The changing epidemiology of the Lassa virus from endemic areas to other parts of West Africa has been reported. However, there have been no documented Lassa fever transmission chains in the Benin Republic. Two outbreaks of Lassa fever (November 2014 and January 2016) in the Benin Republic were characterised by a high number of deaths (more than 50%) among 27 confirmed and other unconfirmed cases.
We report the detection, confirmation and relatedness of the Lassa virus strains from the Benin Republic with other isolates within the West African Sub-region.
A total of 70 blood samples (16 from 2014 and 54 from 2016) from suspected cases with signs and symptoms suggestive of viral haemorrhagic fever were received for molecular analysis at the Centre for Human and Zoonotic Virology, College of Medicine, University of Lagos and the Lagos University Teaching Hospital. With the detection of the Lassa virus RNA by reverse transcriptase polymerase chain reaction, sequencing and phylogenetic analyses were performed using the Sanger dideoxy sequencing technology platform and the MEGA6 software.
S segments of the Lassa virus RNA genome were detected in 5 (7.1%) of the 70 samples analysed. Sequencing and a phylogenetic tree construction confirmed that the strain of Lassa virus had close relationships with strains previously isolated from Nigeria.
We confirmed the presence of the Lassa virus in the Benin Republic, with 2 strains having molecular epidemiological links with Lineage I and II strains from Nigeria. To reduce the likelihood of outbreaks, there is a need for heightened awareness and strengthened surveillance systems about Lassa fever, particularly in the sub-region.
Lassa fever is an acute and often fatal viral haemorrhagic disease caused by an arenavirus called Lassa virus (LASV), an enveloped bi-segmented negative-sensed single-stranded RNA virus, endemic in parts of West Africa. The illness has caused morbidity of about 300 000 persons and has an estimated death rate of 5000 per annum in West Africa.
The incubation period of Lassa fever ranges between 5 and 21 days with an average of about 10 days.
Recently, LASV was discovered to be maintained by multiple rodent reservoirs other than
In the Benin Republic, two outbreaks of Lassa fever (November 2014 and January 2016) were characterised by a high number of deaths (>50%) among confirmed cases.
No ethics approval was obtained. This investigation was performed as part of the Lassa fever public health response in the Benin Republic and Nigeria. It was not considered to be research on human subjects, as documented in Otto et al.
In 2014, a Lassa fever outbreak was reported for the first time in the Tanguiéta and Cobly communes, Atakora Department, north-west Benin Republic. The chain of infection stemmed from a woman who died from Lassa fever 2 days after the delivery of a baby girl. The baby girl took ill 2 weeks after birth and was cared for at Hôpital de Saint Jean de Dieu, where the outbreak was enhanced by nosocomial transmission and eventual transmission within the community. Within a period of 2 weeks (15 October to 04 November), 16 suspected cases with signs and symptoms of VHF and two laboratory confirmed cases, with a case fatality of 56.3%, were recorded in the country in 2014. This included the deaths of four personnel at the healthcare facility with signs and symptoms of VHF. Due to the high case fatality rate within a short period of time, an alarm for the possible outbreak of the Ebola virus was sounded by the health authorities in the Benin Republic. However, all samples were negative for the Ebola virus; LASV was detected instead.
A resurgence of the epidemic was witnessed in several districts in the central and eastern regions of the Benin Republic in 2016 with reports of 54 suspected cases with signs and symptoms of VHF and 16 laboratory confirmed cases, with a case fatality of about 50%. During this outbreak, the communes of Tchaourou (Borgou department) and Djougou (Donga department) along the Nigerian border were the most affected areas. Since the Benin Republic had never identified a case of VHF, blood samples collected from the people who died and suspected cases were sent to a specialised laboratory, the Centre for Human and Zoonotic Virology, College of Medicine, University of Lagos and the Lagos University Teaching Hospital in Lagos, Nigeria, for VHF investigation.
Blood samples collected from different individuals with signs and symptoms of VHF (16 cases during the 2014 outbreak and 54 cases during the 2016 outbreak) were cold-chain-transported in triple-level packaging to the Centre for Human and Zoonotic Virology and Lagos University Teaching Hospital via the Benin Republic Ministry of Health and the World Health Organization. Universal sample and handling precautions were carried out as recommended by the United States Centers for Disease Control and Prevention.
The viral nucleic acid from inactivated sample aliquots (undiluted and 1:10 dilution) were extracted using a mini spin column RNA extraction kit by Qiagen (Qiagen, Germantown, Maryland, United States) in a Class IIA biological safety cabinet according to the manufacturer’s instructions. After the extraction of viral nucleic acid, S segment of the RNA genome, 3` non-coding region and 5` non-coding region of the nucleic acid of LASV (according to Olschlager et al.
Primers used for Lassa, Dengue and yellow fever investigation, Lagos, Nigeria, March 2018.
Virus | Primer name | Primer sequence | Amplicon size base pair |
---|---|---|---|
Lassa fever virus |
36E2 | 5’GTT CTT TGT GCA GGA MAG GGG CAT KGT CAT 3’ | ~ 320 |
LVS-339-rev | 5’ ACC GGG GAT CCTAGG CAT TT 3’ | ||
Dengue fever virus |
DenS | 5’GGA TAG ACC AGA GAT CCT GCT GT 3’ | 79 |
DenAs | 5’ CAT TCC ATT TTC TGG CGT TC 3’ | ||
DenAs+ | 5’ CAG CAT CAT TCC AGG CAC AG 3’ | ||
Yellow fever virus (in-house) | YF fwd | 5’ ATG GCA CTG TTG TGA TGC AG 3’ | 405 |
YF rvs | 5’ AGT TCA AGC CGC CAA ATA GC 3’ |
The positive control used for Lassa assays were previously detected Lassa samples from Irrua, Edo State, Nigeria with accession number GU481078 NIG 08-A47 2008 IRRUA, while those for Dengue and YFV assays were both tissue cultured inactivated samples all from the Virology Unit Laboratory of the Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany through our collaborations.
The specific amplicon band size (320 bp) for LASV was purified using the Jena Bioscience gel extraction kit (Jena, Germany). Purified PCR products were sequenced using 3130xl Applied Biosystems Genetic Analyzer at Genewiz Laboratories in South Plainfield, New Jersey, United States.
Sequence data in FASTA format of the S segment of the RNA genome of other submitted or published LASV genome sequences particularly from Nigeria, Liberia and Sierra Leone were downloaded from the National Center for Biotechnology Information. Downloaded sequences were aligned using the MUSCLE tool of MEGA6 software.
Among the 70 samples, 5 (7.1%) were positive for LASV, while none (0%) was positive for both Yellow fever and Dengue viruses. The expected amplicon band size of approximately 320 base pairs (bp) of the S segment of the RNA genome for LASV was detected by the agarose gel electrophoresis analysis
Reverse transcription polymerase chain reaction detection of S gene fragment of the Lassa virus, Lagos, Nigeria, March 2018. The gel lanes represent neat (undiluted, N) and 1:10 dilutions (D) of the RNA extracts used. Three Nigerian outbreak samples representing lanes S1–S3 (accession numbers: MF317933-35) were run alongside Benin Republic outbreak samples (S4–S5). RNase/DNase free water was used as a negative extraction control (-VE CTRL) while a 2008 outbreak positive sample (GU481078_NIG_08-A47_2008_IRRUA) was used as a positive control (+VE CTRL).
Reverse transcription polymerase chain reaction detection of Dengue virus, Lagos, Nigeria, March 2018. The gel lanes represent neat (undiluted, N) and 1:10 dilutions (D) of the RNA extracts used. Three Nigerian outbreak samples representing lanes S1–S3 (accession numbers: MF317933-35) were run alongside Benin Republic outbreak samples (S4–S5). RNase/DNase free water was used as negative extraction control (-VE CTRL) while a tissue culture inactivated sample of Dengue virus from the Virology Unit Laboratory of the Bernhard Nocht Institute of Tropical Medicine, Germany was used as a positive control (+VE CTRL).
Reverse transcription polymerase chain reaction detection of the yellow fever virus, Lagos, Nigeria, March 2018. The gel lanes represent neat (undiluted, N) and 1:10 dilutions (D) of the RNA extracts used. Three Nigerian outbreak samples representing lanes S1–S3 (accession numbers: MF317933-35) were run alongside Benin Republic outbreak samples (S4–S5). RNase/DNase free water was used as a negative extraction control (-VE CTRL) while a tissue culture inactivated sample of 17D yellow fever strain from the Virology Unit Laboratory of the Bernhard Nocht Institute of Tropical Medicine, Germany, was used as a positive control (+VE CTRL).
Sequence data of the S segment of the RNA genome of LASV were obtained for 2/5 (40%) of the positive samples. The generated nucleotide sequences of the S segment of the RNA genome of the Lassa strains from the Benin Republic showed relatedness with documented LASV strains particularly from Nigeria. Phylogenetic analysis of the sequences of the 2 LASV strains from the Benin Republic with submitted sequences in the GenBank database showed that each of the strains were closely related to Lineage I that covers the 1969 Lassa LP strain and Lineage II which covers strains from Lagos, the eastern states of Nigeria such as the Onitsha strain in 1974, Abakaliki, Irrua, the middle belt and a few northeast central states in Nigeria as shown in
Molecular phylogenetic analysis of the S gene segment of Benin Republic Lassa sequences in comparison with selected Lassa sequences from Nigeria, Liberia and Sierra Leone by the maximum likelihood method, Lagos, Nigeria, March 2018. The numbers at the nodes are bootstrap values. Isolates in green boxes are from the Benin Republic. The isolate in the red box is the positive control of the assay. Lineage I covers the 1969 Lassa LP strain while Lineage II covers strains from Lagos, the eastern states of Nigeria such as the Onitsha strain in 1974, Abakaliki, Irrua, the middle belt and the few northeast central states in Nigeria; Lineage III covers the NIG-CSF-Jos 2000 strain, the western states and northwestern states strains such as GA392 in Zaria, Nigeria; Lineages IV/V cover the Josiah strain of Sierra Leone, and the Liberian strain.
This study confirms the presence of the Lassa virus in the Benin Republic, with 2 isolates having molecular epidemiological links with Lineage-II strains from Nigeria. Large-scale outbreaks of Lassa fever have been reported from Nigeria since 2015 with a frequent and widening geographical spread.
Due to the proximity of the Benin Republic to Nigeria and reports of several Lassa fever outbreaks in Nigeria since 1969, it was expected that a case of Lassa fever should have been reported in the Benin Republic earlier than now due to fluidic inter-border travel between the two countries. Furthermore, reports have shown that migration of the Lassa virus strains out of Nigeria over a long period of time might have contributed to the increased diversity of LASV, with non-Nigerian strains exhibiting improved codon adaptation to the human host, greater viral loads, and increased case fatality rates.
Our phylogenetic analysis of the partial sequences of the glycoprotein region of the Lassa fever virus shows that the 2014 Benin Republic nucleic acid sequence clustered with the Lily Pinneo strain of 1969, whereas the 2016 sequence clustered with the 1974 Onitsha strain. Our findings indicate that the virus may have existed in the
Despite the findings from this study, it is still envisioned that the likelihood of movement of VHFs, particularly LASV, from endemic into non-endemic countries within and beyond the West African sub-region remains a possibility. Increased awareness and surveillance are effective tools in curbing the menace of these agents. Thus, laboratory infrastructure, appropriate facilities, technical proficiency and investigation capacity must be improved for a positive impact on our surveillance mechanisms, diagnosis and identification of infections, clinical case management and the development of new approaches to control Lassa fever outbreaks in the sub-region.
The authors would like to acknowledge and thank all staff members who contributed to the collection and analysis of samples in the laboratory, especially the laboratory scientists Mrs MR Orenolu, Mr RA Anyanwu and Mrs MA Abdullah of the Virology Research Laboratory, Central Research Laboratory, College of Medicine, University of Lagos.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
O.B.S., A.B.J., and S.A.O. were responsible for the conceptualisation and design of the project, performed part of the laboratory work, analysed the data and wrote the manuscript. H.S.B., J.M.A., M.D.S., F.G. and C.F.L. all made conceptual contributions and assisted in writing and preparing the manuscript.
This work was supported by the European Union support for EVD (2014), Bernhard Nocht Institute for Tropical Medicine, Germany and Virology Unit Laboratory, Central Research Laboratory, College of Medicine, University of Lagos, Lagos, Nigeria.
The data are not publicly available due to (restrictions e.g. containing information that could compromise the privacy of research participants). The data that support the findings of this study are available from the corresponding author, upon reasonable request.
Responsibility for the information and views set out in this publication lies entirely with the authors.