ORIGINAL ARTICLE
Griffithsin inhibits Japanese encephalitis virus infection
in vitro and in vivo
Hassan Z. A. Ishag
•
Chen Li
•
Li Huang
•
Ming-xia Sun
•
Fengjuan Wang
•
Bo Ni
•
Thowaiba Malik
•
Pu-yan Chen
•
Xiang Mao
Received: 8 June 2012 / Accepted: 16 August 2012 / Published online: 7 October 2012
Ó Springer-Verlag Wien 2012
Abstract Griffithsin (GRFT) is a broad-spectrum antivi-
ral protein that is effective against several glycosylated
viruses. Here, we have evaluated the in vitro and in vivo
antiviral activities of GRFT against Japanese encephalitis
virus (JEV) infection. In vitro experiments showed that
treatment of JEV with GRFT before inoculation of BHK-
21 cells inhibited infection in a dose-dependent manner,
with 99 % inhibition at 100 lg/ml and a 50 % inhibitory
concentration (IC
50
) of 265 ng/ml (20 nM). Binding assays
suggested that binding of GRFT to JEV virions inhibited
JEV infection. In vivo experiment showed that GRFT
(5 mg/kg) administered intraperitoneally before virus
infection could completely prevent mortality in mice
challenged intraperitoneally with a lethal dose of JEV. Our
study also suggested that GRFT prevents JEV infection at
the entry phase by targeting the virus. Collectively, our
data demonstrate that GRFT is an antiviral agent with
potential application in the development of therapeutics
against JEV or other flavivirus infections.
Introduction
Japanese encephalitis virus (JEV) is a mosquito-borne
virus belonging to the genus of Flavivirus, family of
Flaviviridae. JEV infection causes a broad spectrum of
clinical symptoms, ranging from asymptomatic infections
to fever, encephalitis, and even death. Encephalitis caused
by the infection is estimated to occur in 1 of 20 to 1000
cases [5, 12], with approximately 25 % of encephalitic
patients dying and 50 % of the survivors developing per-
manent neurologic and/or psychiatric sequelae. The JEV
genome is a single-stranded, positive-sense RNA encoding
a polyprotein of approximately 3,400 amino acids that is
cleaved into three structural proteins (capsid [C],
premembrane [prM] and envelope glycoprotein [E]) and
seven nonstructural proteins [23]. The JEV envelope gly-
coprotein (53–55 kDa) contains two potential glycosyla-
tion sites [9] that are important for virus attachment, fusion,
penetration, cell tropism, virulence and attenuation [14].
The JEV prM protein also contains one glycosylation site
that is important for virus release and pathogenesis [13].
Griffithsin (GRFT), a plant-derived antiviral protein, is
isolated from the red alga Griffithsia sp. Due to its lectin
activity, GRFT has been shown to have broad-spectrum
antiviral activity against several enveloped viruses,
including human immunodeficiency virus-1 (HIV-1) [16],
SARS-CoV [27], and hepatitis C virus (HCV) [15].
Recombinant GRFT produced in E. coli or in the Nicotiana
benthamiana expression system does not alter its potent
anti-HIV activity and its safety profile compared to native
GRFT [16, 17]. GRFT is a dimeric lectin with six principal
binding sites that can bind to the SARS-CoV spike gly-
coprotein (S) and prevent virus entry into target cells [27].
Because of the presence of glycans on the JEV virion, we
investigated whether GRFT displays antiviral activity
against JEV infection. Here, we demonstrate that GRFT
inhibits JEV entry into host cells at nanomolar concentra-
tions. Furthermore, we show that the inhibition was due to
the binding ability of GRFT to the glycans on JEV virions.
H. Z. A. Ishag Á C. Li Á L. Huang Á M. Sun Á F. Wang Á B. Ni Á
T. Malik Á P. Chen Á X. Mao (&)
College of Veterinary Medicine, Nanjing Agricultural
University, 1 Weigang, Nanjing 210095,
Jiangsu Province, China
e-mail: xmao@njau.edu.cn
H. Z. A. Ishag
College of Veterinary Science, Nyala University,
Nyala, South Darfur State, Sudan
123
Arch Virol (2013) 158:349–358
DOI 10.1007/s00705-012-1489-2
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