Journal of Medical Virology 84:728–732 (2012)
Genetic Analysis of Hepatitis A Virus Variants
Circulating Among Children Presenting With
Acute Diarrhea in Cameroon
Joseph C. Forbi,1* Simon M. Agwale,1Lucy M. Ndip,2and Mathew D. Esona1
1Clinical Virology Laboratory, Innovative Biotech, Keffi/Abuja, Nigeria
2Department of Biochemistry and Microbiology, Laboratory for Emerging Infectious Diseases, University of Buea,
circulating hepatitis A virus (HAV) associated
with cases of acute diarrhea among children
under 5 years of age in Kumba–Cameroon.
Reverse transcription PCR, sequencing, and
phylogenetic analysis of a 371 bp segment of
the VP1/P2A junction of six isolates obtained
from stool samples showed the exclusive emer-
gence of genetically related HAV subgenotype
IA. All the isolates clustered within a unique
lineage exhibiting a 99.5% nucleotide identity
suggesting infection from a common source.
The Cameroonian HAV isolates did not intermix
or cluster with those from other regions of
Africa and the rest of the world. Tajima’s
neutralization tests using the six sequences
(D ¼ ?1.37; P ¼ 0.016). This is the first descrip-
tion of indigenous HAV genotypes circulating
spread and predominance of HAV/1A infection
in the Kumba area. These findings stress the
need for routine molecular tracking of HAV in-
fection as a contributory cause of acute diar-
rhea in Cameroonian children.
? 2012 Wiley Periodicals, Inc.
J. Med. Virol.
HAV genotype; VP1/P2A junc-
Hepatitis A virus (HAV) is a member of the genus
Hepatovirus in the Picornaviridae family, and has a
positive-sense single-stranded RNA genome approxi-
mately 7.5 kb in length [Cohen et al., 1987]. The
genome codes a large open reading frame (ORF),
which is flanked by 50non-translated region (50NTR)
and 30NTR. The downstream part of 50NTR represents
the internal ribosomal entry site (IRES), which medi-
ates cap-independent translation initiation and is
important for HAV replication [Kanda et al., 2010].
The ORF encodes a polyprotein of approximately
250 kDa that is cleaved co- and post-translationally
into four structural proteins, VP1–VP4 (P1 region),
comprising the viral capsid, and seven non-structural
proteins, 2A–2C (P2 region) and 3A–3D (P3 region)
[Jia et al., 1993; Costa-Mattioli et al., 2003]. HAV has
been shown to possess a single conserved immunogen-
ic neutralization site and isolates from different parts
of the world belong to a single serotype [Stapleton and
Lemon, 1987; Lemon et al., 1992]. Based on analysis
of the 900 nucleotides of the complete VP1 protein, six
HAV genotypes (genotypes I, II, III, IV,V, VI) are now
defined [Costa-Mattioli et al., 2003]. Genotypes I, II,
and III, divided into subtypes A and B, are associated
with human disease. The former genotype VII has
been reclassified within the genotype II clade as
subgenotype IIB [Costa-Mattioli et al., 2003; Lu et al.,
HAV is a hepatotropic virus transmitted through
the faecal-oral route [Cuthbert, 2001]. Children may
get stool on their hands and then touch objects that
other children put into their mouths, thereby exposing
them to HAV. HAV infection has varying epidemiolog-
ical patterns in different parts of the world [Gust,
1992]. In Cameroon, previous studies showed that the
sero-prevalence of antibodies to HAV exceeds 90%
[Stroffolini et al., 1991; Ndumbe et al., 1994]. Given
the high prevalence of HAV infection in Cameroon
and its potential to cause disease outbreaks, it is
remarkable how little is known of the pathogen’s
genetic diversity and molecular epidemiology in the
Conflicts of Interest: None
*Correspondence to: Joseph C. Forbi, PhD, Clinical Virology
Laboratory, Innovative Biotech, Keffi/Abuja, Nigeria
Accepted 31 January 2012
Published online in Wiley Online Library
? 2012 WILEY PERIODICALS, INC.
country. The HAV variants associated with various
disease burden also remains unknown. In addition,
HAV is not a statutory notifiable disease in Cameroon
and universal vaccination against the virus is not
performed in this country. To date, published reports
describing HAV strains circulating in Cameroon are
not available. This information may be important in
establishing the source and transmission events of the
virus and in understanding the molecular epidemiolo-
gy of the virus in Cameroon. In this study, stool
samples were collected from children presenting with
acute diarrhea in a clinic in Kumba, South West
region of Cameroon and the VP1/P2A junction region
were sequenced and aligned with the published
sequences from GenBank to establish phylogenetic
analysis. This report provides new data on the genetic
relatedness of HAV isolates from Cameroon and the
distribution of sub-genotype IA in this part of the
MATERIALS AND METHODS
Stool samples were collected from 78 children under
5 years of age presenting with symptoms of acute
diarrhea at a clinic in Kumba–Cameroon. Kumba is
situated in Meme Division in the South West region
of Cameroon and its geographical coordinates are
4838038 ‘‘North, 9826019’’ East. All experiments were
performed in compliance with relevant laws and insti-
tutional guidelines and in accordance with the ethical
standards of the Declaration of Helsinki. Consent to
participate in the study was obtained from the
parents or the guardians of the children. Permission
to access the patients was obtained from the medical
director of the clinic. The use of samples was approved
by the research/ethics committee of Innovative Bio-
tech. Fecal specimens were diluted 1:10 in phosphate
buffer saline, mixed, centrifuged and the supernatant
generated was stored at ?208C until RNA extraction.
RNA Extraction and PCR Amplification
RNA was extracted from 150 ml of 10% stool sam-
ples by using the total nucleic acid QIAamp Mini
Viral Elute Kit (QIAGEN, Valencia, CA) in accor-
dance with the manufacturer’s protocol. Amplification
of the VP1/P2A fragment was performed by reverse
transcription PCR (RT-PCR) during the first round and
a nested PCR during the second round as described
[Hutin et al., 1999; Nainan et al., 2006].
Sequencing and Phylogenetic Analysis
Nested PCR products were purified and sequenced
using the Jetquick PCR purification kit (Genomed,
Loehne, Germany). Purified products were quantified
with Pico Green (Molecular Probes, Leiden, The
Netherlands) using a Tecan Genios Fluorescence
Reader (Mechelen, Belgium). Ten nanograms DNA
were used for sequencing in both directions with the
(Applied Biosystems, Nieuwerkerk, The Netherlands)
on a capillary sequencer (model 3130 avant, Applied
Biosystems) using the nested PCR primers as se-
quencing primers. The sequencing PCR conditions
were: 1 cycle at 968C for 1 min followed by 25 cycles
of 968C for 10 sec, 508C for 5 sec, and 608C for 2 min.
All sequences generated in this study were collected
web-based nucleotide BLAST program (http://blast.
ncbi.nlm.nih.gov/Blast.cgi) optimized for the blastn al-
gorithm in order to compare our sequences to those
found in the sequence database.
Sequences were aligned with ClustalW and Phylo-
genetic analysis was performed using the Maximum-
Likelihood method and Kimura 2-parameter model as
implemented in MEGA 5 [Kimura, 1980; Tamura
et al., 2011]. Bootstrap values of 50 or above (1,000
replications) to estimate the reliability of the tree
topology are shown in the phylogenetic trees. The tree
with the highest log likelihood is shown.
Overall mean genetic distances were calculated
using the distance matrix in MEGA 5 [Tamura et al.,
2011]. Statistical test for population neutrality was
estimated using the Tajima statistical test conducted
in Mega 5 [Tajima, 1989; Nei and Kumar, 2000].
A negative Tajima’s D signifies an excess of low fre-
quency polymorphisms, indicating population size
The nucleotide sequence data reported in this paper
have been deposited in the GenBank nucleotide
sequence database with accession numbers JQ434459
Dye Terminator v.3.1 cycle sequencingkit
Six out of 78 fecal samples were positive for HAV
RNA. Phylogenetic analysis (MEGA version 5) of a
371 nucleotide long segment of the VP1/P2A sequen-
ces obtained from the six HAV RNA positive patients
in this study and 50 sequences downloaded from
GenBank showed that all the six new samples sequen-
ces clustered together to form a distinguished and
unique branch of genetically related HAV genotype IA
group (Fig. 1). Thus, results from this study further
confirm the predominance of HAV genotype 1 in most
parts of the world [Costa-Mattioli et al., 2003; Nainan
et al., 2006]. Results from the BLAST program found
that none of the HAV/IA isolates in this study were
identical to any sequence in the NCBI database thus
revealing their uniqueness.
The HAV isolates in this study did not intermix
with other HAV1/A sequences from other regions of
Africa, predominately from Tunisia obtained from the
GenBank. Also, they were not similar to HAV/IA
sequences found in other parts of the world. The six
HAV sequences isolated in this study were almost
identical to each other; clustering in one branch of
phylogenetic tree (Fig. 1). The mean genetic distance
among all genotype IA sequences generated in this
HAV Genotype 1A in Cameroon729
J. Med. Virol. DOI 10.1002/jmv
study was 0.5%. The nearly 100% (exact ¼ 99.5%) ge-
nome homology suggest a common or single source of
this infection in these children. Tajima test statistics
rejected the null hypothesis of population neutrality
and constant population size (D ¼ ?1.37; P ¼ 0.016).
Diarrhea is an important cause of disease and death
among children in developing countries [Guerrant
et al., 1990]. In Cameroon, diarrhea is very common
among children under 5 years of age [IRC and
USAID, 2008]. The role of HAV as an element in
diarrhea and its evolution has not been documented
in Cameroon. It is interesting that a previous sero-
where this study was conducted found that overall
IgG anti-HAV prevalence was 96.9%, reaching 100%
by the age of 11 years [Stroffolini et al., 1991].
Analysis of the HAV genotypes within a defined
Fig. 1. Maximum-likelihood tree based on Kimura 2-parameter model on 371 nucleotides of the HAV
VP1/P2A fragment. The tree with the highest log likelihood is shown. The tree was constructed using
the six sequences from this study and 50 sequences representing HAV genotypes I, II, and III from
various parts of the world obtained from the GenBank. GenBank strains have been identified by their
accession numbers. New strains from Cameroon are indicated with filled dark triangles. The numbers
at the nodes are the percentages of 1,000 bootstrap replicates higher than 70%.
730Forbi et al.
J. Med. Virol. DOI 10.1002/jmv
population is a useful tool for the study of the evolu-
tion of HAV infection in different geographic regions
transmission chains, and sources of infection.
HAV isolates obtained from six children presenting
with diarrheal disease from a clinic in Kumba–
Cameroon were analyzed in the present study. Al-
though these isolates were obtained from the same
clinic, they were sporadic cases and an epidemiologi-
cal link could not be established among cases. The iso-
lates were therefore considered to be representative of
strains circulating in the general population. Based
on the VP1/P2A region, all the six Cameroon isolates
clustered with genotype IA viruses and were therefore
classified as HAV/IA. This study corroborates the
notion of the worldwide distribution of HAV/IA
[Costa-Mattioli et al., 2003; Nainan et al., 2006]. The
exclusive finding of unique HAV/IA in this study
reveals that this strain of the virus exhibits a fitness
advantage in this area and plays a role in the aetiol-
ogy of diarrheal diseases among children in Came-
roon. Therefore clinicians need to consider HAV
infection in children presenting with diarrhea. This
also underscores the need to implement a country-
wide molecular epidemiological
infection and training of laboratory personnel in the
molecular diagnosis of this infection. An important
insight obtained from analysis of the phylogenetic re-
construction was the absence of clustering between
the Cameroonian HAV/IA strains with those from
other African countries and the rest of the world
(Fig. 1), suggesting that the ancestral sequences of
HAV/IA probably existed for a long period of time
enough to allow for speciation to occur. Furthermore,
the very close nucleotide homology (99.5%) observed
from the six HAV/IA strains in this study suggest a
community-wide spread of HAV/IA infection possibly
from a common source in the Kumba area since the
cases investigated were sporadic and thought to rep-
resent stains circulating in the entire region. Identifi-
cation of the source or sources of infection would
greatly limit the spread of HAV infection and also
contribute to the understanding of the epidemiology of
HAV infection in the region.
Contrary to this study findings, a French epidemio-
logical survey reported the detection of HAV genotype
IIA among French travelers to Cameroon [Desbois
et al., 2010] and concluded that they were imported
by the travelers returning from Cameroon. This is
suggestive of a genotype switch in the epidemiology of
HAV infection in Cameroon or a reflection of the
circulation of different HAV genotypes in the different
ecological zones of the country. Given the epidemiolog-
ical scenarios of the French study [Desbois et al.,
2010] it is difficult to assume that the travelers actu-
ally acquired the HAV/IIA infection from Cameroon.
Traveling makes the actual source of infection very
uncertain. The travelers probably had other opportu-
nities to acquire infection during the course of their
air travel and could also have visited other countries
and this makes the source of infection even more diffi-
cult to trace. In this context, the French study
remains only speculative.
This study was conducted among children under
5 years of age. Finding active HAV /IA infection in
this age group may be an indication that children in
this area probably play a role in maintaining and
transmitting the virus within the population. The
exclusive circulation of HAV/IA in a region of high
endemicity might be related to the young age of expo-
sure to HAV in Cameroon or an adaptation of HAV/IA
strain to the local population. This brings to light the
need for surveillance among young children and possi-
bly early intervention should be considered in this
subpopulation. HAV vaccine is not widely used in
Cameroon, the occurrence of active HAV infection
among children in Kumba implies that susceptible
populations for HAV infection exist in the country.
The introduction of the vaccine especially among
young children in this region might be important.
HAV is common in regions with compromised sanita-
tion [Poovorawan et al., 2002]. In developed countries,
routine HAV vaccination, improved sanitation, and
water supply had resulted in decline of HAV infection.
HAV strains associated with acute diarrheal diseases
among children in Cameroon were characterized.
Detecting high anti-HAV antibodies and finding active
cases (RNA positive) presupposes an expansion of
HAV infection in the country. A rejection of the null
hypotheses of population neutrality and constant size
provides additional support to this. A possible limita-
tion of this study is that the findings were restricted
to a specific geographical location where stool samples
were obtained. Additional molecular epidemiology
studies from other regions of the country will allow
for a better understanding of HAV genotype distribu-
tion and evolution in the entire country. To our
knowledge, this study presents the first report on the
distribution of HAV genotype in Cameroon.
Thanks to the staff of the clinic, parents, and guard-
ians of the children who participated in this study.
The supportfrom Benardine
Germany) is also acknowledged. The ideas in this
study are those of the authors and do not necessarily
represent the views of the institutions where they
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