Journal of Clinical Virology 62 (2015) 80–83

Contents lists available at ScienceDirect

Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Short Communication

Circulating vaccine-derived polioviruses in the Extreme North region

of Cameroon
Marie Claire Endegue-Zanga a,1 , Serge Alain Sadeuh-Mba a,1 , Jane Iber b , Cara Burns b ,
Marcellin Nimpa-Mengouo c , Maurice Demanou a , Guy Vernet a , François-Xavier Etoa d ,
Richard Njouom a,∗
a
Centre Pasteur of Cameroon, P.O. Box 1274, Yaoundé, Cameroon
b
Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
c
WHO Cameroon Country Office, P.O. Box 155, Yaoundé, Cameroon
d
The University of Yaoundé I, P.O. Box 337, Yaoundé, Cameroon

a r t i c l e i n f o a b s t r a c t

Article history: Background: The World Health Organization (WHO) poliovirus eradication program includes careful
Received 21 May 2014 surveillance of acute-flaccid paralysis (AFP) and mass and routine immunization with oral polio vaccine
Received in revised form (OPV). In populations with low vaccine coverage, the live-attenuated Sabin strains, OPV types 1, 2 and
21 November 2014
3, can evolve into virulent vaccine-derived polioviruses (VDPVs) and circulate in the community. Until
Accepted 23 November 2014
recently, circulating VDPVs (cVDPVs) had not been reported in Cameroon despite the fact that VDPV2
outbreaks have occurred in nearby countries.
Keywords:
Objectives: This study aimed to characterize virus isolates from four AFP patients infected with cVDPV2
Vaccine-derived poliovirus
Acute-flaccid paralysis
in the Extreme North region of Cameroon in 2013.
Poliomyelitis Study design: The complete VP1 region of the four VDPV strains was sequenced and the relationships with
Cameroon cVDPVs from neighboring countries were investigated.
Results: All four patients were infected by cVDPV2 strains showing 1.2–2.0% nucleotide difference com-
pared to the reference Sabin 2 VP1 sequence. Phylogenetic analysis indicated that the VDPV strains were
genetically linked to cVDPV2 lineages of the recent Chad cVDPV2 outbreak.
Conclusions: The circulation of pathogenic VDPVs suggests that there are localized immunization gaps
in some districts like Makary, Mada and Kolofata in Cameroon. To avoid poliomyelitis outbreaks in
Cameroon, especially in the districts close to neighboring countries with ongoing cVDPV outbreaks, high
polio vaccine coverage is essential.
© 2014 Elsevier B.V. All rights reserved.

1. Background used in mass vaccination campaigns is made up of live attenu-

The Global Polio Eradication Initiative (PEI) is based on extensive
vaccination campaigns with the oral polio vaccine (OPV) and effi-
cient surveillance of acute flaccid paralysis (AFP) cases to determine
the wild or vaccine-derived origin of isolated polioviruses (PVs).
This strategy has led to a tremendous reduction in poliomyeli-
tis incidence, from an estimated 350,000 in 1988 to less than
500 cases reported in 2013 (http://www.polioeradication.org/
Dataandmonitoring/Poliothisweek.aspx). Trivalent OPV (tOPV)
∗ Corresponding author. Tel.: +237 99 65 47 67; fax: +237 22 23 15 64.
E-mail addresses: njouom@pasteur-yaounde.org, njouom@yahoo.com
(R. Njouom).
1
These authors contributed equally to this work.
ated viruses (Sabin strains of serotypes 1, 2 and 3) that are able
to multiply to high titers in the gastrointestinal tract, thus induc-
ing strong mucosal immunity. In addition to tOPV, bivalent OPV
(bOPV) [composed of Sabin 1 and 3] or monovalent OPV (mOPV)
[composed of Sabin 1 or 3] are used during Supplemental Immu-
nization Activities (SIAs). bOPV, mOPV1 and mOPV3 are more
effective in inducing immunity to types 1 and 3 because of the
absence of interference by Sabin 2. However, low vaccine cov-
erage allows for circulation and genetic drift of OPV strains and
the emergence of pathogenic, vaccine-derived PVs (VDPVs). VDPVs
are vaccine-related isolates that differ by >0.6% (VDPV serotype 2,
VDPV2) or >1.0% (VDPV serotype 1 and 3, VDPV1 and VDPV3) in
nucleotide (nt) identity compared to the corresponding OPV strain
in the full-length sequence of the genomic region encoding the
major viral capsid protein, VP1 (∼900 nt) [1,2]. In addition to the

http://dx.doi.org/10.1016/j.jcv.2014.11.027
1386-6532/© 2014 Elsevier B.V. All rights reserved.
 M.C. Endegue-Zanga et al. / Journal of Clinical Virology 62 (2015) 80–83
Fig. 1. Map indicating the location of the 2013 type 2 circulating vaccine-derived
polioviruses (cVDPVs) at the borders of Nigeria, Chad and Cameroon. The circles
represent the cVDPVs, which are genetically linked to circulating VDPV2s originating
in Chad. cVPDVs found in the Extreme North province of Cameroon originated from
the districts of Makary, Mada, and Kolofata.
nt divergence criteria, circulating VDPV (cVDPV) specifically refers
to VDPV strains for which epidemiologic and genetic evidence for
person–person transmission in the community has been estab-
lished. AFP surveillance and immunizations with OPV led to the
interruption of transmission of indigenous wild PVs in Cameroon
in 1999. In subsequent years, wild PV importations were reported in
the Extreme North region of Cameroon [3] without re-established
transmission. This part of Cameroon is a narrow region flanked by
Nigeria and Chad with extensive human movement across the bor-
ders of the three countries (Fig. 1). Until recently, cVDPVs had not
been reported in Cameroon despite the fact that VDPV2 outbreaks
have occurred in nearby countries [2].
2. Objectives
From May to August 2013, VDPV2 was isolated from stool spec-
imens from four AFP cases originating from three districts of the
Extreme North region of Cameroon. We describe the character-
ization of the VDPV2 originating from these patients and their
relationships with cVDPVs isolated in the neighboring countries.
3. Study design
3.1. Patients
All four AFP cases originated from the Extreme North region of
Cameroon. The cases from Makary district had a history of immu-
nization with trivalent OPV while other cases had no history of
immunization with OPV containing type 2 (Table 1).
3.2. Specimens, virus isolation and intratypic differentiation
Stool samples were collected from each patient within 14 days
of the onset of AFP; they were stored at +4 ◦ C and subsequently sent
to Centre Pasteur of Cameroon (CPC). Virus isolation and intratypic
81
differentiation (ITD) of the isolates by real time RT-PCR (rRT-PCR)
amplification were carried out according to standard WHO proce-
dures [4,5].
3.3. Sequencing and phylogenetic analyses
Isolates were sent to the Centers for Disease Control and Preven-
tion (Atlanta, USA) for sequencing, according to WHO guidelines.
Sequencing was performed as described previously [6]. Full-length
sequences of the VP1 capsid region were compared to the reference
Sabin sequence. A phylogenetic radial tree was reconstructed by the
neighbor-joining (NJ) method using MEGA version 5 bioinformatics
software [7] with the Kimura two parameter algorithm for genetic
distance determination. Full-length VP1 sequences were deposited
in Genbank under accession numbers KP143045–KP143072 if they
had not been deposited previously.
4. Results
Virus isolation on L20B cells followed by ITD showed that all
four patients were infected with strains derived from Sabin 2.
The VDPV rRT-PCR screening assay flagged them for sequencing.
The sequencing of the genomic region encoding the VP1 cap-
sid protein of the isolates from these patients confirmed that
they had >0.6% nucleotides (nt) divergence from the Sabin 2
VP1 sequence and were therefore classified as VDPV2s: viruses
CAE1318981 (18 nt substitutions), CAE1318982 (13 nt substitut-
ions), CAE1318983 (12 nt substitutions), and CAE1318984 (11 nt
substitutions) (Table 1). PVs accumulate nt substitutions at an
estimated overall rate of about 1.1% per year [8]. With 1.2–2.0% dif-
ferences in nucleotide identity compared to the Sabin 2 strain, the
studied VDPVs had been circulating and/or replicating for longer
than 1 year.
We investigated the phylogenetic relatedness among the
Cameroon VDPVs and those originating from ongoing outbreaks
in neighboring countries [8–10]. VDPV sequences from Kolofata
(CAE1318983 and CAE1318984) were closely related to each other
at 99.9% and both were genetically linked to viruses from the recent
Chad outbreak (Fig. 2). The closest matching sequences were from
cVDPV2 from northern Nigeria that had been imported previously
imported from Chad [11] into Nigeria. Strains from the districts
of Makary and Mada were also closely related to each other
(99.45% nt identity). They were genetically linked to a cVDPV2
detected in eastern N’djamena in July 2012 during an outbreak
in Chad (http://www.polioeradication.org/Dataandmonitoring/
Poliothisweek/Circulatingvaccinederivedpoliovirus.aspx) (Fig. 2).
Overall, our findings indicated that the studied patients were
infected by circulating strains genetically linked to viruses from
the Chad outbreak.
5. Discussion
Multiple VDPV2 emergences have been documented in north-
ern states of Nigeria where they have been associated with large
outbreaks from 2005 to the present [9,10,12]. cVDPVs were also
reported in Chad during 2012 to 2013 [13,14]. Low polio vaccine
coverage is the main factor favoring the emergence and circulation
of VDPVs [2,15]. The national polio vaccine coverage rate was esti-
mated to be about 80% from 2007 to 2010 [16]. Our finding of VDPVs
that have been circulating for more than 1 year in the population
without detection suggests that there were immunization gaps in
some districts in the Extreme North of Cameroon in 2013.
The first patient was incompletely immunized with tOPV (2 of 4
required doses) while the three others had never received tOPV
containing type 2 OPV, but they had received bOPV containing
82 M.C. Endegue-Zanga et al. / Journal of Clinical Virology 62 (2015) 80–83
Table 1
Description of the four acute-flaccid paralysis cases associated to vaccine-derived from the Extreme North region of Cameroon.
Patients Type 2 VDPV strains Age (months) Sex District OPV historya
1 CAE1318981 36 M Makary 2 tOPV
2 CAE1318982 24 M Mada 2 bOPV
3 CAE1318983 23 F Kolofata No
4 CAE1318984 10 M Kolofata No
a
tOPV, trivalent oral polio vaccine for routine immunization; bOPV, bivalent oral polio vaccine (Sabin 1 and 3) during supplemental immunization activities.
types 1 and 3 (Table 1). With the disappearence of poliomyelitis in
most countries, some populations no longer perceive the need for
polio immunization. This leads to a decline of vaccine coverage and
vulnerability to wild polio importations or VDPV emergence in pre-
viously polio-free countries [17–20]. Field investigations in 2013
showed that only 50% of children <5 years old were immunized
with trivalent OPV in routine immunizations in the Extreme North
region (national Expanded Program of Immunization (EPI) annual
report). According to the national EPI annual reports, only bivalent
OPV (types 1 and 3) was administered during local immunization
campaigns from 2010 to 2012 to supplement routine coverage with
tOPV in Cameroon. Thus, the immunization against type 2 PVs was
based only on routine immunization. Routine immunization cov-
erage rates were low enough that the population immunity in the
Extreme North region of Cameroon was inadequate. The immuno-
logical gap favored the circulation of VDPV2 in some districts of
the Extreme North region of Cameroon. The lowest paralytic case-
to-infection ratio among wild polioviruses is estimated at 1:1800
for PV2 among the three poliovirus serotypes [21]. Assuming that
this ratio for VDPV2s is similar to that for WPV2, we estimate
that thousands of cVDPV2 infections have occurred in Northern
Fig. 2. Phylogenetic Radial Tree of Cameroon (CAE), Chad (CHA), Democratic
Republic of Congo (RDC) and Nigeria (NIE) serotype 2 VDPV polioviruses (rooted
to Sabin 2) from 2011 to 2013. Branch labels give country code followed by year of
onset and sequence identifier, district location of AFP case, onset date of paralysis,
and number of nucleotide differences from Sabin 2.
Last OPV (date) Onset date Specimen date VP1 nucleotide differences
to Sabin 2, n (%)
27/04/2013 09/05/2013 12 and 13/05/2013 18 (2.0)
10/05/2013 27/05/2013 08 and 09/06/2013 13 (1.4)
n/a 17/07/2013 23 and 24/07/2013 12 (1.3)
n/a 12/08/2013 18 and 19/08/2013 11 (1.2)
Cameroon and/or neighboring countries during this outbreak. In
response to the detection of these VDPVs, immunization campaigns
with tOPV were conducted in the Extreme North region in July and
August of 2013. To be effective, tOPV rather than bOPV, should be
used for some SIA activities in Cameroon, especially in the Extreme
North region, as long as there is proven circulation of VDPV2 in
neighboring countries.
Gaps in population immunity to PV2 are not the only vulnera-
bility in Cameroon. Some districts in other regions of the country
may also have low vaccine coverage. Four wild type 1 PVs related
to Chad viruses were identified in southern Cameroon (West
region) in 2013, where no wild PV had been detected for the prior
3 years (http://www.polioeradication.org/Dataandmonitoring/
Poliothisweek/Polioinfecteddistricts.aspx). Our findings indicated
that cVDPV2 strains from all four patients were genetically linked
to VDPVs from the Chad outbreak of 2012 and 2013 (Fig. 2).
The movements of the populations across the borders of Chad,
Cameroon and Nigeria contribute to the circulation of polioviruses
between countries. Efforts to strengthen surveillance and to ensure
high-quality routine and supplemental immunization activities in
Cameroon need to continue in order to stop PV transmission, limit
circulation from importation events, and prevent emergence of
VDPVs.
Conflict of interest
None.
Funding
This work was supported by a WHO grant (TSA 2014/409463-0),
Technical Service Agreement (TSA) 2013.
Ethical approval
Stool specimens were collected with the approval of the
Cameroonian health authorities within the frame of the poliomyeli-
tis surveillance in Cameroon.
Acknowledgments
We are grateful to Daniel Kamga Njile and Arice Michel Tabon-
fack for their commitment in processing samples, and the EPI and
members of the WHO Global Poliovirus Laboratory Network for
their collaboration and support.
The findings and conclusions in this report are those of the
author(s) and do not necessarily represent the official position of
the Centers for Disease Control and Prevention.
References
[1] CDC. Update on vaccine-derived polioviruses – worldwide July 2009–March
2011. MMWR Morb Mortal Wkly Rep 2011;60:846–50.
[2] Diop OM, Burns CC, Wassilak SG, Kew OM. Update on vaccine-derived
polioviruses – worldwide, July 2012–December 2013. MMWR Morb Mortal
Wkly Rep 2014;63:242–8.
 M.C. Endegue-Zanga et al. / Journal of Clinical Virology 62 (2015) 80–83
[3] Arita I, Francis D. Safe landing for global polio eradication: a perspective. Vac-
cine 2011;29:8827–34.
[4] Anonymous. Isolation and identification of polioviruses. In: WHO, editor. Polio
laboratory manual. Geneva, Switzerland: World Health Organization; 2004. p.
87–100.
[5] Kilpatrick DR, Ching K, Iber J, Chen Q, Yang SJ, De L, et al. Identification of
vaccine-derived polioviruses using dual-stage real-time RT-PCR. J Virol Meth-
ods 2014;197:25–8.
[6] Kilpatrick DR, Iber JC, Chen Q, Ching K, Yang SJ, De L, et al. Poliovirus serotype-
specific VP1 sequencing primers. J Virol Methods 2011;174:128–30.
[7] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molec-
ular evolutionary genetics analysis using maximum likelihood, evolutionary
distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731–9.
[8] Jorba J, Campagnoli R, De L, Kew O. Calibration of multiple poliovirus molecular
clocks covering an extended evolutionary range. J Virol 2008;82:4429–40.
[9] Burns CC, Shaw J, Jorba J, Bukbuk D, Adu F, Gumede N, et al. Multiple indepen-
dent emergences of type 2 vaccine-derived polioviruses during a large outbreak
in northern Nigeria. J Virol 2013;87:4907–22.
[10] CDC. Progress toward poliomyelitis eradication – Nigeria, January
2012–September 2013. MMWR Morb Mortal Wkly Rep 2013;62:1009–13.
[11] Sheikh MA, Makokha F, Hussein AM, Mohamed G, Mach O, Humayun K, et al.
Combined use of inactivated and oral poliovirus vaccines in refugee camps and
surrounding communities – Kenya, December 2013. MMWR Morb Mortal Wkly
Rep 2014;63:237–41.
[12] Wassilak S, Pate MA, Wannemuehler K, Jenks J, Burns C, Chenoweth P,
et al. Outbreak of type 2 vaccine-derived poliovirus in Nigeria: emergence
83
and widespread circulation in an underimmunized population. J Infect Dis
2011;203:898–909.
[13] CDC. Progress toward poliomyelitis eradication – Chad January 2011–August
2012. MMWR Morb Mortal Wkly Rep 2012;61:858–62.
[14] WHO. Update on vaccine-derived polioviruses detected worldwide April
2011–June 2012. Wkly Epidemiol Rec 2012;87:358–68.
[15] Duintjer Tebbens RJ, Pallansch MA, Kim JH, Burns CC, Kew OM, Oberste
MS, et al. Oral poliovirus vaccine evolution and insights relevant to model-
ing the risks of circulating vaccine-derived polioviruses (cVDPVs). Risk Anal
2013;33:680–702.
[16] WHO-UNICEF. “Immunization summary”. Report, the 2012 edition; 2012.
[17] CDC. Outbreaks following wild poliovirus importations – Europe, Africa,
and Asia, January 2009–September 2010. MMWR Morb Mortal Wkly Rep
2010;59:1393–9.
[18] Gregory CJ, Ndiaye S, Patel M, Hakizamana E, Wannemuehler K, Ndinga E, et al.
Investigation of elevated case-fatality rate in poliomyelitis outbreak in Pointe
Noire Republic of Congo, 2010. Clin Infect Dis 2012;55:1299–306.
[19] Joffret ML, Jegouic S, Bessaud M, Balanant J, Tran C, Caro V, et al. Common and
diverse features of cocirculating type 2 and 3 recombinant vaccine-derived
polioviruses isolated from patients with poliomyelitis and healthy children. J
Infect Dis 2012;205:1363–73.
[20] Razafindratsimandresy R, Joffret ML, Rabemanantsoa S, Andriamamonjy S,
Heraud JM, Delpeyroux F. Reemergence of recombinant vaccine-derived
polioviruses in healthy children, Madagascar; 2013.
[21] Nathanson N, Kew OM. From emergence to eradication: the epidemiology of
poliomyelitis deconstructed. Am J Epidemiol 2010;172:1213–29.