unique sequences sorted into 15 lineages (A–O). Mean pairwise distances (p-distances) were <0.017 within lineages, and bootstrap values were >70%. Scale bar indicates substitutions per site. CAR, Central African Republic; SNS, Smithburn neurotropic strain.

Ms Grobbelaar is a medical scientist in the Special Pathogens Unit at the National Institute for Communicable Diseases in Sandringham, South Africa. Her research interests are viral hemorrhagic fevers and arboviruses.

ReferencesPepin M, Bouloy M, Bird BH, Kemp A, Paweska J. Rift Valley fever (Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnosis and prevention. Vet Res. 2010;41:61 10.1051/vetres/201003321188836Schmaljohn CS. Bunyaviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, et al. Fields virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers; 1996 p. 1447–71.Battles JK, Dalrymple JM. Genetic variation among geographic strains of Rift Valley fever virus. Am J Trop Med Hyg. 1988;39:617312462795Sall AA. de A Zanotto, Zeller HG, Digoutte JP, Thiongane Y, Bouloy M. Variability of the NS(S) protein among Rift Valley fever isolates. J Gen Virol. 1997;78:285389367372Sall AA, De A, Zanotto PM, Sene OK, Zeller HG, Digoutte JP, Genetic reassortment of Rift Valley fever virus in nature. J Virol. 1999;73:819620010482570Bird BH, Khristova ML, Rollin PE, Ksiazek TG, Nichol ST. Complete genome analysis of 33 ecologically and biologically diverse Rift Valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry. J Virol. 2007;81:280516 10.1128/JVI.02095-0617192303Bird BH, Githinji JW, Macharia JM, Kasiiti JL, Muriithi RM, Gacheru SG, Multiple virus lineages sharing common recent ancestry were associated with a large Rift Valley fever outbreak among livestock in Kenya during 2006–2007. J Virol. 2008;82:1115266 10.1128/JVI.01519-0818786992Vialat P, Billecocq A, Kohl A, Bouloy M. The S segment of Rift Valley fever Phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice. J Virol. 2000;74:153843 10.1128/JVI.74.3.1538-1543.200010627566Woods CW, Karpati AM, Grein T, McCarthy N, Gaturuku P, Muchiri E, An outbreak of Rift Valley fever in northeastern Kenya, 1997–98. Emerg Infect Dis. 2002;8:13844 10.3201/eid0802.01002311897064Shoemaker T, Boulianne C, Vincent MJ, Pezzanite L, Al Qahtani MM, Al Mazrou Y, Genetic analysis of viruses associated with emergence of Rift Valley fever in Saudi Arabia and Yemen, 2000–2001. Emerg Infect Dis. 2002;8:14152012498657Miller BR, Godsey MS, Crabtree MB, Savage HM, Al Mazrao Y, Al Jeffri MH, Isolation and characterization of Rift Valley fver virus from Aedes vexans arabiensis, Kingdom of Saudi Arabia. Emerg Infect Dis. 2002;8:1492412498669Botros B, Omar A, Elian K, Mohamed G, Soliman A, Salib A, Adverse response of non-indigenous cattle of European breeds to live attenuated Smithburn Rift Valley Fever vaccine. J Med Virol. 2006;78:78791 10.1002/jmv.2062416628582Faye O, Diallo M, Diop D, Bezaid E, Ba H, Niang M, Rift Valley fever outbreak with east-central African virus lineage in Mauritania, 2003. Emerg Infect Dis. 2007;13:10162318214173Andriamandimby SF, Randrianarivo-Solofoniaina AE, Jeanmaire EM, Ravololomanana L, Razafimanantsoa LT, Rakotojoelinandrasana T, Rift Valley fever during rainy seasons, Madagascar, 2008 and 2009. Emerg Infect Dis. 2010;16:9637020507747Jupp PG, Grobbelaar AA, Leman PA, Kemp A, Dunton RF, Burkot TR, Experimental detection of Rift Valley fever by reverse transcription–polymerase chain reaction assay in large samples of mosquitoes. J Med Entomol. 2000;37:46771 10.1603/0022-2585(2000)037[0467:EDORVF]2.0.CO;215535594Collett MS, Purchio AF, Keegan K, Frazier S, Hays W, Anderson DK, Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus. Virology. 1985;144:22845 10.1016/0042-6822(85)90320-42998042Hall TA. User friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 1999;14:958Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:15969 10.1093/molbev/msm09217488738Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol. 2007;7:214 10.1186/1471-2148-7-21417996036Heath L, van der Walt E, Varsani A, Martin DP. Recombination patterns in aphthoviruses mirror those found in other picornaviruses. J Virol. 2006;80:1182732 10.1128/JVI.01100-0616971423Hillis DM, Bull JJ. An emperical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol (Stevenage). 1993;42:18292Gear JH. Clinical aspects of African viral hemorrhagic fevers. Rev Infect Dis. 1989;11(Suppl 4):S77782 10.1093/clinids/11.Supplement_4.S7772665013Hussein NA, Chizyuka RZ, Ksiazek TG, Scott RM, Boulos BAM. Epizootic of Rift Valley fever in Zambia, 1985. Vet Rec. 1987;121:111 10.1136/vr.121.5.1112889285Smithburn KC. Rift Valley fever: the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine. Br J Exp Pathol. 1949;30:11618128091van Velden DJ, Meyer JD, Oliver J, Gear JH, McIntosh B. Rift Valley fever affecting humans in South Africa. S Afr Med J. 1977;51:86771561445Kaschula VR. The propagation and modification of strains of Rift Valley fever viruses in embryonated eggs and their use as immunizing agents for domestic ruminants [DVSc thesis]. Pretoria (South Africa): University of Pretoria; 1953Barnard BJ. Rift Valley fever vaccine: antibody and immune response in cattle to a live and an inactivated vaccine. J S Afr Vet Assoc. 1979;50:1557551199Annual Report of the Director, Onderstepoort Veterinary Institute, 1953–1986. Onderstepoort (South Africa): The Institute; 1986Swanepoel R, Coetzer JAW. Rift Valley fever. In: Coetzer JA, Tustin RC, editors. Infectious diseases of livestock. 2nd ed. Cape Town (South Africa): Oxford University Press; 2004 p. 1037–70.Coackley W, Pini A, Gosden D. Experimental infection of cattle with pantropic Rift Valley fever virus. Res Vet Sci. 1967;8:3994056064502Capstick PB, Gosden D. Neutralizing antibody response of sheep to pantropic and neurotropic Rift Valley fever virus. Nature. 1962;195:5834 10.1038/195583a013876399Report A. Department of Veterinary Services, Kenya, 1951–1970. Nairobi (Kenya): The Department; 1970Coetzer JA, Barnard BJ. Hydrops amnii in sheep associated with hydranencephaly and arthrogryposis with Wesselsbron disease and Rift Valley fever viruses as aetiological agents. Onderstepoort J Vet Res. 1977;44:11926351506Davies FG. Rift Valley fever in Kenya. Proceedings of the 49th General Session of the Office International Des Epizooties. Paris: The Office; 1981Swanepoel R. Wesselsbron virus disease. In: Monath TP, editor. The arboviruses: epidemiology and ecology. Vol. 5. Boca Raton (FL): CRC Press; 1988 p. 31–57.Turell MJ, Saluzzo J-F, Tammariello RF, Smith JF. Generation and transmission of Rift Valley fever reassortants by the mosquito Culex pipiens. J Gen Virol. 1990;71:230712 10.1099/0022-1317-71-10-23072230736Swanepoel R. Observations on Rift Valley fever in Zimbabwe. Contributions to Epidemiology and Biostatistics. 1981;3:8391Battles JK, Dalrymple JM. Nucleic acid sequence changes associated with reduced mouse virulence in monoclonal antibody: escape mutants of Rift valley fever virus. In: Abstracts of the 36th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Los Angeles, November 29–December 3, 1987. Deerfield (IL): American Society of Tropical Medicine and Hygiene; 1987Swanepoel R, Erasmus BJ, Williams R, Taylor MB. Encephalitis and chorioretinitis associated with neurotropic African horsesickness virus infection in laboratory workers. Part III. Virological and serological investigations. S Afr Med J. 1992;81:458611574748Hayes EB. Is it time for a new yellow fever vaccine? Vaccine. 2010;28:80736 10.1016/j.vaccine.2010.10.01520971115