Eidolon helvum Bats in Zambia
In the past 10 years, a lot of attention has been given to bats as reservoirs of emerging
zoonotic viruses. This has been as a result of the high detection rate of previously unknown
viral sequences in bats coupled with the emergence of pathogens, such as Hendra, Nipah, Severe
acute respiratory syndrome (SARS)-Corona, Ebola and Marburg viruses, all of which are highly
virulent and pose a great zoonotic risk [2, 3, 8, 9, 17]. Bats, being
the only flying mammals with ancient evolutionary origins and long life span, are capable of
covering great distances during migrations, rendering them suitable hosts and reservoirs for
various viruses. Paramyxoviruses from the family Paramyxoviridae have been
implicated in several human epidemics and mortalities [6, 10, 19]. Several studies have indicated bats as potential natural reservoirs of
Paramyxoviruses, such as Henipavirus-, Respirovirus- and Morbillivirus-related viruses [1, 4]. This
undoubtedly presents a threat to the health of the human population in areas where human
beings live in close proximity to fruit bat species.
In Zambia, straw-colored fruit bats (Eidolon helvum) annually converge in
Kasanka National Park (KNP).
In this study, we investigated the presence of paramyxoviruses in the Eidolon
helvum bats captured over a period of 4 years (2008–2011) from KNP (S15:34.688
E28:16.513). During that period, a total of 312 spleen samples were collected from the same
number of bats (Table 1). Appropriate research permits and hunting licenses were obtained from the
Zambia Wildlife Authority (ZAWA).
Total RNA was isolated from spleen tissues using TRIzol (Life Technologies, Carlsbad, CA,
U.S.A.) according to the manufacturer’s instructions. A semi-nested broad spectrum RT-PCR
targeting the paramyxovirus polymerase (L) gene was used to screen total RNA
samples (n=312) for paramyxoviruses using PAR-F1, PAR-F2 and PAR-R primers and PCR conditions
described by Tong et al.. A total
of 25 samples out of 312 bat spleens (8%) were positive for paramyxoviruses on semi-nested
PCR. The positive products (584 bp) were then purified using the monofas purification kit (GL
sciences, Tokyo, Japan), according to the manufacturer’s instructions. The purified PCR
products were then subjected to Cycle sequencing reactions using the Big Dye Terminator v3.1
system (Life Technologies) and the PAR-F2 and PAR-R inner primers. Ethanol precipitation was
used to remove the labeled dNTPs from cycle sequence products and subjected to electrophoresis
in the ABI 3130 genetic analyzer (Life Technologies). Phylogenetic analysis was performed
using reference sequences and positive samples by aligning all sequences using ClustalW1.6
followed by the creation of a MEGA file format created using MEGA ver.5.2. The neighbor joining method was used to generate the
phylograms with a 1,000 bootstrap replicate
confidence level. To compute the evolutionary
distances, the Maximum Composite Likelihood method was used with the number of base
substitutions per site as units.
Samples AB853101, AB853102, AB853104, AB853105 and AB853094 showed a nucleotide homology of
73% with the Nipah virus (AF212302), while AB853106 and AB853096 had a nucleotide homology of
74% with the unclassified Bat paramyxovirus (Bat PV) (JN648087) from Ghana. The relatively low
nucleotide homology might indicate that these sequences originate from novel paramyxoviruses.
The samples from Zambia formed clusters with the Henipavirus-related viruses and with the
unclassified Bat paramyxoviruses (Fig. 1). Within the Henipavirus-related virus cluster, two groups (A and B) were observed.
Group A comprised novel Zambian strains closely related to the Nipah (NC002728, FN86955 and
AF212302) and Hendra (AF017149 and NC001906) viral sequences, while Group B comprised a
cluster of Zambian strains, in close relationship with an unclassified Bat PV from Ghana
(JN648085) and Cedar virus (JQ001776) (Fig. 1). The
remaining Zambian strains, including the novel AB853106 sequence, formed a cluster with the
unclassified Bat PV sequences from Ghana (JN648078, JN648081, JN648087 and JN648089) and Congo
Brazzaville (HE647835 and HE647837) (Fig. 1). The
close relatedness of the viral sequence from Ghana and Congo Brazzaville strains with those
from Zambia might imply the ability of bats to harbor and transmit similar viruses over long
and diverse geographical distances. This is facilitated by their ability to migrate, covering
thousands of kilometers to their hibernation and feeding sites. Along their migratory path, they interact directly or indirectly with
several terrestrial mammalian species in different geographical locations, thus enhancing the
interspecies transmission of viruses. Humans can
become exposed to these viruses through environmental contamination with urine and feces from
bats. Although paramyxovirus infections derived from bats have been reported in humans in
Bangladesh, none have been reported in Africa. The
absence of cases might be as a result of under-reporting. As such continued surveillance and
assessment of the zoonotic risk posed by these viruses still remains important.
In order to isolate the detected viruses, spleens from PCR positive bats were homogenized in
minimum essential media (MEM) followed by centrifugation at 1,000 × g for 3
min. The supernatant was then applied to Vero E6 cell with 70–80% growth confluence. The Vero
E6 cells were cultured in MEM with 2% fetal bovine serum (FBS) and 2% antibiotic-antimycotic
(Life Technologies). The inoculated Vero E6 cell cultures were then incubated at 37°C for 21
days, coupled with cell passage and microscopic examination. However, after several passages,
cytopathic effects were not observed. We also performed semi-nested RT-PCR to detect
paramyxovirus RNA in the supernatants of the inoculated cells. However, no positive signals
were detected. Isolation of paramyxoviruses using Vero E6 cells has successfully been reported
[18], implying that Vero E6 cell lines might be
suitable for isolation of paramyxoviruses. In this study, the small amount of virus in the
supernatant or the failure to successfully remove the virus from infected cells using the
freeze and thaw technique might be responsible for the absence of cytopathic effects in the
cell culture. In a study by Wilkinson, out of 8
positive samples, virus isolation was only successfully carried out in two samples.
Furthermore, serological examination of bat sera may provide important information about their
exposure to specific infections. Unfortunately, however, our study did not carry out any
serological test.
In conclusion, we report the identification of novel Henipavirus-related (n=5) and unrelated
(n=2) viruses in fruit bats from the KNP using RT-PCR. The viruses identified in this study
were shown to originate from wide geographical areas, and their presence in fruit bat species
might pose a public health risk and as such, continued surveillance of these viruses in fruit
bats in essential.