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Molecular Epidemiology of Paramyxoviruses in Frugivorous

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.