Chapter 10
Production of Zika Virus Virus-Like Particles
Atichat Kuadkitkan, Suwipa Ramphan, Suchin Worawichawong,
Wannapa Sornjai, Nitwara Wikan, and Duncan R. Smith
Abstract
Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the
world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a
nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the
nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus
and thus present a similar antigenic profile. These properties make them good candidates for vaccine
development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins
into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP
can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation
of the VLP is achieved through western blotting and electron microscopy.
Key words Flavivirus, Structural proteins, Eukaryotic expression vector, Transfection, Centrifugation, Zika, Virus-like particles, Vaccine
1 Introduction
Mosquito transmitted viruses impose a significant public health
burden in many tropical and subtropical countries [1]. While
human pathogenic mosquito transmitted viruses belong to several
families, the largest health burden worldwide results from viruses
belonging to the family Flaviviridae, genus Flavivirus. A total of
53 viral species are recognized in this genus [2], and the genus
includes viruses such as dengue virus, yellow fever virus, Japanese
encephalitis virus, and West Nile virus. Over the last few years, one
member of the genus Flavivirus, Zika virus (ZIKV), has garnered
considerable public attention as a result of the rapid emergence of
this virus and its transmission around much of the world. ZIKV was
first isolated in 1947 [3], although following the original identification of ZIKV there were only a handful of reported cases of
human infection from Africa and Asia over the next 60 years (as reviewed elsewhere [4]). A large outbreak of ZIKV infections in
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
183
Production of Zika Virus Virus-Like Particles
Atichat Kuadkitkan, Suwipa Ramphan, Suchin Worawichawong,
Wannapa Sornjai, Nitwara Wikan, and Duncan R. Smith
Abstract
Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the
world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a
nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the
nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus
and thus present a similar antigenic profile. These properties make them good candidates for vaccine
development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins
into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP
can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation
of the VLP is achieved through western blotting and electron microscopy.
Key words Flavivirus, Structural proteins, Eukaryotic expression vector, Transfection, Centrifugation, Zika, Virus-like particles, Vaccine
1 Introduction
Mosquito transmitted viruses impose a significant public health
burden in many tropical and subtropical countries [1]. While
human pathogenic mosquito transmitted viruses belong to several
families, the largest health burden worldwide results from viruses
belonging to the family Flaviviridae, genus Flavivirus. A total of
53 viral species are recognized in this genus [2], and the genus
includes viruses such as dengue virus, yellow fever virus, Japanese
encephalitis virus, and West Nile virus. Over the last few years, one
member of the genus Flavivirus, Zika virus (ZIKV), has garnered
considerable public attention as a result of the rapid emergence of
this virus and its transmission around much of the world. ZIKV was
first isolated in 1947 [3], although following the original identification of ZIKV there were only a handful of reported cases of
human infection from Africa and Asia over the next 60 years (as reviewed elsewhere [4]). A large outbreak of ZIKV infections in
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
183