Arbovirus vaccines; opportunities for the baculovirus-insect cell expression system

Journal of Invertebrate Pathology 107 (2011) S16–S30

Contents lists available at ScienceDirect

Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip

Minireview

Arbovirus vaccines; opportunities for the baculovirus-insect cell expression system Stefan W. Metz, Gorben P. Pijlman ⇑ Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

a r t i c l e

i n f o

Keywords: Arbovirus Subunit vaccine Virus-like particles Baculovirus Insect cells

a b s t r a c t The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging group of medically important viral pathogens that are transmitted to humans and animals via arthropod vectors, mostly mosquitoes, ticks or midges. Few arboviral vaccines are currently available, but there is a growing need for safe and effective vaccines against some highly pathogenic arboviruses such as Chikungunya, dengue, West Nile, Rift Valley fever and Bluetongue viruses. This comprehensive review discusses the biology and current state of the art in vaccine development for arboviruses belonging to the families Togaviridae, Flaviviridae, Bunyaviridae and Reoviridae and the potential of the baculovirus-insect cell expression system for vaccine antigen production The members of three of these four arbovirus families have enveloped virions and display immunodominant glycoproteins with a complex structure at their surface. Baculovirus expression of viral antigens often leads to correctly folded and processed (glyco)proteins able to induce protective immunity in animal models and humans. As arboviruses occupy a unique position in the virosphere in that they also actively replicate in arthropod cells, the baculovirus-insect cell expression system is well suited to produce arboviral proteins with correct folding and post-translational processing. The opportunities for recombinant baculoviruses to aid in the development of safe and effective subunit and virus-like particle vaccines against arboviral diseases are discussed. Ó 2011 Elsevier Inc. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medically important and emerging arboviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Togaviridae – genus Alphavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Flaviviridae – genus Flavivirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Bunyaviridae – genera Orthobunyavirus, Phlebovirus and Nairovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Reoviridae – genus Orbivirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arbovirus vaccinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Alphavirus vaccinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Flavivirus vaccinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Bunyaviridae vaccinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Reoviridae vaccinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

S16 S18 S18 S22 S22 S23 S23 S23 S24 S25 S26 S26 S27 S27 S27

1. Introduction

⇑ Corresponding author. Fax: +31 317 484820. E-mail address: [email protected] (G.P. Pijlman). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.05.002

Baculoviruses have been used since the early 1980s to express transgenes in cultured insect cells (Summers and Smith, 1987). This now well-established technology is capable of generating high yields of heterologous protein and can be easily scaled up to large

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

volume insect-cell bioreactors. The baculovirus-insect cell expression system has resulted in the production of thousands of proteins used for scientific purposes, but also for the development of therapeutic peptides and (subunit) vaccines. A wide variety of viral antigens has been expressed for this purpose by recombinant baculoviruses, ranging from relatively simple viral coat proteins to complex, secreted and/or glycosylated subunits (van Oers, 2006). Expression in insect cells is ideally suited to safely produce proteins with complex folding and post-translational processing (e.g. glycosylation) found exclusively in higher eukaryotes. Furthermore, baculoviruses do not replicate in mammals and recombinants can therefore be constructed and produced with relative safety. Because baculoviruses are insect viruses with optimal virus replication in the range of 25–30 °C, the expression system is also of high value for production of proteins derived from insects or other ectothermic animals that may require lower temperatures for optimal biological activity. In fact, there are many pathogenic mammalian viruses that have an additional, active replication cycle in insects or other arthropods. These so-called arthropod-borne (arbo-)viruses are transmitted to humans and/or other mammals by blood-feeding arthropod vectors such as mosquitoes, ticks, midges or sandflies. In contrast to most other mammalian viruses, arboviruses have a very wide temperature window for viral RNA replication, ranging from approximately 10–40 °C. Arbovirus replication in warm-blooded or endothermic hosts, dependent on the host species, occurs around 37 °C, whereas virus replication in the ectothermic, arthropod vector takes place at the ambient environmental temperatures. The baculovirus-insect cell expression system is an attractive platform to produce these proteins. Arboviruses are found in four families of RNA viruses: Togaviridae, Flaviviridae, Bunyaviridae and Reoviridae (Fig. 1, Table 1). Not all viruses belonging to these families are arboviruses by definition. Some are not transmitted by arthropod vectors (e.g. Hepatitis C virus in the family of Flaviviridae), whereas others are vectored by arthropods but infect plants (e.g. Tomato spotted wilt virus in the family of Bunyaviridae). Highly pathogenic arboviruses that are transmitted by mosquitoes include Chikungunya virus (CHIKV), Dengue virus (DENV), West Nile virus (WNV) and Crimean Congo hemorrhagic fever virus (CCHFV). They have a significant worldwide impact on human health by causing a variety of diseases including (hemorrhagic) fever, hepatitis and encephalitis, leading to hundreds of thousands of deaths each year (Whitehead et al., 2007). Epidemics of animal arboviruses, such as Venezuelan equine encephalitis virus (VEEV) and Bluetongue virus (BTV) are on the increase as well and can cause dramatic losses of livestock in short periods of time, especially in cases when no vaccines or antiviral

S17

treatments are available (Paessler and Weaver, 2009; Savini et al., 2008). In a literature study covering an extensive range of 1407 human pathogens, it was found that out of all 208 viral diseases, 77 were emerging viruses, of which approximately 50 were zoonotic (Woolhouse and Gowtage-Sequeria, 2005). The vast majority of these zoonotic viruses are not transmitted directly between humans and include many arboviruses circulating in animals. It is clear from these numbers that focus should be shifted towards the interdisciplinary approach of human and animal health. The One Health Initiative (www.onehealthinitiative.com) acknowledges that 70% of emerging infections are vector-borne or zoonotic and therefore strives to unite the fields of human and veterinary medicine. The threat of arboviral diseases is further illustrated by the recent EmZoo Research Program report by the Dutch National Institute for Public Health and the Environment (RIVM). They presented a priority list of 25 zoonotic pathogens important for the Netherlands, where 6 out of 14 non-endemic zoonotic pathogens were arboviruses (Havelaar et al., 2010). The threat of emerging vector-borne viral diseases has been generally recognized and many arboviral vaccines are now in clinical development or commercially available (Table 2). Increased global trade, transport and travel in combination with changing climate and increasing population density are considered important factors in the emergence and increased incidence of arbovirus-related illnesses worldwide, particularly in the temperate regions of Western Europe and North America. In some cases, arboviruses are introduced into areas with naïve host and vector populations, the so-called virgin-soil territories, with the striking example of WNV introduction in the USA in 1999. In subsequent years, WNV was spread over the entire North American continent by native birds and mosquitoes and caused mortality in thousands of humans, horses and birds (Murray et al., 2010). In other examples, the insect vectors colonized new habitats allowing for associated arboviruses to invade new territory. A dramatic, recent example of global vector distribution is the Asian tiger mosquito, Aedes albopictus, which originated in Asia but is now endemic in large parts of the USA and Southern Europe (Paupy et al., 2009). The recent arbovirus epidemic in Italy of CHIKV in 2007 involved multiple human cases and was a direct consequence of the introduction and establishment of Ae. albopictus in the temperate climate of Italy and Southern France. In this particular case, it only took a single individual to introduce CHIKV from an epidemic area in India to initiate a novel arboviral epidemic in Italy (Angelini et al., 2008). Most recently, in September 2010, two autochthonous cases of DENV and two cases of CHIKV in the South of France were reported,

Fig. 1. Arbovirus particles. Virion images generated from Protein Data Bank data (A) Chikungunya virus (based on Sindbis EM density, PDB ID: 2XFB), (B) Dengue virus (PDB ID: 1K4R) and (D) Bluetongue virus (PDB ID: 2BTV). Images were created with VMD (www.ks.uiuc.edu/Research/vmd/). (C) Rift valley fever virus particle. Image was created with UCSF Chimera (www.cgl.ucsf.edu/chimera/) using EM-density map data from EMDB database (RVFV ID 5124). Images were kindly provided by Jean-Yves Sgro.

S18

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

Table 1 Arboviruses known to cause disease in humans or animals. Family/genus

Species

Vector

Host

Clinical symptoms

Geographical distribution

Togaviridae Alphavirus

Eastern equine encephalitis virus (EEEV)

Mosquito

Culiseta sp.

Human, equine Human, equine

Febrile illness, encephalitis Febrile illness, encephalitis

N/S-America

Western equine encephalitis virus (WEEV)

Mosquito

Culex sp.

Venezuelan equine encephalitis virus (VEEV) Sindbis virus (SINV)

Mosquito

Culiseta sp. Culex sp.

Human, equine Human, bird

Febrile illness, encephalitis Febrile illness, arthritis

N/S-America

Mosquito

Culex sp.

Febrile illness, arthritis

Aedes sp. Anopheles sp. Culex sp.

Human, rodent Human Human Human

Africa, Asia, Australia, Asia, Europe Africa, Asia

Semliki Forest virus (SFV)

Mosquito

Aedes sp.

Chikungunya virus (CHIKV) O’nyong nyong virus (ONNV) Ross River virus (RRV)

Mosquito Mosquito Mosquito

Febrile illness, arthritis Febrile illness, arthritis Febrile illness, arthritis

Africa, Asia, Europe Africa Australia

Dengue 1–4 (DENV)

Mosquito

Aedes sp.

Human

Yellow fever virus (YFV)

Mosquito

Aedes sp.

Human

N/S-America, Asia, Australia, Africa N/S-America, Africa

Japanese encephalitis virus (JEV)

Mosquito

Culex sp.

Human

West Nile virus (WNV)

Mosquito

Culex sp.

St.Louis encephalitis virus (SLEV) Tick-borne encephalitis virus (TBEV)

Mosquito Tick

Culex sp. Ixodes sp.

Human, equine Human Human

Febrile illness, hemorrhagic fever Hepatitis, hemorrhagic fever Febrile illness, encephalitis Febrile illness, encephalitis Encephalitis Encephalitis

Tick

Hemorrhagic fever

Africa, Asia, Europe

Mosquito Mosquito

Hyalomma sp. Aedes sp. Aedes sp.

Human

Orthobunyavirus Phlebovirus

Crimean-Congo hemorrhagic fever virus (CCHFV) La Crosse virus (LACV) Rift valley fever virus (RVFV)

Human Human, ruminant

Encephalitis Hemorrhagic fever, encephalitis

N-America Africa

Reoviridae Orbivirus

Bluetongue virus (BTV)

Midge

Culicoides sp.

Ruminants

Febrile illness, cyanosis

African horse-sickness virus (AHSV) Colorado tick fever virus (CTFV)

Midge Tick

Culicoides sp. Dermacentor sp.

Equine Human

Respiratory failure, fever Febrile illness, malaise

N/S-America, Africa, Asia, Europe Africa, M-East, Europe N-America

Flaviviridae Flavivirus

Bunyaviridae Nairovirus

N/S-America

SE Asia N-America, Africa, M-East, Europe N/S-America Europe, Asia

Data according to the latest update of the Centers for Disease Control and Prevention (CDC) website (2010).

most likely the result of arbovirus circulation in the established, local population of Ae. albopictus (Gould et al., 2010). These occurrences exemplify the ongoing threat associated with the continuous expanding distribution of Ae. albopictus and the associated pathogenic arboviruses. Although development is ongoing, no licensed vaccines or antivirals are available for the prevention or treatment of either DENV or CHIKV infections. The question is, what is the best solution to fight arbovirus epidemics? Should the focus be on vector control or is the money better spent on the development of vaccines and antiviral drugs? In the short term, vector control may provide some relief and can be used to contain small scale epidemics. However, recent attempts to control larger mosquito populations in Italy by using insecticides have proven difficult (Cavrini et al., 2009). Most likely, a balanced combination of a change in human behavior (e.g. protective clothing, reduction of mosquito breeding places in urban areas), vector control and the use of antivirals and vaccines will provide the best solution to limit arbovirus epidemics. Vaccines are probably the most effective tools to protect humans and livestock against arboviral disease, although this will unfortunately not stop arbovirus transmission when other reservoir animals are involved, e.g. wild birds as in the case of WNV. At present, only a few arboviral vaccines are licensed for use in humans and are directed against Yellow fever virus (YFV), Japanese encephalitis virus (JEV) and Tick-borne encephalitis virus (TBEV) (family Flaviviridae). For animals, WNV, RVFV and BTV vaccines are currently available.

The baculovirus-insect cell expression system is a safe and efficient method for large-scale expression of heterologous proteins in eukaryotic cells (Kost et al., 2005), which has led to many commercial veterinary vaccines (van Oers, 2006). The recent release of a human vaccine against cervical cancer (Cervarix, GlaxoSmithKline) (Paavonen and Lehtinen, 2008) is expected to pave the way for novel vaccine development using this baculovirus-insect cell platform technology. In this review, we will focus on arboviral vaccines. As arboviruses can efficiently replicate in cells of arthropod origin, the folding and glycosylation patterns of arboviral proteins produced in baculovirus-infected insect cells are often correct. We will describe the four arbovirus families, the architecture of their virions and their associated immunogenic proteins, demonstrate the state-of-the art in vaccine development and discuss the challenges ahead. More specifically, we will provide insight into the potential of the baculovirus expression system as a fast and robust platform technology to generate subunit and virus-like particle vaccines against arboviral diseases.

2. Medically important and emerging arboviruses 2.1. Togaviridae – genus Alphavirus The family Togaviridae encompasses a large group of enveloped plus strand RNA viruses and is divided into the genera Alphavirus

S19

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30 Table 2 Vaccines and vaccination studies targeting arbovirus infections. Family/genus Togaviridae Alphavirus

Species

Vaccine strategy

Production/ expression system

Antigen(s)

References

Venezuelan equine encephalitis virus (VEEV)

Inactivated virus

Chicken embryo

Whole virus

Randall et al. (1949)

a

DNA Subunit(s)

Ross River virus (RRV) Chikungunya virus (CHIKV)

Inactivated virus Inactivated virus Live-attenuated Chimeric-vector DNA c

VLP subunit

BHK21 -cells, HEK293-cells pES expression vector Baculovirus expression Vero-cells GMKb-cells GMK-cells BHK21-cells, HEK293-cells pVax1 expression vector 239T-cells

Hart et al. (2000) E1 and E2

Dupuy et al. (2009)

C–E3–E2–6K– E1 6K–E1

Hodgson et al. (1999)

Whole virus Whole virus Whole virus C–E3–E2–6 K– E1 C, E1 and E2

Kistner et al. (2007) Harrison et al. (1971) Levitt et al. (1986) Wang et al. (2008, 2011)

C–E3–E2–6 K– E1

Mallilankaraman et al. (2011) and Muthumani et al. (2008) Akahata et al. (2010)

Flaviviridae Flavivirus

Dengue virus (DENV)

Live-attenuated

Mouse brain, PDKd-cells

Whole virus

Chimeric-vector DNA

BHK21-cells pkCMVintPolyli, pVR1012 VEE-replicon expression Baculovirus expression

prM–E prM–E

VRPe Subunit(s)

Japanese encephalitis virus (JEV)

VLP subunit

Sf9f cells

Inactivated virus

Chicken embryo fibroblasts BHK21-cells Vero-cells

Chimeric-vector Subunits(s)

VLP subunit

Baculovirus expression Baculovirus expression Sf9 cells

Yellow fever virus (YFV)

Live-attenuated (17D)

Chicken embryo

West Nile virus (WNV)

Inactivated virus InnovatorÒ Duvaxyn WNVÒ (Europe) Inactivated virus

VLP subunit

prM–E

Kanesa-Thasan et al. (2003), Kitchener et al. (2006) and Sabin and Schlesinger (1945) Edelman et al. (2003) Guirakhoo et al. (2006) Kochel et al. (1997) and Raviprakash et al. (2000) Chen et al. (2007)

E truncated

Eckels et al. (1994) and Staropoli et al. (1997)

prM–E prM–E

Delenda et al. (1994) and Kelly et al. (2000) Kuwahara and Konishi (2010)

Whole virus

(Kyoto Biken Laboratories, Japan)

ChimeriVax technology E truncated prM–E

Lili et al. (2003) Sanofi-Pasteur, France Li et al. (2009) Konishi et al. (1992)

prM–E

Kuwahara and Konishi (2010)

Whole virus

Bugher and Smith (1944)

Whole virus

Fort Dodge Animal Health, Pfizer Animal Health, USA Razumov et al. (1994)

Mouse brain

Whole virus

Rec. canarypox virus RecombitekÒ Chimeric-vector PreveNile™

vCP2017, CEFcells Vero-cells

prM–E

Kimron Veterinary Institute (Israel) Samina et al. (2005) Merial, France Minke et al. (2004)

Chimeric-vector

Vero-cells

Chimeric-vector

WN/DEN4– 3’delta30 BHK-cells

Single-cycle vaccine RepliVax WNÒ DNA

DNA DNA Subunit(s) Subunits(s) VLP subunit Bacteriophage VLP

VRCWNVDNA020–00VP pKUN1 SRIPsg Insect larvae Baculovirus expression CHO-cells BL21 DE3 cells

prM–E

Intervet, Schering Plough, USA Razumov et al. (1994)

ChimeriVax technology prM–E

Acambis, Sanofi Pasteur, France Hall and Khromykh (2007) NIAID/NIH, USA Razumov et al. (1994)

prM–E

Nelson et al. (2010)

prM–E

NIAID/NIH, USA Razumov et al. (1994)

Whole virus Whole virus E-domain III E truncated

Hall et al. (2003) Chang et al. (2008) Alonso-Padilla et al. (2011) Bonafe et al. (2009) and Chu et al. (2007)

C–prM–E E-domain III

Ohtaki et al. (2010) Spohn et al. (2010) (continued on next page)

S20

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

Table 2 (continued) Family/genus

Species

Vaccine strategy carrier VLP subunit

Production/ expression system

Antigen(s)

References

Baculovirus expression

C–prM–E

Qiao et al. (2004)

prM–E Tick-borne encephalitis virus (TBEV)

Inactivated virus FSME- ImmuneÒ Inactivated virus EncepurÒ Adult Inactivated virus Inactivated virus EnceVirÒ Subunit(s) Subunit(s)

CECh-cells

Whole virus

Baxter Vaccine AG, Austria

CEC-cells

Whole virus

CEC-cells CEC-cells

Whole virus Whole virus

Bock et al. (1990) Novartis Vaccines, Germany RAMSci, Russia Microgen, Russia

Baculovirus expression Baculovirus expression

C–E

Gomez et al. (2003)

prM–E

Liu et al. (2005)

Chicken embryo/ Mouse brain Mouse brain/HPFicells pWRG7077 expression vector Baculovirus expression Drosophila S2-cells 293T-cells Drosophila S2-cells

Whole virus Whole virus

Eddy et al. (1981; Randall et al. (1962) and Barnard and Botha (1977) Morrill et al. (1991)

GN–GC

Spik et al. (2006)

M-segment GC GN N–GN–GC GN–GC

Schmaljohn et al. (1989) de Boer et al. (2010) Naslund et al. (2009) de Boer et al. (2010)

Bunyaviridae Phlebovirus

Rift valley fever virus (RVFV)

Inactivated virus Live-attenuated DNA Subunit(s) Subunit(s) VLP subunit VLP subunit

Nairovirus

Crimean-Congo hemorrhagic fever virus (CCHFV)

DNA

pWRG7077 expression vector

GN–GC

Spik et al. (2006)

Orthobunyavirus

La Crosse virus (LACV)

DNA

pVR1012 expression vector Baculovirus expression

GN–GC

Schuh et al. (1999)

GC

Pekosz et al. (1995)

Whole virus

Merial, France

Whole virus

CZ Veterinaria S.A, Spain

Whole virus

Fort Dodge Animal Health, Pfizer Animal Health, USA Razumov et al. (1994) Intervet, Schering Plough, USA Razumov et al. (1994) Dungu et al. (2004) and Patta et al. (2004) (French et al. (1990) and Roy et al. (1994)

Subunit(s)

Reoviridae Orbivirus

Bluetongue virus(BTV)

Inactivated virus BTVPUR ALSap™8 Inactivated virus BLUEVACÒ 8 Inactivated virus ZulvacÒ8 Inactivated virus Bovilis BTV8Ò Live-attenuated VLP subunit VLP subunit

African horse-sickness virus (AHSV)

Subunit(s) VLP subunit

Whole virus Chicken embryo Baculovirus expression Baculovirus expression

Whole virus VP3–VP7– VP2–VP5 VP2–VP5

Baculovirus expression Baculovirus expression

VP2–VP5

Roy et al. (1996)

VP3–VP7– VP2–VP5

Roy and Sutton (1998)

Stewart et al. (2010)

Commercially available vaccines are indicated in bold. a Baby hamster kidney cells. b Green monkey kidney cells. c Virus like particle. d Primary dog kidney cells. e Virus replicon particle. f Spodoptera frugiperda cells. g Single-round infectious particle. h Chick embryo cells. i Human diploid fibroblasts.

and Rubivirus. The enveloped virions are approximately 70 nm in diameter and spherical in appearance (Andrewes, 1952; Kuhn, 2007) (Fig. 2A). Only the alphaviruses are arthropod-borne and they use mosquito vectors for viral transmission (Strauss and Strauss, 1994) (Table 1). Alphaviruses are an emerging threat to human health with recent epidemics of CHIKV as a striking example. Rubella virus, so far the only member of the Rubivirus genus,

has no known arthropod vector and is transmitted by aerosol (Kuhn, 2007). Based on their geographical distribution, alphaviruses are divided into the New World alphaviruses, which include Western- (WEEV), Eastern- (EEEV) and, Venezuelan equine encephalitis virus (VEEV) and the Old World (OW) alphaviruses, Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV) and CHIKV. The majority of the NW alphaviruses are mainly

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

S21

Fig. 2. Schematic representation of (A) Togaviridae, (B) Flaviviridae, (C) Bunyaviridae, (D) Reoviridae cross-sections and ER-membrane organisation of their respective glycoproteins. N- and C-terminal ends are indicated. Dark arrows represent cleavage sites for S (host signalases), F (furin-like proteases) and P (viral serine protease). Glycosylation sites are indicated by blue chains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

found and broadly distributed throughout the American continents and are known to cause encephalitis in humans (Zacks and Paess-

ler, 2010). The OW alphaviruses are genetically and structurally very similar to the NW alphaviruses yet only sporadically cause se-

S22

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

vere neurological symptoms. The major clinical symptoms caused by e.g. CHIKV are febrile illness and severe long lasting joint pain and arthritis (Solignat et al., 2009). Alphaviruses (Fig. 1A) have a single-stranded, positive-sense RNA genome of approximately 11.8 kb (Khan et al., 2002). The RNA is encapsidated in a nucleocapsid, which is enveloped by a host-derived lipid bilayer supporting eighty trimeric glycoprotein-spikes, that facilitate cell receptor recognition and cell entry via low pH-dependent endocytosis (Fig. 2A). The genome contains two open reading frames encoding the non-structural polyprotein and the structural polyprotein (Strauss et al., 1984). The non-structural proteins (nsP1-4) regulate viral RNA replication and are directly translated from the 5’end of the genome. The structural proteins (Capsid or C, E3, E2, 6K, E1) constitute the virion and are translated from a subgenomic mRNA located in the 30 segment of the genome. During translation of the structural polyprotein, the capsid (C) is autocatalytically cleaved and the remaining envelope cassette is translocated to the ER by signal sequences in E3 and 6K (Fig. 2A). Proteolytic processing by host signalases cleaves 6K at the N-and C-terminal end, yielding E3E2 or precursor E2 (PE2), 6K and E1 (Kuhn, 2007). This processing enables the formation of heterodimers of PE2 and E1, three of which are assembled into trimers in the early Golgi compartment. Subsequently, E3 is released from PE2 by furin-dependent maturation in the trans-Golgi system. Furin cleavage is not a prerequisite for virion assembly, but incomplete processing will result in mature virions with impaired fusion properties (Strauss and Strauss, 1994). The mature heterotrimers are displayed at the cell-surface as trimeric spikes and the viral RNA is encapsidated in nucleocapsids composed of multiple copies of C (Weiss et al., 1989). Virion budding is most likely regulated upon the interaction between the intracellular domain of E2 with a single C molecule within the nucleocapsid. The nucleocapsid buds out from the cell, taking along the hosts plasma membrane and the trimeric spikes (Garoff et al., 2004). During an alphavirus infection, neutralizing antibodies are mainly directed against E2 and to a lesser extent to E1, making them primary targets for subunit vaccine development (Hunt et al., 2010; Strauss et al., 1991; Vrati et al., 1988). 2.2. Flaviviridae – genus Flavivirus The Flaviviridae family comprises three genera (Flavivirus, Hepacivirus and Pestivirus) and represents a large group of viruses that are associated with disease and mortality in humans and animals. Symptoms of infection may range from fever and general malaise, to hemorrhagic fever and fatal encephalitis. Only the Flavivirus genus contains arboviruses like DENV, WNV, JEV, YFV, and TBEV, which are transmitted by mosquitoes or ticks (Mukhopadhyay et al., 2005). The arthropod vector becomes infected when feeding on infected hosts. In the case of WNV, the hosts that develop viraemia sufficiently high to transmit the virus to blood-feeding mosquitoes are mainly birds. WNV is transmitted by the mosquitoes to other susceptible hosts such as humans and horses. These mammalian hosts are considered dead-end hosts, since the resulting viral load is not sufficient for further transmission (Weaver and Barrett, 2004). Other flaviviruses e.g. DENV have an urban replication cycle involving only humans and mosquitoes (Gubler et al., 2007). Flaviviruses (Fig. 1B) have an unsegmented, positive-strand RNA genome of approximately 10.8-kb, which is encapsidated in a nucleocapsid that is surrounded by 180 copies of two glycoproteins, anchored in a host-derived lipid bilayer (Lindenbach and Rice, 2001) (Fig. 2B). The genome has a single open reading frame (ORF) coding for one polyprotein. The 5’-end of the genome encodes three structural proteins: Capsid (C), precursor membrane

(prM) and envelope (E). The non-structural proteins NS1, -2A, 2B, -3, -4A, -4B and -5 are encoded on the remainder of the genome and are essential for viral RNA replication, which occurs in the cytoplasm of the infected cell (Lindenbach and Rice, 2001). After initial infection and release of the genomic RNA, the genome is translated into a single polyprotein, which is inserted into to the ER membrane. Signal sequences cause the polyprotein to translocate NS1, prM and E into the ER lumen, whereas C, NS3 and NS5 localize to the cytoplasm (Fig. 2B) (Westaway et al., 2003). NS2A/ B and NS4A/B are transmembrane proteins and span the ER membrane multiple times. The polyprotein is co- and post-translationally cleaved by host signalases (ER lumen) and by the viral protease NS3 with its cofactor NS2B (cytoplasm) (Falgout et al., 1991; Perera and Kuhn, 2008). Following initial proteolytic cleavage, C localizes to the cytoplasm, but remains associated with the ER. Shortly after translation, prM and E will form stable heterodimers in the ER lumen (Lorenz et al., 2002; Mukhopadhyay et al., 2005). The NS1 and prM proteins are glycosylated in the ER lumen. The E protein however, is only glycosylated in certain flavivirus species including DENV (Kelly et al., 2000). Flavivirus assembly starts with the encapsidation of viral RNA leading to the formation of the nucleocapsids. These nucleocapsids are thought to be short lived and bud directly into the ER. This process is regulated by the membrane-bound capsid proteins and the prM–E heterodimers in the ER lumen and initially results in the generation of immature viral particles (Zhang et al., 2003). The immature particles are structurally different from mature viral particles as they are covered with prM–E spikes, composed of three interacting prM–E heterodimers (Li et al., 2008). During the transport to the trans-Golgi, acidification causes conformational changes within the prM–E heterodimers and flattens the surface. This enables host-dependent furin cleavage on prM, resulting in mature particles, composed of a nucleocapsid and a lipid bilayer envelope exposing M-E spikes (Yu et al., 2008). In addition to the release of mature viral particles, the production of a second, socalled subviral particle is often observed during flavivirus infections. These smaller, smooth particles are assembled in the ER and undergo similar post-translational processing as the immature viral particles. Subviral particles, however, lack a nucleocapsid and consist only of a lipid membrane and the two prM and E glycoproteins and are alternatively called prM–E particles (Lorenz et al., 2003; Mukhopadhyay et al., 2005). The flavivirus E protein is considered to be the immunodominant epitope of flaviviruses, as it induces the production of neutralizing antibodies (Lindenbach and Rice, 2001). 2.3. Bunyaviridae – genera Orthobunyavirus, Phlebovirus and Nairovirus The Bunyaviridae family is one of the largest groups of animal viruses, with over 300 virus species, almost all of which are transmitted by arthropods. However, only the viruses replicating in arthropods and causing disease in animals are regarded as arboviruses. The Bunyaviridae family members are classified into five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus and Tospovirus (Table 1) (Elliott, 1997). Orthobunyaviruses and phleboviruses are in general transmitted by mosquitoes and midges, whereas nairoviruses and phleboviruses are transmitted by sandflies and ticks. Tospoviruses are the only plant-infecting members of the Bunyaviridae and are therefore not called arboviruses, yet they are transmitted by thrips vectors, in which they actively replicate similar to arboviruses in other arthropods (Elliott, 1990). Hantaviruses, in contrast, do not have an arthropod vector, but persist in rodents and are transmitted to human via aerosol (Schmaljohn and Hjelle, 1997). The arboviruses in the family Bunyaviridae are widespread over all continents and cause severe diseases with

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

S23

symptoms including headaches, chills, abdominal pains, encephalitis, hemorrhagic fever and fatal respiratory syndrome in humans and animals (Swanepoel, 1995). Viruses such as La Crosse virus (LACV), Rift Valley fever virus (RVFV) and Crimean-Congo hemorrhagic fever virus (CCHFV) have gained much attention in recent years due to increased prevalence and severe clinical manifestations in humans. The spherical virus particle (Fig. 1C) of approximately 100 nm has a viral envelope consisting of a host derived lipid bilayer in which glycoprotein spikes are anchored (Fig. 2C) (Murphy et al., 1973; Pettersson et al., 1971; Pettersson and Von Bonsdorff, 1987; Schmaljohn and Hooper, 2001; Smith and Pifat, 1982; Talmon et al., 1987; Whitehouse, 2004). All bunya-, phlebo- and nairoviruses have a segmented negative-strand RNA genome. The genome is divided into three RNA segments: small (S), medium (M) and large (L). The L-segment encodes the L-protein that functions as the RNA dependent RNA polymerase (RdRP) (Endres et al., 1989). The M-segment encodes a polyprotein that is co- and posttranslationally processed into the viral glycoproteins Gn and Gc and the non-structural protein NSm (Lees et al., 1986). The S-segment of bunya- and phleboviruses encodes the nucleocapsid protein N and a non-structural protein called NSs. The S-segment of the nairoviruses, however, only encodes the N-protein (Elliott, 1990). The multifunctional N protein forms ribonucleoprotein (RNP) complexes with the three viral RNA-segments, and the L protein associates with all three RNA-segments. The N protein is likely involved in linking the RNPs to the envelope proteins, but the exact mechanism by which RNA segment complexes are incorporated in a coordinated fashion into the viral particles, is unclear (Kaukinen et al., 2005). The two glycoproteins Gn and Gc are essential in the formation and maturation of progeny viral particles. In the ER, Gn and Gc are cotranslationally cleaved from the same precursor by signalases (Fig. 2C). Gn is glycosylated at one site, where the amount of Gc glycosylation sites may vary between species, but is conserved within serotypes (Gonzalez-Scarano, 1985). Heterodimerization between Gn and Gc occurs rapidly after translation and most likely takes place in the ER. Only newly synthesized Gc and mature Gn are able to form heterodimers. The Gn and Gc from the same precursor molecule will therefore not associate into one dimeric spike (Persson and Pettersson, 1991). The Gn/Gc dimers are subsequently transported to the Golgi apparatus, where maturation of viral particles occurs and virions bud into Golgi vesicles. Subsequent transportation to the plasma membrane and exocytosis releases the viral particles in the extracellular space (Elliott, 1990). Next to their implication in virus maturation, the glycoproteins are important in virulence, neutralization of the virus, cell fusion and receptor recognition. It is believed that Gc is involved in all processes and serves as the major antigenic determinant of the virus, although Gn and N are also immunogenic (Najjar et al., 1985; Pekosz et al., 1995; Sundin et al., 1987).

vector and is widely distributed over all continents except Antartica. Climatic changes have caused the vector to spread to more temperate regions, thereby drastically altering the global distribution of BTV (Wilson and Mellor, 2008). Bluetongue disease is characterized by several clinical symptoms such as excessive salivation, face and tongue swelling and cyanosis, the typical blue coloration of the tongue. The disease is mostly self-resolved, but in some cases mortality rates can be as high as 30% (Maclachlan et al., 2009). BTV (Fig. 1D) is a non-enveloped virus with a capsid composed of two protein shells, that encapsidate the ten-segmented dsRNA genome (Mertens et al., 2004; Roy et al., 1990). Each RNA segment encodes one of the three non-structural proteins (NS1, NS2 and Ns3/NS3a) or one of the seven structural proteins (VP1–VP7) (Roy, 1989) The dsRNA segments VP1, -4 and -6 code for the transcription complex, which is incorporated within the inner capsid shell (Fig. 2D). This inner shell is a complex structure built up from 260 trimers of VP7, which is supported by a layer of 120 copies of VP3 (Prasad et al., 1992). The inner capsid is surrounded by the outer capsid shell, composed of VP2 and VP5, which regulate cell recognition and cell entry, respectively. Furthermore, VP2 is highly variable and induces a neutralizing antibody response. Therefore, VP2 is considered to be the major antigenic determinant among BTV serotypes (Roy, 2008b). VP5 is a globular protein, of which 360 copies are positioned on top of the VP7 trimers. The VP2 protein is sail-shaped and has triskelion spikes that are protruding from the outer shell (Hassan et al., 2001; Hassan and Roy, 1999; Roy, 2008b). After infection, the BTV particle is swiftly uncoated by removal of its outer protein shell, thereby releasing the transcription core particle. BTV encodes its own RdRP, helicase and capping enzymes, VP1, VP6 and VP4, respectively. The inner capsid acts as a capsule, that delivers the intact replicase complex to the cytoplasm. Transcription of the dsRNA segments is activated during the removal of VP2 and VP5, and initial synthesis of capped mRNA takes place within the inner capsid itself (Mertens et al., 2004). The capped mRNAs are then released from the capsid, after which they are translated in the host cytoplasm. Newly translated viral proteins interact with the viral mRNAs within so-called viral inclusion bodies (VIB), that are believed to be the sites of dsRNA synthesis and mRNA production (Brookes et al., 1993). The VIB are mainly composed of NS2, which regulates core protein recruitment. However, it still remains unclear how newly synthesized dsRNA is encapsidated into progeny virions (Roy, 2008a). Maturation of VP2 and VP5 is independent from inner capsid formation and seems to be associated with NS3, which is important for virus maturation and release (Hyatt et al., 1993). The addition of the outer capsid proteins takes place after the inner capsid, containing the dsRNA and the replication proteins, has been released from the VIB. The progeny particles are released from the host through cell-lysis or by membrane penetration.

2.4. Reoviridae – genus Orbivirus

3. Arbovirus vaccinology

The Reoviridae family is a large, double-stranded RNA (dsRNA) virus group, that encompasses nine different genera. The Orbivirus genus contains clinically significant arboviruses that are widespread in nature and use ticks, midges, mosquitoes and sandflies as transmission vectors (Gorman, 1979; Verwoerd et al., 1979). Important and well known orbiviruses are Bluetongue virus (BTV) and African horse-sickness virus (AHSV). BTV is the orbivirus type species and etiological agent of bluetongue in ruminants and has a high impact on animal health and the economy. With over 24 different serotypes (Chaignat et al., 2009), BTV infects mostly sheep, and in some cases cattle and wild-stock such as white-tailed deer (Maclachlan et al., 2009). BTV uses midges as a transmission

3.1. Alphavirus vaccinology Currently, there are no specific treatments (antiviral) or commercial vaccines for alphavirus infections. However, the emergence of e.g. CHIKV or VEEV and the clinical manifestation of disease in humans and animals have triggered vaccine development. The first vaccine prototypes were formalin-inactivated viruses (Harrison et al., 1971; Randall et al., 1949). Although efficacious, these vaccines presented the serious risk of incomplete inactivation as shown by the isolation of infectious virus from vaccine preparations (Sutton and Brooke, 1954). Another drawback is that production of the virus must take place in costly, high containment

S24

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

facilities to prevent accidental release of live virus into the environment. As an alternative, live-attenuated virus vaccines have been developed (Berge et al., 1961; Levitt et al., 1986), some of which are still manufactured and used in Mexico and Colombia (Paessler and Weaver, 2009). A live-attenuated virus CHIKV vaccine, developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID), was tested in phase II clinical trials and elicited a protective immune response. However, 8% of tested volunteers developed transient arthralgia (Edelman et al., 2000; Levitt et al., 1986; Thiboutot et al., 2010). Similar results were found after the use of the attenuated VEEV vaccine TC-83 in humans. TC-83 induced seroconversion in about 80% of all test-cases, but clinical symptoms developed in a substantial number of volunteers (Paessler and Weaver, 2009). The subsequently developed V3526 strain appeared to be safer and more immunogenic in mice and non-human primates, compared to TC-83 (Hart et al., 2000; Ludwig et al., 2001; Pratt et al., 2003). Nonetheless, the prospect of live-(attenuated) alphavirus vaccine development is still questionable due to vaccine-induced clinical manifestations and the undeniable risk of transmission by mosquito vectors and possible reversion to pathogenicity. Optimized and alternative approaches such as DNA vaccination and chimeric vector-based vaccines are in development, and produce robust neutralizing antibody responses and generate full protection against lethal challenge (Dupuy et al., 2009; Muthumani et al., 2008; Paessler et al., 2003; Tiwari et al., 2009; Wang et al., 2007, 2008). The baculovirus-insect cell expression system has been used for alphavirus protein analysis and for the development of serodiagnostic tools and subunit vaccines. Several studies have shown that baculovirus-mediated expression of alphavirus structural proteins C, E3, E2, 6K and E1, results in correctly processed and mature (glyco)proteins in insect cells (Cho et al., 2008; Favre et al., 1993; Hodgson et al., 1999; Oker-Blom and Summers, 1989). Although the baculovirus expression system serves as an elegant platform for subunit vaccine development and available data are promising, this is still a fairly uncharted area and few studies have focused on the immunogenicity of alphavirus subunits produced in insect cells. Recombinant baculoviruses expressing the full VEEV structural cassette (C–E3–E2–6K–E1) generated products that were antigenically indistinguishable from wildtype viral proteins. Immunization of mice with lysates of infected insect cells yielded VEEV neutralizing antibodies and protected mice from lethal challenge (Hodgson et al., 1999). Individual expression of VEEV E1 and E2 with their respective signal peptides 6K and E3, however, resulted in incorrect glycoprotein processing (Fig. 2A). Nevertheless, the VEEV 6KE1 construct provided full protection against challenge (Hodgson et al., 1999). Recently, CHIKV E1 and E2 were co-expressed with 6K and E3, respectively, and yielded mature and correctly processed glycoproteins. Moreover, deletion of the C-terminal transmembrane domain of CHIKV E1 and E2, allowed glycosylation, furin cleavage and secretion from insect cells (Metz et al., unpublished results). However, the immunogenicity of baculovirus-expressed CHIKV E1, and E2 has yet to be tested. Recent studies have shown promising results in the development of a CHIKV VLP-based vaccine. The complete structural cassettes of different CHIKV strains were expressed via plasmid DNA transfection of 293T human kidney cells, thereby producing CHIKV VLPs. Rhesus macaques were subsequently immunized with VLPs, eliciting a neutralizing antibody response against homologous and heterologous strains and protection against lethal CHIKV challenge (Akahata et al., 2010). These results open the way for production of alphavirus VLPs by using recombinant baculovirus infected insect cells (Fig. 3), which could possibly lead to the development of safe and efficacious vaccines.

3.2. Flavivirus vaccinology Vaccination remains the most effective measure for protection against flaviviruses, because vector control and antiviral development have proven difficult and these measures are insufficient (Barrett, 2001). Three human vaccines are commercially available against YFV, TBEV and JEV infections. Although there are several licensed equine WNV vaccines (e.g. Innovator by Fort Dodge and PreveNileÒ by Intervet) (Table 2) there are no human vaccines registered for the prevention of WNV, DENV and St. Louis encephalitis virus (SLEV) infections. The YFV-17D vaccine is a very effective attenuated-virus vaccine that is licensed worldwide. It was developed in 1944 (Bugher and Smith, 1944) and is produced in embryonated chicken eggs. A single dose-vaccination is believed to provide lifelong protection (Pugachev et al., 2003). New generation flavivirus vaccines build upon the success of YFV-17D and use its viral backbone to express homologous structural proteins from for example DENV, JEV and WNV. This so-called ChimeriVax technology (Hall and Khromykh, 2007) is currently undergoing clinical testing and could potentially lead to the development of highly effective and safe flavivirus vaccines (Guy et al., 2010). In 1976, Baxter Bioscience introduced the purified formalininactivated TBEV vaccine FSME-ImmuneÒ. Two other inactivated vaccines against this disease have been introduced and licensed since: One by the Academy of Medical Sciences, Moscow and the other, Encepur by Behringwerke AG, Germany (Bock et al., 1990; Harabacz et al., 1992; Popov et al., 1985). Two vaccines are widely used against JEV infections. The formalin-inactivated vaccine from Biken (Japan) is globally available (Schiøler et al., 2007), whereas the attenuated JEV vaccine (SA14-14-2) is produced and licensed in China and South Korea (Lili et al., 2003). Recently, the formalin inactivated whole virus vaccine IC51 and ChimeriVax-JE, by Novartis and Sanofi Pasteur, respectively, have been licensed in the US and the EU. The ChimeriVax-JE vaccine is composed of a freezedried chimeric virus, which has the structural proteins prM and E of the JEV SA14-14-2 strain combined with the non-structural proteins of the YFV-17D vaccine strain. A single dose induces longlasting protection with an efficacy of 99% (Kollaritsch et al., 2009; Monath et al., 2002). Recombinant baculovirus expression of antigens in insect cells has been used for the development of subunit vaccines against flavivirus infections. Due to the absence of an effective vaccine, DENV and WNV have been major targets for both subunit and VLP vaccine development. The flavivirus E protein was successfully expressed and processed in insect cells using recombinant baculoviruses, as was the E2 glycoprotein of Classical swine fever virus (genus Pestivirus) (Bouma et al., 1999), which is the major component of the Porcilis PestiÒ subunit vaccine for swine (Intervet). The C-terminal transmembrane domain of the DENV-2 E protein was deleted, and the E-mutant was fused to the maltose-binding protein, resulting in correct processing through the Golgi, protein-secretion and allowing for affinity purification (Staropoli et al., 1996). Truncation of the individual E protein is a prerequisite for secretion in insect cells. Multiple studies have shown that antigenicity is retained after truncation of E. Moreover, these subunit candidates have elicited neutralizing antibodies and completely or partially protected mice from lethal DENV challenges (Delenda et al., 1994; Eckels et al., 1994; Staropoli et al., 1997; Velzing et al., 1999). More recently, full length DENV-2 E protein was co-expressed with an upstream prM translocation signal. E was found to form aggregates which reacted strongly with a series of monoclonal antibodies specific for native E, suggesting correct folding and exposure of functional epitopes. In addition, the E-aggregates induced high titer neutralizing antibody responses in mice (Kelly et al., 2000). Similar results were obtained for WNV-E and JEV-E expressed by recombinant baculoviruses (Bonafe et al., 2009; Chu et al., 2007; Li et al., 2009), showing that

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

S25

Fig. 3. Arbovirus VLP formation and maturation in insect cells after recombinant baculovirus expression. The heterologous genes encoding the structural proteins of (2–8) Toga-, (9–12) Bunya-, (13–17) Flavi- and (18–20) Reoviridae are transcribed in the nucleus. (1) The Togaviridae envelope glycoproteins E1 and E2 are transported to the ER, complex into heterodimers (2) and are transported to the Golgi (3 and 4) where three heterodimers complex into trimeric spikes and furin processing takes place. (5) The mature trimeric spikes are transported to the plasma membrane and are exposed on the surface of the cell. (6) The capsid protein assembles into nucleocapsids in the cytoplasm. (7) The nucleocapsids bud out from the plasma membrane, taking along the trimeric spikes anchored in the lipid bilayer. (8) Bunyaviridae glycoproteins Gn and Gc are transported to the ER and complex into heterodimers. (9) The dimers are transported to the Golgi, where they assemble into new viral particles (10). (11) The particles containing membrane vesicles bud out from the plasma membrane. (12) Flaviviridae glycoproteins M and E are transported into the ER and form heterodimers. (13) The capsid protein assembles into nucleocapsids in the cytoplasm and buds into the ER (14). In addition, subviral particles are formed, that lack nucleocapsids (15). Both viral particles are transported through the Golgi (16) in membrane vesicles which eventually fuse with the plasma membrane. 17) All Reoviridae structural proteins are translated in the cytoplasm (18, 19) First, the inner capsid is assembled from VP3 and VP7 (18), after which the particle matures with the anchoring of VP5 and VP2 (19). (20) The particle is released from the cell through cell lysis or membrane penetration.

the recombinant baculovirus insect cell expression system represents a good approach for the production of stable and effective subunit vaccines against flavivirus infections (Fig. 3). However, recent studies have shown that the functionality of induced neutralizing antibodies is still higher after immunization with inactivated WNV virions. The neutralizing response focuses more efficiently on the exposed epitopes on E when it is embedded in virus particles. Subunits also display other protein surfaces, which are cryptic in mature virus particles (Zlatkovic et al., 2010). This might indicate that virus-like particles (VLP) are able to induce a more efficacious neutralizing antibody response, compared to E-subunits. Similar to other eukaryotic expression systems, recombinant baculoviruses have been used to produce flavivirus VLPs and subviral like particles (sVLP), by expressing CprME or prME, respectively. (Konishi and Fujii, 2002; Konishi et al., 1992; Ohtaki et al., 2010; Qiao et al., 2004; Sugrue et al., 1997). Mice immunized with WNV sVLPs produced in insect cells, developed neutralizing antibody responses and did not show any morbidity, mortality, viraemia or viral RNA in the spleen and brain after lethal challenge with WNV (Qiao et al., 2004).

3.3. Bunyaviridae vaccinology The development of safe and effective vaccines targeting LACV, RVFV and CCHFV is required for the protection of humans, horses and ruminants. Currently, there are no specific treatments or licensed vaccines available for LACV, CCHFV and RVFV. Over the last decades, RVFV has been a major target for vaccine development, due to serious public health and bio-security threats. Several formalin-inactivated RVFV vaccines have been developed which elicit a protective immune response in humans and ruminants (Barnard and Botha, 1977; Eddy et al., 1981; Niklasson, 1982; Pittman et al., 1999; Randall et al., 1962). Different from live-attenuated vaccines, inactivated vaccines usually require adjuvants and repeated immunization (boost injections) to induce immunological memory against RVFV infections. The first (genetically) modified live-virus vaccine (MLV) has been used for vaccination applications, but the risk of reversion appeared too high for non-endemic countries (Ikegami and Makino, 2009). Two other highly attenuated live vaccines are also in development. The MP-12 vaccine virus is attenuated in all three RNA

S26

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

segments and is currently in phase III clinical trials in humans (Morrill et al., 1991; Peters, 1997). In addition, a reassortant vaccine strain R566 is under development for veterinary purposes. R566 carries the low virulent S-segment of clone 13 of the 74HB59 strain, and the M and L-segments of MP-12 (Kortekaas et al., 2010). There are few data available on the development of vaccines targeting LACV and CCHFV. Although LACV and CCHFV are emerging pathogens, development has been hampered by the lack of interest from the industry and absence of suitable animal models. Recently, adequate animal challenge models have been developed for these two viruses and can be used to thoroughly test novel vaccines (Bente et al., 2010; Hefti et al., 1999). These include a thermo-chemical inactivated CCHFV vaccine, a DNA vaccine encoding CCHFV-Gn and -Gc, a LACV-Gc subunit vaccine and a LACV-Gc, -Gn DNA vaccine (Kortekaas et al., 2010; Pekosz et al., 1995; Schuh et al., 1999; Spik et al., 2006). Little research has been performed on the use of the recombinant baculovirus expression system for subunit vaccine development. However, preliminary studies with RVFV and LACV are promising. Recombinant baculovirus expression of Gc and Gn in insect cells results in correctly processed and functionally active glycoproteins that retained their fusogenic properties (Liu et al., 2008). A single inoculation with lysates from cells infected with a recombinant baculovirus expressing the RVFV M-segment induced neutralizing antibody responses that protected mice from lethal challenge (Schmaljohn et al., 1989). Furthermore, it was shown that individual RVFV-Gn and LACV-Gc, expressed by recombinant baculoviruses, were able to induce a protective neutralizing antibody response (Pekosz et al., 1995; Schmaljohn et al., 1989). The expression of soluble RVFV-Gc in a Drosophila expression system resulted in full protection to lethal RVFV challenge (de Boer et al., 2010). More recent studies have focused on the generation of VLPs for bunyavirus vaccine purposes. Co-expression of RVFV-N, -Gc, and Gn results in the formation of VLPs from baculovirus infected insect cells (Liu et al., 2008). VLP immunization in mice elicited a high titer neutralizing antibody response and mice were fully protected against subsequent challenge (Naslund et al., 2009). Generating VLPs by expressing all three structural proteins is disadvantageous for the differentiation of vaccinated from infected individuals, as antibodies are raised against all three proteins after both wildtype infection and VLP immunization. A promising finding is that VLPs can be generated by the expression of glycoproteins Gn and Gc without the need for N. These VLPs were shown to generate protective neutralizing antibodies (de Boer et al., 2010). The production of subunits or VLPs using the baculovirus insect cell expression system (Fig. 3) has some major advantages over inactivated or live-attenuated vaccines targeting RVFV, LACV or CCHFV. Live virus production must be performed under Biosafety Level 3 or higher conditions. Furthermore, the absence of the N-protein in the vaccine preparation provides a useful marker for differentiation between infected and vaccinated individuals or animals using a serodiagnostic test to detect an antibody response against N. 3.4. Reoviridae vaccinology Several vaccination strategies have been developed to protect ruminants and equines from BTV and AHSV infections. These strategies include inactivated virus formulations, VLPs produced in insect cells, live-attenuated vaccines and recombinant vaccinia or canarypox virus vaccines (Chiam et al., 2009; Guthrie et al., 2009; Savini et al., 2008). The live-attenuated or modified live virus vaccines (MLV) have been used since affected countries undertook vaccination of livestock, to prevent further spread of BTV and to allow for safe international movement of animals. Attenuated by se-

rial passages through tissue culture or embryonated chicken eggs, MLVs stimulate a strong protective immune response after single inoculation and prevent clinical Bluetongue disease (Dungu et al., 2004; Patta et al., 2004). Despite the relatively low production costs and high efficacy, MLVs involve major potential disadvantages. There is a high risk of under-attenuation, which may cause depressed milk production, abortion and abnormalities in offspring. In addition, MLVs can potentially be spread by vectors, with the associated increased risk of reversion and reassortment with wildtype virus (Savini et al., 2008). The first inactivated vaccines became available in 2005, and were used during the most recent vaccination campaigns in Europe. Like most arbovirus epidemics, BTV outbreaks are highly unpredictable, and the prevalence of more than 24 different serotypes complicates the development of a one-size-fits-all BTV vaccine. Inactivated vaccines have now been developed against BTV serotypes 1, 2, 4, 8, and 9. All are highly efficacious and induced full and significant protection against BTV infection, clinical signs and viraemia. In most cases, two doses of inactivated vaccine were needed to protect sheep for a period of 12 months (Savini et al., 2008), which signifies one of the disadvantages of using inactivated vaccines. This, in addition to high production costs and the large amount of antigen required, makes the use of inactivated vaccines problematic. The recombinant baculovirus-insect cell expression system has been shown to be an elegant method for BTV and AHSV protein expression, to study virion structure, protein function and to develop diagnostic tools and subunit vaccines (Hassan et al., 2001; Hassan and Roy, 1999; Maree and Paweska, 2005; Roy, 1990, 1996; Roy et al., 1996). The formulation of an effective subunit vaccine using this system might clear most of the problems mentioned above. Immunogenicity of AHSV subunits has been tested for both outer capsid proteins VP2 and VP5 produced in insect cells. Combinations of VP2–VP5 and VP2 alone were able to induce a protective immune response against clinical disease and AHSV infections (Roy et al., 1996), suggesting that neutralizing epitopes are conserved after expression by recombinant baculoviruses. Moreover, co-expression of the AHSV inner capsid proteins VP3 and VP7 by recombinant baculoviruses, resulted in the formation of core-like particles which structurally resemble empty AHSV cores (French and Roy, 1990; Maree and Paweska, 2005). It is believed that these core-particles are critical scaffolds for the correct conformational presentation of the outer-capsid proteins VP2 and VP5. These double-shelled VLPs are able to induce a high titer neutralizing antibody response (French et al., 1990). Even at low doses, BTV VP2/VP5 VLPs were found to be more immunogenic than VP2 or VP5 subunits (Roy, 1992) and in some cases offered cross-protection against heterologous strains (Roy et al., 1994). Recently, an improved strategy for rapid BTV VLPs production has been presented. A baculovirus expressing VP3 of BTV-10 and VP7 of BTV-17, was used as a basis for insertion of VP2 and VP5 from different serotypes. This strategy was demonstrated to be fast in production, safe, and highly effective in sheep (Stewart et al., 2010). During natural infection, specific antibodies can also be detected against BTV NS1 and NS2 (Adkinson et al., 1988) and this characteristic can be used to differentiate infected from vaccinated animals (Fig. 3).

4. Concluding remarks Baculovirus technology offers a convenient and robust platform to produce high amounts of complex recombinant proteins in insect cell bioreactors, which is especially useful for the production of emergency vaccines (Cox and Hashimoto, 2011). Recombinant

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

baculoviruses are easily generated using well-established protocols and commercial kits, although many improvements to the stability and performance of the expression vectors still need to be made (van Oers, 2011). In comparison to other insect cell expression systems, such as the Drosophila S2 expression system, baculoviruses may be preferred for production of antigens in large culture volumes. For example, baculovirus production in insect cells does not require the time-consuming establishment, and sequent characterization and qualification of (semi-)stable S2 cell lines and the use of chemical inducers to initiate transgene mRNA transcription. Drosophila S2 cells have also been reported to be persistently infected with small RNA viruses (Flynt et al., 2009), which potentially makes this system less attractive for human vaccine production. Potential drawbacks of the baculovirus-insect cell system include its lytic nature and the fact that, in the case of human vaccines, budded baculoviruses must be separated from the produced subunits or VLPs. New technologies (Marek et al., 2011) are currently in development to address the latter problem (Galibert and Merten, 2011). Individual recombinant proteins for scientific research and those that are used as subunit vaccines have been produced for a long time. More recently, complex structures including VLPs derived from non-enveloped as well as enveloped viruses are generated in cultured cells of both mammalian and insect origin. VLPs are generally more immunogenic than their subunit counterparts (van Oers, 2006), but may also be more difficult to produce and/ or purify. The successes obtained in VLP generation of BTV with baculovirus expression open the way to develop similar vaccines for other, non-arboviral, members in the family Reoviridae. VLPs of the other arbovirus families contain two or more viral (glyco)proteins anchored in a host-derived envelope (Fig. 1B). It is interesting to note that for arboviruses from the families Flaviviridae and Bunyaviridae, VLPs can be made by expression of glycoproteins in the absence of the nucleocapsid protein (de Boer et al., 2010; Konishi et al., 1992; Kuwahara and Konishi, 2010; Qiao et al., 2004). Flavivirus subviral or prME particles have been shown to be immunogenic, and sometimes give higher immune responses than the corresponding nucleocapsid-containing VLPs (Qiao et al., 2004). For the Togaviridae, VLP formation without C expression has not been reported, which is not entirely unexpected taking into account that the direct protein–protein interaction between E2 and C is required for virion assembly (Kuhn, 2007). However, new studies have shown that CHIKV and SINV trimeric spikes can also be efficiently produced via the replacement of 6 K by a short peptide linker (Li et al., 2010; Voss et al., 2010). These complex yet stable polypeptides were secreted from the cells and were suitable for crystallization and structure analysis by X-ray diffraction. These artificial spikes adopted a similar conformation to the trimeric spikes found on the surface of alphavirus virions. They can be considered a compromise between true subunits and VLPs and may provide a promising novel approach for alphavirus vaccine design. Recent research showed that reasonable amounts of CHIKV VLPs can be produced by plasmid DNA transfection of mammalian cells (Akahata et al., 2010). Similarly, DENV and JEV subviral particles were efficiently produced via plasmid DNA transfection of cultured cells, with higher yields obtained in Sf21 insect cells as compared to mammalian cells (Kuwahara and Konishi, 2010). However, large scale plasmid DNA transfection in bioreactor settings may be a major challenge. Baculovirus vectors may in these cases provide an attractive alternative, taking into account the following consideration. Insect cells are dramatically transformed by baculovirus infection, have enlarged nuclei and are specialized to support nuclear baculovirus DNA replication and virion production. Small amounts of only two baculoviral glycoproteins, i.e. GP37 and GP64 (Wang

S27

et al., 2010), are produced to be incorporated in the envelope of budded viruses. In this respect, the environment in baculovirus-infected cells is very different from the cellular transformation induced by enveloped arboviruses, which replicate their viral RNA in virus-induced, proliferating membraneous structures in the cytoplasm of infected cells (Mackenzie, 2005). Arbovirus replication is accompanied by high level expression of complex viral glycoproteins in the ER, in order to produce virions that are heavily decorated with envelope glycoproteins. Many examples illustrate that complex heterologous glycoproteins can be successfully expressed at high levels in baculovirus-infected cells, including those of arboviruses (Table 2) (van Oers, 2006), yet solving this apparent discrepancy in the needs for baculovirus replication vs. arbovirus glycoprotein production in the same cell is a key point for future technological improvements. Co-expression from the baculovirus expression vector of ER-resident chaperones, foldases, or ER proliferating genes may further boost the potential for arbovirus glycoprotein production (Van Oers, 2011). Currently, most baculovirus expression constructs use the very late polyhedrin and/or p10 promoters. Provided that expression levels are high enough, putative beneficial effects of earlier transgene expression from baculoviral early or late (e.g. AcMNPV ie-1 or p6.9) promoters may be anticipated (Van Oers, 2011). This is especially valuable for those arboviral (glyco)protein subunits and VLPs that require extensive post-translational processing and/or secretion. Further studies will be needed to address whether or not this is an avenue that will lead to improved production processes. The near future will show whether the baculovirus-insect cell expression system will live up to the expectation that novel, safe and highly effective vaccines can indeed be successfully developed to help in the fight against emerging arboviral diseases. Acknowledgments The authors would like to thank Jean-Yves Sgro for providing structural images of the arbovirus virions. We also thank Just M. Vlak and Monique M. van Oers for helpful suggestions to improve the manuscript. Conflicts of Interest The following authors report no conflict of interest: Stefan W. Metz and Gorben P. Pijlman. References Adkinson, M., Stott, J., Osburn, B., 1988. Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection. Vet. Microbiol. 16, 231–241. Akahata, W., Yang, Z.Y., Andersen, H., Sun, S., Holdaway, H.A., Kong, W.P., Lewis, M.G., Higgs, S., Rossmann, M.G., Rao, S., Nabel, G.J., 2010. A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection. Nat. Med. 16, 334–338. Alonso-Padilla, J., de Oya, N., Blazquez, A., Escribano-Romero, E., Escribano, J., Saiz, J., 2011. Recombinant West Nile virus envelope protein E and domain III expressed in insect larvae protects mice against West Nile disease. Vaccine 29, 1830–1835. Andrewes, C., 1952. Classification and nomenclature of viruses. Ann. Rev. Microbiol. 6, 119–138. Angelini, P., Macini, P., Finarelli, A., Po, C., Venturelli, C., Bellini, R., Dottori, M., 2008. Chikungunya epidemic outbreak in Emilia-Romagna (Italy) during summer 2007. Parassitologia 50, 97–98. Barnard, B.J., Botha, M.J., 1977. An inactivated rift valley fever vaccine. J.S. Afr. Vet. Assoc. 48, 45–48. Barrett, A.D., 2001. Current status of flavivirus vaccines. Ann. N. Y. Acad. Sci. 951, 262–271. Bente, D.A., Alimonti, J.B., Shieh, W.J., Camus, G., Stroher, U., Zaki, S., Jones, S.M., 2010. Pathogenesis and immune response of Crimean–Congo hemorrhagic fever virus in a STAT-1 knockout mouse model. J. Virol. 84, 11089–11100.

S28

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

Berge, T., Banks, I., Tigertt, W., 1961. Attenuation of Venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea-pig heart cells. Am. J. Epidemiol. 73, 209–218. Bock, H.L., Klockmann, U., Jungst, C., Schindel-Kunzel, F., Theobald, K., Zerban, R., 1990. A new vaccine against tick-borne encephalitis: initial trial in man including a dose-response study. Vaccine 8, 22–24. Bonafe, N., Rininger, J.A., Chubet, R.G., Foellmer, H.G., Fader, S., Anderson, J.F., Bushmich, S.L., Anthony, K., Ledizet, M., Fikrig, E., Koski, R.A., Kaplan, P., 2009. A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells. Vaccine 27, 213–222. Bouma, A., de Smit, A.J., de Kluijver, E.P., Terpstra, C., Moormann, R.J., 1999. Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus. Vet. Microbiol. 66, 101–114. Brookes, S.M., Hyatt, A.D., Eaton, B.T., 1993. Characterization of virus inclusion bodies in bluetongue virus-infected cells. J. Gen. Virol. 74, 525–530. Bugher, J., Smith, H., 1944. Antigenicity of yellow fever vaccine virus (17D) following fifty-seven subcultures in homologous immune serum. Am. J. Epidemiol. 39, 52–57. Cavrini, F., Gaibani, P., Pierro, A.M., Rossini, G., Landini, M.P., Sambri, V., 2009. Chikungunya: an emerging and spreading arthropod-borne viral disease. J. Infect. Dev. Ctries. 3, 744–752. Chaignat, V., Worwa, G., Scherrer, N., Hilbe, M., Ehrensperger, F., Batten, C., Cortyen, M., Hofmann, M., Thuer, B., 2009. Toggenburg Orbivirus, a new bluetongue virus: initial detection, first observations in field and experimental infection of goats and sheep. Vet. Microbiol. 138, 11–19. Chang, D., Liu, W., Anraku, I., Clark, D., Pollitt, C., Suhrbier, A., Hall, R., Khromykh, A., 2008. Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus. Nat. Biotechnol. 26, 571–577. Chen, L., Ewing, D., Subramanian, H., Block, K., Rayner, J., Alterson, K.D., Sedegah, M., Hayes, C., Porter, K., Raviprakash, K., 2007. A heterologous DNA primeVenezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques. J. Virol. 81, 11634–11639. Chiam, R., Sharp, E., Maan, S., Rao, S., Mertens, P., Blacklaws, B., Davis-Poynter, N., Wood, J., Castillo-Olivares, J., 2009. Induction of antibody responses to African horse sickness virus (AHSV) in ponies after vaccination with recombinant modified vaccinia Ankara (MVA). PLoS ONE 4, e5997. Cho, B., Jeon, B.Y., Kim, J., Noh, J., Kim, J., Park, M., Park, S., 2008. Expression and evaluation of Chikungunya Virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection. Yonsei Med. J. 49, 828–835. Chu, J., Chiang, C., Ng, M., 2007. Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection. J. Immunol. 178, 2699. Cox, M.M.J., Hashimoto, Y., 2011. A fast track influenza virus vaccine produced in insect cells. J. Invertebr. Pathol. 107, S31. de Boer, S.M., Kortekaas, J., Antonis, A.F., Kant, J., van Oploo, J.L., Rottier, P.J., Moormann, R.J., Bosch, B.J., 2010. Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge. Vaccine 28, 2330–2339. Delenda, C., Frenkiel, M.P., Deubel, V., 1994. Protective efficacy in mice of a secreted form of recombinant dengue-2 virus envelope protein produced in baculovirus infected insect cells. Arch. Virol. 139, 197–207. Dungu, B., Gerdes, T., Smit, T., 2004. The use of vaccination in the control of bluetongue in southern Africa. Vet. Ital. 40, 616–622. Dupuy, L.C., Locher, C.P., Paidhungat, M., Richards, M.J., Lind, C.M., Bakken, R., Parker, M.D., Whalen, R.G., Schmaljohn, C.S., 2009. Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine. Vaccine 27, 4152–4160. Eckels, K.H., Dubois, D.R., Summers, P.L., Schlesinger, J.J., Shelly, M., Cohen, S., Zhang, Y.M., Lai, C.J., Kurane, I., Rothman, A., et al., 1994. Immunization of monkeys with baculovirus-dengue type-4 recombinants containing envelope and nonstructural proteins: evidence of priming and partial protection. Am. J. Trop. Med. Hyg. 50, 472–478. Eddy, G., Peters, C., Meadors, G., Cole, F. Jr., 1981. Rift Valley Fever Vaccine for Humans1. Karger, p. 124. Edelman, R., Tacket, C.O., Wasserman, S.S., Bodison, S.A., Perry, J.G., Mangiafico, J.A., 2000. Phase II safety and immunogenicity study of live Chikungunya virus vaccine TSI-GSD-218. Am. J. Trop. Med. Hyg. 62, 681–685. Edelman, R., Wasserman, S.S., Bodison, S.A., Putnak, R.J., Eckels, K.H., Tang, D., KanesaThasan, N., Vaughn, D.W., Innis, B.L., Sun, W., 2003. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am. J. Trop. Med. Hyg. 69, 48–60. Elliott, R., 1990. Molecular biology of the Bunyaviridae. J. Gen. Virol. 71, 501. Elliott, R., 1997. Emerging viruses: the Bunyaviridae. Mol. Med. 3, 572–577. Endres, M., Jacoby, D., Janssen, R., Gonzalez-Scarano, F., Nathanson, N., 1989. The large viral RNA segment of California serogroup bunyaviruses encodes the large viral protein. J. Gen. Virol. 70, 223–228. Falgout, B., Pethel, M., Zhang, Y.M., Lai, C.J., 1991. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J. Virol. 65, 2467–2475. Favre, D., Studer, E., Nishimura, T., Weitz, M., Michel, M.R., 1993. Semliki Forest virus capsid protein expressed by a baculovirus recombinant. Arch. Virol. 132, 307–319. Flynt, A., Liu, N., Martin, R., Lai, E.C., 2009. Dicing of viral replication intermediates during silencing of latent Drosophila viruses. Proc. Natl. Acad. Sci. USA 106, 5270–5275. French, T.J., Roy, P., 1990. Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J. Virol. 64, 1530–1536.

French, T.J., Marshall, J.J., Roy, P., 1990. Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins. J. Virol. 64, 5695–5700. Galibert, L., Merten, O.-W., 2011. Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases. J. Invertebr. Pathol. 107, S80. Garoff, H., Sjoberg, M., Cheng, R.H., 2004. Budding of alphaviruses. Virus Res. 106, 103–116. Gomez, I., Marx, F., Saurwein-Teissl, M., Gould, E., Grubeck-Loebenstein, B., 2003. Characterization of tick-borne encephalitis virus-specific human T lymphocyte responses by stimulation with structural TBEV proteins expressed in a recombinant baculovirus. Viral Immunol. 16, 407–414. Gonzalez-Scarano, F., 1985. La Crosse virus G1 glycoprotein undergoes a conformational change at the pH of fusion. Virology 140, 209–216. Gorman, B.M., 1979. Variation in orbiviruses. J. Gen. Virol. 44, 1–15. Gould, E., Gallian, P., De Lamballerie, X., Charrel, R., 2010. First cases of autochthonous dengue fever and Chikungunya fever in France. From bad dream to reality! Clin. Microbiol. Infect. 16, 1702–1704. Gubler, D., Kuno, G., Markoff, L., 2007. Flaviviruses. Fields Virology, fifth ed. Lippincott Williams & Wilkins, Philadelphia, pp. 1153–1252. Guirakhoo, F., Kitchener, S., Morrison, D., Forrat, R., McCarthy, K., Nichols, R., Yoksan, S., Duan, X., Ermak, T.H., Kanesa-Thasan, N., Bedford, P., Lang, J., Quentin-Millet, M.J., Monath, T.P., 2006. Live attenuated chimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vaccine: phase I clinical trial for safety and immunogenicity: effect of yellow fever pre-immunity in induction of cross neutralizing antibody responses to all 4 dengue serotypes. Hum. Vaccine 2, 60–67. Guthrie, A.J., Quan, M., Lourens, C.W., Audonnet, J.C., Minke, J.M., Yao, J., He, L., Nordgren, R., Gardner, I.A., Maclachlan, N.J., 2009. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus. Vaccine 27, 4434–4438. Guy, B., Guirakhoo, F., Barban, V., Higgs, S., Monath, T.P., Lang, J., 2010. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine 28, 632–649. Hall, R., Khromykh, A., 2007. ChimeriVax-West Nile vaccine. Curr. Opin. Mol. Ther. 9, 498. Hall, R., Nisbet, D., Pham, K., Pyke, A., Smith, G., Khromykh, A., 2003. DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus. Proc. Natl. Acad. Sci. USA 100, 10460–10464. Harabacz, I., Bock, H., Jungst, C., Klockmann, U., Praus, M., Weber, R., 1992. A randomized phase II study of a new tick-borne encephalitis vaccine using three different doses and two immunization regimens. Vaccine 10, 145–150. Harrison, V.R., Eckels, K.H., Bartelloni, P.J., Hampton, C., 1971. Production and evaluation of a formalin-killed Chikungunya vaccine. J. Immunol. 107, 643–647. Hart, M., Caswell-Stephan, K., Bakken, R., Tammariello, R., Pratt, W., Davis, N., Johnston, R., Smith, J., Steele, K., 2000. Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate. Vaccine 18, 3067–3075. Hassan, S.S., Roy, P., 1999. Expression and functional characterization of bluetongue virus VP2 protein: role in cell entry. J. Virol. 73, 9832–9842. Hassan, S.H., Wirblich, C., Forzan, M., Roy, P., 2001. Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization. J. Virol. 75, 8356–8367. Havelaar, A.H., van Rosse, F., Bucura, C., Toetenel, M.A., Haagsma, J.A., Kurowicka, D., Heesterbeek, J.H., Speybroeck, N., Langelaar, M.F., van der Giessen, J.W., Cooke, R.M., Braks, M.A., 2010. Prioritizing emerging zoonoses in the Netherlands. PLoS ONE 5, e13965. Hefti, H.P., Frese, M., Landis, H., Di Paolo, C., Aguzzi, A., Haller, O., Pavlovic, J., 1999. Human MxA protein protects mice lacking a functional alpha/beta interferon system against La crosse virus and other lethal viral infections. J. Virol. 73, 6984–6991. Hodgson, L.A., Ludwig, G.V., Smith, J.F., 1999. Expression, processing, and immunogenicity of the structural proteins of Venezuelan equine encephalitis virus from recombinant baculovirus vectors. Vaccine 17, 1151–1160. Hunt, A.R., Frederickson, S., Maruyama, T., Roehrig, J.T., Blair, C.D., 2010. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity. PLoS Negl. Trop. Dis. 4, e739. Hyatt, A.D., Zhao, Y., Roy, P., 1993. Release of bluetongue virus-like particles from insect cells is mediated by BTV nonstructural protein NS3/NS3A. Virology 193, 592–603. Ikegami, T., Makino, S., 2009. Rift valley fever vaccines. Vaccine 27 (Suppl. 4), D69– D72. Kanesa-Thasan, N., Edelman, R., Tacket, C.O., Wasserman, S.S., Vaughn, D.W., Coster, T.S., Kim-Ahn, G.J., Dubois, D.R., Putnak, J.R., King, A., Summers, P.L., Innis, B.L., Eckels, K.H., Hoke Jr., C.H., 2003. Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines. Am. J. Trop. Med. Hyg. 69, 17–23. Kaukinen, P., Vaheri, A., Plyusnin, A., 2005. Hantavirus nucleocapsid protein: a multifunctional molecule with both housekeeping and ambassadorial duties. Arch.Virol. 150, 1693–1713. Kelly, E.P., Greene, J.J., King, A.D., Innis, B.L., 2000. Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice. Vaccine 18, 2549–2559. Khan, A.H., Morita, K., Parquet Md Mdel, C., Hasebe, F., Mathenge, E.G., Igarashi, A., 2002. Complete nucleotide sequence of Chikungunya virus and evidence for an internal polyadenylation site. J. Gen. Virol. 83, 3075–3084.

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30 Kistner, O., Barrett, N., Bruhmann, A., Reiter, M., Mundt, W., Savidis-Dacho, H., Schober-Bendixen, S., Dorner, F., Aaskov, J., 2007. The preclinical testing of a formaldehyde inactivated Ross River virus vaccine designed for use in humans. Vaccine 25, 4845–4852. Kitchener, S., Nissen, M., Nasveld, P., Forrat, R., Yoksan, S., Lang, J., Saluzzo, J.F., 2006. Immunogenicity and safety of two live-attenuated tetravalent dengue vaccine formulations in healthy Australian adults. Vaccine 24, 1238–1241. Kochel, T., Wu, S.J., Raviprakash, K., Hobart, P., Hoffman, S., Porter, K., Hayes, C., 1997. Inoculation of plasmids expressing the dengue-2 envelope gene elicit neutralizing antibodies in mice. Vaccine 15, 547–552. Kollaritsch, H., Paulke-Korinek, M., Dubischar-Kastner, K., 2009. IC51 Japanese encephalitis vaccine. Expert. Opin. Biol. Ther. 9, 921–931. Konishi, E., Fujii, A., 2002. Dengue type 2 virus subviral extracellular particles produced by a stably transfected mammalian cell line and their evaluation for a subunit vaccine. Vaccine 20, 1058–1067. Konishi, E., Pincus, S., Paoletti, E., Shope, R.E., Burrage, T., Mason, P.W., 1992. Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188, 714–720. Kortekaas, J., Ergonul, O., Moormann, R.J., 2010. Interventions against West Nile virus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus: where are we? Vector Borne Zoonotic Dis. 10, 709–718. Kost, T.A., Condreay, J.P., Jarvis, D.L., 2005. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol. 23, 567–575. Kuhn, R.J., 2007. Togaviridae: The Viruses and Their Replication. Lippincott, Williams and Wilkins, Philadelphia, PA, pp. 1001–1022. Kuwahara, M., Konishi, E., 2010. Evaluation of extracellular subviral particles of Dengue Virus type 2 and Japanese encephalitis virus produced by Spodoptera frugiperda cells for use as vaccine and diagnostic antigens. Clin.Vaccine Immunol. 17, 1560. Lees, J., Pringle, C., Elliott, R., 1986. Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of structural features in the Bunyavirus glycoprotein gene product. Virology 148, 1–14. Levitt, N.H., Ramsburg, H.H., Hasty, S.E., Repik, P.M., Cole Jr., F.E., Lupton, H.W., 1986. Development of an attenuated strain of Chikungunya virus for use in vaccine production. Vaccine 4, 157–162. Li, L., Lok, S.M., Yu, I.M., Zhang, Y., Kuhn, R.J., Chen, J., Rossmann, M.G., 2008. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319, 1830–1834. Li, Y., Ye, J., Cao, S., Xiao, S., Zhao, Q., Liu, X., Jin, M., Chen, H., 2009. Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice. J. Gene Med. 11, 57–65. Li, L., Jose, J., Xiang, Y., Kuhn, R.J., Rossmann, M.G., 2010. Structural changes of envelope proteins during alphavirus fusion. Nature 468, 705–708. Lili, J., Zhiwei, W., Yongxin, Y., 2003. Protection of SA14-14-2 live attenuated Japanese encephalitis vaccine against the wild-type JE viruses. Chin. Med. J. 116, 941–943. Lindenbach, B., Rice, C., 2001. Flaviviridae: the viruses and their replication. Fields Virol. 1, 991–1041. Liu, Y., Si, B., Hu, Y., Zhang, Y., Yang, Y., Zhu, Q., 2005. Expression of tick-borne encephalitis virus prM–E protein in insect cells and studies on its antigenicity. Chin. J. Exp. Clin. Virol. 19, 335–339. Liu, L., Celma, C.C., Roy, P., 2008. Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins. Virol. J. 5, 82. Lorenz, I.C., Allison, S.L., Heinz, F.X., Helenius, A., 2002. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J. Virol. 76, 5480–5491. Lorenz, I.C., Kartenbeck, J., Mezzacasa, A., Allison, S.L., Heinz, F.X., Helenius, A., 2003. Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus. J. Virol. 77, 4370–4382. Ludwig, G.V., Turell, M.J., Vogel, P., Kondig, J.P., Kell, W.K., Smith, J.F., Pratt, W.D., 2001. Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice. Am. J. Trop. Med. Hyg. 64, 49–55. Mackenzie, J., 2005. Wrapping things up about virus RNA replication. Traffic 6, 967– 977. Maclachlan, N.J., Drew, C.P., Darpel, K.E., Worwa, G., 2009. The pathology and pathogenesis of bluetongue. J. Comp. Pathol. 141, 1–16. Mallilankaraman, K., Shedlock, D.J., Bao, H., Kawalekar, O.U., Fagone, P., Ramanathan, A.A., Ferraro, B., Stabenow, J., Vijayachari, P., Sundaram, S.G., Muruganandam, N., Sarangan, G., Srikanth, P., Khan, A.S., Lewis, M.G., Kim, J.J., Sardesai, N.Y., Muthumani, K., Weiner, D.B., 2011. A DNA vaccine against Chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates. PLoS Negl. Trop. Dis. 5, e928. Maree, S., Paweska, J.T., 2005. Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera. J. Virol. Meth. 125, 55–65. Marek, M., van Oers, M.M., Devaraj, F., Vlak, J.M., Merten, O.W., 2011. Engineering of baculovirus vectors for the manufacture of virion free biopharmaceuticals. Biotechnol. Bioeng. 108, 1056–1067. Mertens, P.P., Diprose, J., Maan, S., Singh, K.P., Attoui, H., Samuel, A.R., 2004. Bluetongue virus replication, molecular and structural biology. Vet. Ital. 40, 426–437. Minke, J., Siger, L., Karaca, K., Austgen, L., Gordy, P., Bowen, R., Renshaw, R., Loosmore, S., Audonnet, J., Nordgren, B., 2004. Recombinant canarypoxvirus

S29

vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge. Arch. Virol. Suppl. 18, 221–230. Monath, T.P., McCarthy, K., Bedford, P., Johnson, C.T., Nichols, R., Yoksan, S., Marchesani, R., Knauber, M., Wells, K.H., Arroyo, J., Guirakhoo, F., 2002. Clinical proof of principle for ChimeriVax: recombinant live, attenuated vaccines against flavivirus infections. Vaccine 20, 1004–1018. Morrill, J.C., Carpenter, L., Taylor, D., Ramsburg, H.H., Quance, J., Peters, C.J., 1991. Further evaluation of a mutagen-attenuated Rift Valley fever vaccine in sheep. Vaccine 9, 35–41. Mukhopadhyay, S., Kuhn, R.J., Rossmann, M.G., 2005. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3, 13–22. Murphy, F.A., Harrison, A.K., Whitfield, S.G., 1973. Bunyaviridae: morphologic and morphogenetic similarities of Bunyamwera serologic supergroup viruses and several other arthropod-borne viruses. Intervirology 1, 297–316. Murray, K., Mertens, E., Desprès, P., 2010. West Nile virus and its emergence in the United States of America. Vet. Res. 41, 67–81. Muthumani, K., Lankaraman, K.M., Laddy, D.J., Sundaram, S.G., Chung, C.W., Sako, E., Wu, L., Khan, A., Sardesai, N., Kim, J.J., Vijayachari, P., Weiner, D.B., 2008. Immunogenicity of novel consensus-based DNA vaccines against Chikungunya virus. Vaccine 26, 5128–5134. Najjar, J.A., Gentsch, J.R., Nathanson, N., Gonzalez-Scarano, F., 1985. Epitopes of the G1 glycoprotein of La Crosse virus form overlapping clusters within a single antigenic site. Virology 144, 426–432. Naslund, J., Lagerqvist, N., Habjan, M., Lundkvist, A., Evander, M., Ahlm, C., Weber, F., Bucht, G., 2009. Vaccination with virus-like particles protects mice from lethal infection of Rift Valley Fever Virus. Virology 385, 409–415. Nelson, M., Winkelmann, E., Ma, Y., Xia, J., Mason, P., Bourne, N., Milligan, G., 2010. Immunogenicity of RepliVAX WN, a novel single-cycle West Nile virus vaccine. Vaccine 29, 174–182. Niklasson, B., 1982. Rift Valley fever virus vaccine trial: study of side-effects in humans. Scand. J. Infect. Dis. 14, 105–109. Ohtaki, N., Takahashi, H., Kaneko, K., Gomi, Y., Ishikawa, T., Higashi, Y., Kurata, T., Sata, T., Kojima, A., 2010. Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate. Vaccine 28, 6588–6596. Oker-Blom, C., Summers, M., 1989. Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector. J. Virol. 63, 1256–1264. Paavonen, J., Lehtinen, M., 2008. Introducing human papillomavirus vaccines – questions remain. Ann. Med. 40, 162–166. Paessler, S., Weaver, S.C., 2009. Vaccines for Venezuelan equine encephalitis. Vaccine 27 (Suppl. 4), D80–D85. Paessler, S., Fayzulin, R.Z., Anishchenko, M., Greene, I.P., Weaver, S.C., Frolov, I., 2003. Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic. J. Virol. 77, 9278–9286. Patta, C., Giovannini, A., Rolesu, S., Nannini, D., Savini, G., Calistri, P., Santucci, U., Caporale, V., 2004. Bluetongue vaccination in Europe: the Italian experience. Vet. Ital. 40, 601–610. Paupy, C., Delatte, H., Bagny, L., Corbel, V., Fontenille, D., 2009. Aedes albopictus, an arbovirus vector: from the darkness to the light. Microbes Infect. 11, 1177– 1185. Pekosz, A., Griot, C., Stillmock, K., Nathanson, N., Gonzalez-Scarano, F., 1995. Protection from La Crosse virus encephalitis with recombinant glycoproteins: role of neutralizing anti-G1 antibodies. J. Virol. 69, 3475–3481. Perera, R., Kuhn, R.J., 2008. Structural proteomics of dengue virus. Curr. Opin. Microbiol. 11, 369–377. Persson, R., Pettersson, R.F., 1991. Formation and intracellular transport of a heterodimeric viral spike protein complex. J. Cell. Biol. 112, 257–266. Peters, C., 1997. Emergence of Rift Valley Fever. Factors in the Emergence of Arbovirus Diseases. Elsevier, Paris, pp. 253–264. Pettersson, R., Von Bonsdorff, C., 1987. Bunyaviridae. Perspect. Med. Virol. 3, 147– 157. Pettersson, R., Kaariainen, L., von Bonsdorff, C.H., Oker-Blom, N., 1971. Structural components of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 46, 721–729. Pittman, P., Liu, C., Cannon, T., Makuch, R., Mangiafico, J., Gibbs, P., Peters, C., 1999. Immunogenicity of an inactivated Rift Valley fever vaccine in humans: a 12year experience. Vaccine 18, 181–189. Popov, O.V., Sumarokov, A.A., Shkol’nik, R., El’bert, L.B., Vorob’eva, M.S., 1985. Reactogenicity and antigenic activity of a chromatographic cultured purified and concentrated inactivated dried vaccine against tick-borne encephalitis. Zh. Mikrobiol. Epidemiol. Immunobiol., pp. 34–39. Prasad, B.V., Yamaguchi, S., Roy, P., 1992. Three-dimensional structure of singleshelled bluetongue virus. J. Virol. 66, 2135–2142. Pratt, W.D., Davis, N.L., Johnston, R.E., Smith, J.F., 2003. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine 21, 3854–3862. Pugachev, K., Guirakhoo, F., Trent, D., Monath, T., 2003. Traditional and novel approaches to flavivirus vaccines. Int. J. Parasitol. 33, 567–582. Qiao, M., Ashok, M., Bernard, K.A., Palacios, G., Zhou, Z.H., Lipkin, W.I., Liang, T.J., 2004. Induction of sterilizing immunity against West Nile Virus (WNV), by immunization with WNV-like particles produced in insect cells. J. Infect. Dis. 190, 2104–2108. Randall, R., Maurer, F.D., Smadel, J.E., 1949. Immunization of laboratory workers with purified Venezuelan equine encephalomyelitis vaccine. J. Immunol. 63, 313–318.

S30

S.W. Metz, G.P. Pijlman / Journal of Invertebrate Pathology 107 (2011) S16–S30

Randall, R., Gibbs Jr., C.J., Aulisio, C.G., Binn, L.N., Harrison, V.R., 1962. The development of a formalin-killed Rift Valley fever virus vaccine for use in man. J. Immunol. 89, 660–671. Raviprakash, K., Kochel, T.J., Ewing, D., Simmons, M., Phillips, I., Hayes, C.G., Porter, K.R., 2000. Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein. Vaccine 18, 2426–2434. Razumov, I.A., Khusainova, A.D., Agapov, E.V., Gaidamovich, S., Pereboev, A.V., Kolykhalov, A.A., Netesov, S.V., Loktev, V.B., 1994. Analysis of the hemagglutination activity domains of the Venezuelan equine encephalomyelitis and eastern equine encephalomyelitis viruses. Intervirology 37, 356–360. Roy, P., 1989. Bluetongue virus genetics and genome structure. Virus Res. 13, 179– 206. Roy, P., 1990. Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus. FEMS Microbiol. Immunol. 2, 223–234. Roy, P., 1992. Bluetongue virus proteins. J. Gen. Virol. 73, 3051–3064. Roy, P., 1996. Multiple gene expression in baculovirus system. Third generation vaccines for bluetongue disease and African horsesickness disease. Ann. N. Y. Acad. Sci. 791, 318–332. Roy, P., 2008a. Bluetongue virus: dissection of the polymerase complex. J. Gen. Virol. 89, 1789–1804. Roy, P., 2008b. Functional mapping of bluetongue virus proteins and their interactions with host proteins during virus replication. Cell Biochem. Biophys. 50, 143–157. Roy, P., Sutton, G., 1998. New generation of African horse sickness virus vaccines based on structural and molecular studies of the virus particles. Arch. Virol. Supplementum. 14, 177. Roy, P., Marshall, J.J., French, T.J., 1990. Structure of the bluetongue virus genome and its encoded proteins. Curr. Top. Microbiol. Immunol. 162, 43–87. Roy, P., Bishop, D.H., LeBlois, H., Erasmus, B.J., 1994. Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection. Vaccine 12, 805–811. Roy, P., Bishop, D.H., Howard, S., Aitchison, H., Erasmus, B., 1996. Recombinant baculovirus-synthesized African horsesickness virus (AHSV) outer-capsid protein VP2 provides protection against virulent AHSV challenge. J. Gen. Virol. 77, 2053–2057. Sabin, A., Schlesinger, R., 1945. Production of immunity to dengue with virus modified by propagation in mice. Science 101, 640. Samina, I., Khinich, Y., Simanov, M., Malkinson, M., 2005. An inactivated West Nile virus vaccine for domestic geese-efficacy study and a summary of 4 years of field application. Vaccine 23, 4955–4958. Savini, G., MacLachlan, N.J., Sanchez-Vizcaino, J.M., Zientara, S., 2008. Vaccines against bluetongue in Europe. Comp. Immunol. Microbiol. Infect. Dis. 31, 101–120. Schiøler, K., Samuel, M., Wai, K., 2007. Vaccines for Preventing Japanese Encephalitis. Status and Date, vol. 1. John Wiley & Sons, pp. 1–49. Schmaljohn, C., Hjelle, B., 1997. Hantaviruses: a global disease problem. Emerg. Infect. Dis. 3, 95–104. Schmaljohn, C., Hooper, J., 2001. Bunyaviridae: the viruses and their replication. Fund. Virol. 2001, 1581–1602. Schmaljohn, C.S., Parker, M.D., Ennis, W.H., Dalrymple, J.M., Collett, M.S., Suzich, J.A., Schmaljohn, A.L., 1989. Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins. Virology 170, 184–192. Schuh, T., Schultz, J., Moelling, K., Pavlovic, J., 1999. DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells. Hum. Gene Ther. 10, 1649–1658. Smith, J.F., Pifat, D.Y., 1982. Morphogenesis of sandfly viruses (Bunyaviridae family). Virology 121, 61–81. Solignat, M., Gay, B., Higgs, S., Briant, L., Devaux, C., 2009. Replication cycle of Chikungunya: a re-emerging arbovirus. Virology 393, 183–197. Spik, K., Shurtleff, A., McElroy, A.K., Guttieri, M.C., Hooper, J.W., SchmalJohn, C., 2006. Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus. Vaccine 24, 4657–4666. Spohn, G., Jennings, G., Martina, B., Keller, I., Beck, M., Pumpens, P., Osterhaus, A., Bachmann, M., 2010. A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice. Virol. J. 7, 146. Staropoli, I., Clement, J.M., Frenkiel, M.P., Hofnung, M., Deubel, V., 1996. Dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein. J. Virol. Methods 56, 179–189. Staropoli, I., Frenkiel, M.P., Megret, F., Deubel, V., 1997. Affinity-purified dengue-2 virus envelope glycoprotein induces neutralizing antibodies and protective immunity in mice. Vaccine 15, 1946–1954. Stewart, M., Bhatia, Y., Athmaran, T.N., Noad, R., Gastaldi, C., Dubois, E., Russo, P., Thiery, R., Sailleau, C., Breard, E., Zientara, S., Roy, P., 2010. Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus. Vaccine 28, 3047–3054. Strauss, J.H., Strauss, E.G., 1994. The alphaviruses: gene expression, replication, and evolution. Microbiol. Mol. Biol. Rev. 58, 491–562.

Strauss, E.G., Rice, C.M., Strauss, J.H., 1984. Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology 133, 92–110. Strauss, E.G., Stec, D.S., Schmaljohn, A.L., Strauss, J.H., 1991. Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants. J. Virol. 65, 4654–4664. Sugrue, R.J., Fu, J., Howe, J., Chan, Y.C., 1997. Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles. J. Gen. Virol. 78, 1861–1866. Summers, M.D., Smith, G.E., 1987. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Bulletin B-Texas Agricultural Experiment Station, USA. Sundin, D.R., Beaty, B.J., Nathanson, N., Gonzalez-Scarano, F., 1987. A G1 glycoprotein epitope of La Crosse virus: a determinant of infection of Aedes triseriatus. Science 235, 591–593. Sutton, L., Brooke, C., 1954. Venezuelan equine encephalomyelitis due to vaccination in man. JAMA 155, 1473. Swanepoel, R., 1995. Bunyaviridae. Princ. Pract. Clin. Virol. 3, 517–554. Talmon, Y., Prasad, B.V., Clerx, J.P., Wang, G.J., Chiu, W., Hewlett, M.J., 1987. Electron microscopy of vitrified-hydrated La Crosse virus. J. Virol. 61, 2319–2321. Thiboutot, M.M., Kannan, S., Kawalekar, O.U., Shedlock, D.J., Khan, A.S., Sarangan, G., Srikanth, P., Weiner, D.B., Muthumani, K., 2010. Chikungunya: a potentially emerging epidemic? PLoS Negl. Trop. Dis. 4, e623. Tiwari, M., Parida, M., Santhosh, S.R., Khan, M., Dash, P.K., Rao, P.V., 2009. Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus. Vaccine 27, 2513–2522. van Oers, M.M., 2006. Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv. Virus Res. 68, 193–253. van Oers, M.M., 2011. Opportunities and challenges for the baculovirus expression system. J. Invertebr. Pathol. 107, S3. Velzing, J., Groen, J., Drouet, M.T., van Amerongen, G., Copra, C., Osterhaus, A.D., Deubel, V., 1999. Induction of protective immunity against Dengue virus type 2: comparison of candidate live attenuated and recombinant vaccines. Vaccine 17, 1312–1320. Verwoerd, D., Huismans, H., Erasmus, B., 1979. Comprehensive Virology, vol. 14. Academic Press, New York. Voss, J.E., Vaney, M.C., Duquerroy, S., Vonrhein, C., Girard-Blanc, C., Crublet, E., Thompson, A., Bricogne, G., Rey, F.A., 2010. Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 468, 709–712. Vrati, S., Fernon, C.A., Dalgarno, L., Weir, R.C., 1988. Location of a major antigenic site involved in Ross River virus neutralization. Virology 162, 346–353. Wang, E., Petrakova, O., Adams, A.P., Aguilar, P.V., Kang, W., Paessler, S., Volk, S.M., Frolov, I., Weaver, S.C., 2007. Chimeric Sindbis/eastern equine encephalitis vaccine candidates are highly attenuated and immunogenic in mice. Vaccine 25, 7573–7581. Wang, E., Volkova, E., Adams, A.P., Forrester, N., Xiao, S.Y., Frolov, I., Weaver, S.C., 2008. Chimeric alphavirus vaccine candidates for Chikungunya. Vaccine 26, 5030–5039. Wang, R., Deng, F., Hou, D., Zhao, Y., Guo, L., Wang, H., Hu, Z., 2010. Proteomics of the Autographa californica nucleopolyhedrovirus budded virions. J. Virol. 84, 7233– 7242. Wang, D., Suhrbier, A., Penn-Nicholson, A., Woraratanadharm, J., Gardner, J., Luo, M., Le, T.T., Anraku, I., Sakalian, M., Einfeld, D., Dong, J.Y., 2011. A complex adenovirus vaccine against chikungunya virus provides complete protection against viraemia and arthritis. Vaccine 29, 2803–2809. Weaver, S., Barrett, A., 2004. Transmission cycles, host range, evolution and emergence of arboviral disease. Nat. Rev. Microbiol. 2, 789–801. Weiss, B., Nitschko, H., Ghattas, I., Wright, R., Schlesinger, S., 1989. Evidence for specificity in the encapsidation of Sindbis virus RNAs. J. Virol. 63, 5310–5318. Westaway, E.G., Mackenzie, J.M., Khromykh, A.A., 2003. Kunjin RNA replication and applications of Kunjin replicons. Adv. Virus Res. 59, 99–140. Whitehead, S.S., Blaney, J.E., Durbin, A.P., Murphy, B.R., 2007. Prospects for a dengue virus vaccine. Nat. Rev. Microbiol. 5, 518–528. Whitehouse, C.A., 2004. Crimean-Congo hemorrhagic fever. Antiviral Res. 64, 145– 160. Wilson, A., Mellor, P., 2008. Bluetongue in Europe: vectors, epidemiology and climate change. Parasitol. Res. 103, 69–77. Woolhouse, M.E., Gowtage-Sequeria, S., 2005. Host range and emerging and reemerging pathogens. Emerg. Infect. Dis. 11, 1842–1847. Yu, I.M., Zhang, W., Holdaway, H.A., Li, L., Kostyuchenko, V.A., Chipman, P.R., Kuhn, R.J., Rossmann, M.G., Chen, J., 2008. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834–1837. Zacks, M.A., Paessler, S., 2010. Encephalitic alphaviruses. Vet. Microbiol. 140, 281– 286. Zhang, Y., Corver, J., Chipman, P.R., Zhang, W., Pletnev, S.V., Sedlak, D., Baker, T.S., Strauss, J.H., Kuhn, R.J., Rossmann, M.G., 2003. Structures of immature flavivirus particles. EMBO J. 22, 2604–2613. Zlatkovic, J., Stiasny, K., Heinz, F., 2010. Immunodominance and functional activities of antibody responses to inactivated west nile virus and recombinant subunit vaccines in mice. J. Virol. 85, 1994–2003.