Co-housing of Rift Valley Fever Virus Infected Lambs with Immunocompetent or Immunosuppressed Lambs Does Not Result in Virus Transmission.

Rift Valley fever virus (RVFV) is transmitted among susceptible animals by mosquito vectors. Although the virus can be isolated from nasal and oral swabs of infected animals and is known to be highly infectious when administered experimentally via oral or respiratory route, horizontal transmission of the virus is only sporadically reported in literature. We considered that immunosuppression resulting from stressful conditions in the field may increase the susceptibility to horizontally transmitted RVFV. Additionally, we reasoned that horizontal transmission may induce immune responses that could affect the susceptibility of contact-exposed animals to subsequent infection via mosquito vectors. To address these two hypotheses, viremic lambs were brought into contact with sentinel lambs. One group of sentinel lambs was treated with the immunosuppressive synthetic glucocorticosteroid dexamethasone and monitored for signs of disease and presence of virus in the blood and target organs. Another group of contact-exposed sentinel lambs remained untreated for three weeks and was subsequently challenged with RVFV. We found that none of the dexamethasone-treated contact-exposed lambs developed detectable viremia, antibody responses or significant increases in cytokine mRNA levels. Susceptibility of immunocompetent lambs to RVFV infection was not influenced by previous contact-exposure. Our results are discussed in light of previous findings.

Vector independent transmission of the vector-borne bluetongue virus.

Bluetongue is an economically important disease of ruminants. The causative agent, Bluetongue virus (BTV), is mainly transmitted by insect vectors. This review focuses on vector-free BTV transmission, and its epizootic and economic consequences. Vector-free transmission can either be vertical, from dam to fetus, or horizontal via direct contract. For several BTV-serotypes, vertical (transplacental) transmission has been described, resulting in severe congenital malformations. Transplacental transmission had been mainly associated with live vaccine strains. Yet, the European BTV-8 strain demonstrated a high incidence of transplacental transmission in natural circumstances. The relevance of transplacental transmission for the epizootiology is considered limited, especially in enzootic areas. However, transplacental transmission can have a substantial economic impact due to the loss of progeny. Inactivated vaccines have demonstrated to prevent transplacental transmission. Vector-free horizontal transmission has also been demonstrated. Since direct horizontal transmission requires close contact of animals, it is considered only relevant for within-farm spreading of BTV. The genetic determinants which enable vector-free transmission are present in virus strains circulating in the field. More research into the genetic changes which enable vector-free transmission is essential to better evaluate the risks associated with outbreaks of new BTV serotypes and to design more appropriate control measures.

Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice.

Crimean-Congo hemorrhagic fever virus is a tick-borne bunyavirus of the Nairovirus genus that causes hemorrhagic fever in humans with high case fatality. Here, we report the development of subunit vaccines and their efficacy in signal transducer and activator of transcription 1 (STAT1) knockout mice. Ectodomains of the structural glycoproteins Gn and Gc were produced using a Drosophila insect cell-based expression system. A single vaccination of STAT129 mice with adjuvanted Gn or Gc ectodomains induced neutralizing antibody responses, which were boosted by a second vaccination. Despite these antibody responses, mice were not protected from a CCHFV challenge infection. These results suggest that neutralizing antibodies against CCHFV do not correlate with protection of STAT1 knockout mice.

Nonspreading Rift Valley Fever Virus Infection of Human Dendritic Cells Results in Downregulation of CD83 and Full Maturation of Bystander Cells.

Vaccines based on nonspreading Rift Valley fever virus (NSR) induce strong humoral and robust cellular immune responses with pronounced Th1 polarisation. The present work was aimed to gain insight into the molecular basis of NSR-mediated immunity. Recent studies have demonstrated that wild-type Rift Valley fever virus efficiently targets and replicates in dendritic cells (DCs). We found that NSR infection of cultured human DCs results in maturation of DCs, characterized by surface upregulation of CD40, CD80, CD86, MHC-I and MHC-II and secretion of the proinflammatory cytokines IFN-ß, IL-6 and TNF. Interestingly, expression of the most prominent marker of DC maturation, CD83, was consistently downregulated at 24 hours post infection. Remarkably, NSR infection also completely abrogated CD83 upregulation by LPS. Downregulation of CD83 was not associated with reduced mRNA levels or impaired CD83 mRNA transport from the nucleus and could not be prevented by inhibition of the proteasome or endocytic degradation pathways, suggesting that suppression occurs at the translational level. In contrast to infected cells, bystander DCs displayed full maturation as evidenced by upregulation of CD83. Our results indicate that bystander DCs play an important role in NSR-mediated immunity.

Four-segmented Rift Valley fever virus induces sterile immunity in sheep after a single vaccination.

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family, causes recurrent outbreaks with severe disease in ruminants and occasionally humans. The virus comprises a segmented genome consisting of a small (S), medium (M) and large (L) RNA segment of negative polarity. The M-segment encodes a glycoprotein precursor (GPC) protein that is co-translationally cleaved into Gn and Gc, which are required for virus entry and fusion. Recently we developed a four-segmented RVFV (RVFV-4s) by splitting the M-genome segment, and used this virus to study RVFV genome packaging. Here we evaluated the potential of a RVFV-4s variant lacking the NSs gene (4s-ΔNSs) to induce protective immunity in sheep. Groups of seven lambs were either mock-vaccinated or vaccinated with 10(5) or 10(6) tissue culture infective dose (TCID50) of 4s-ΔNSs via the intramuscular (IM) or subcutaneous (SC) route. Three weeks post-vaccination all lambs were challenged with wild-type RVFV. Mock-vaccinated lambs developed high fever and high viremia within 2 days post-challenge and three animals eventually succumbed to the infection. In contrast, none of the 4s-ΔNSs vaccinated animals developed clinical signs during the course of the experiment. Vaccination with 10(5) TCID50 via the IM route provided sterile immunity, whereas a 10(6) dose was required to induce sterile immunity via SC vaccination. Protection was strongly correlated with the presence of RVFV neutralizing antibodies. This study shows that 4s-ΔNSs is able to induce sterile immunity in the natural target species after a single vaccination, preferably administrated via the IM route.

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA.

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA). BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine. Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses. Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.

Paramyxovirus-based production of Rift Valley fever virus replicon particles.

Replicon-particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR), and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) were able to produce high levels of NSR particles. In addition, using reverse genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains two amino acid substitutions in, respectively, Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.

No significant differences in the breadth of the foot-and-mouth disease serotype A vaccine induced antibody responses in cattle, using different adjuvants, mixed antigens and different routes of administration.

Inactivated whole virus foot-and-mouth disease (FMD) vaccines are used worldwide for protection against FMD, but not all vaccines induce protection against all genetic variants of the same FMD virus serotype. The aim of this study is to investigate whether the "breadth" of the antibody response against different strains of the same FMD virus serotype in cattle could be improved by using a different adjuvant, a mix of antigens and/or different routes of administration. To this end, six groups of five cattle were vaccinated with different FMD virus serotype A strain vaccines formulated with Montanide ISA 206 VG adjuvant. Antibody responses for homologous and heterologous cross-reactivity against a panel of 10 different FMD virus serotype A strains were tested by a liquid-phase blocking ELISA. Results of cattle vaccinated with ISA 206 VG adjuvanted vaccine were compared with results obtained in a previous study using aluminium hydroxide-saponin adjuvant. No significant effect of adjuvant on the breadth of the antibody response was observed, neither for mixing of antigens nor for the route of administration (subcutaneous vs. intradermal). Comparison of antigen payload, however, increased both homologous and heterologous titres; a 10-fold higher antigen dose resulted in approximately four times higher titres against all tested strains. Our study shows that breadth of the antibody response depends mainly on the vaccine strain; we therefore propose that, for vaccine preparation, only FMD virus strains are selected that, among other important characteristics, will induce a wide antibody response to different field strains.

Creation of Rift Valley fever viruses with four-segmented genomes reveals flexibility in bunyavirus genome packaging.

UNLABELLED: Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines. IMPORTANCE: Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.

Comparison of test methodologies for foot-and-mouth disease virus serotype A vaccine matching.

Vaccination has been one of the most important interventions in disease prevention and control. The impact of vaccination largely depends on the quality and suitability of the chosen vaccine. To determine the suitability of a vaccine strain, antigenic matching is usually studied by in vitro analysis. In this study, we performed three in vitro test methods to determine which one gives the lowest variability and the highest discriminatory capacity. Binary ethylenimine inactivated vaccines, prepared from 10 different foot-and-mouth disease (FMD) virus serotype A strains, were used to vaccinate cattle (5 animals for each strain). The antibody titers in blood serum samples 3 weeks postvaccination (w.p.v.) were determined by a virus neutralization test, neutralization index test, and liquid-phase blocking enzyme-linked immunosorbent assay (ELISA). The titers were then used to calculate relationship coefficient (r1) values. These r1 values were compared to the genetic lineage using receiver operating characteristic (ROC) analysis. In the two neutralization test methods, the median titers observed against the test strains differed considerably, and the sera of the vaccinated animals did not always show the highest titers against their respective homologous virus strains. When the titers were corrected for test strain effect (scaling), the variability (standard error of the mean per vaccinated group) increased because the results were on a different scale, but the discriminatory capacity improved. An ROC analysis of the r1 value calculated on both observed and scaled titers showed that only r1 values of the liquid-phase blocking ELISA gave a consistent statistically significant result. Under the conditions of the present study, the liquid-phase blocking ELISA showed less variation and still had a higher discriminatory capacity than the other tests.

Transplacental transmission of Bluetongue virus serotype 1 and serotype 8 in sheep: virological and pathological findings.

The Bluetongue virus serotype 8 (BTV-8) strain, which emerged in Europe in 2006, had an unusually high ability to cause foetal infection in pregnant ruminants. Other serotypes of BTV had already been present in Europe for more than a decade, but transplacental transmission of these strains had never been demonstrated. To determine whether transplacental transmission is a unique feature of BTV-8 we compared the incidence and pathological consequences of transplacental transmission of BTV-8 to that of BTV-1. Nine pregnant ewes were infected with either BTV-8 or BTV-1. The BTV strains used for the infection were field strains isolated on embryonated chicken eggs and passaged twice on mammalian cells. Blood samples were taken to monitor the viraemia in the ewes. Four weeks after the infection, the foetuses were examined for pathological changes and for the presence of BTV. BTV-8 could be demonstrated in 12 foetuses (43%) from 5 ewes (56%). %). BTV-1 was detected in 14 foetuses (82%) from 6 ewes (67%). Pathological changes were mainly found in the central nervous system. In the BTV-8 group, lympho-histiocytic infiltrates, gliosis and slight vacuolation of the neuropil were found. BTV-1 infection induced a severe necrotizing encephalopathy and severe meningitis, with macroscopic hydranencephaly or porencephaly in 8 foetuses. In our experimental setting, using low passaged virus strains, BTV-1 was able to induce transplacental transmission to a higher incidence compared to BTV-8, causing more severe pathology.

A single vaccination with an improved nonspreading Rift Valley fever virus vaccine provides sterile immunity in lambs.

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 10(6.3) TCID50/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.

Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.

Creation of a nonspreading Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.

Parenteral vaccination of mammalian livestock with Newcastle disease virus-based vector vaccines offers optimal efficacy and safety.

Newcastle disease virus (NDV) is an avian virus that is being evaluated as a vaccine vector for the delivery of foreign genes in mammals. The use of NDV as a vaccine vector in these species offers two major advantages. First, NDV is highly attenuated in mammals, rendering its use inherently safe. Second, mammals lack pre-existing NDV immunity, which minimizes the risk of vaccination failure. NDV-vector vaccines are generally administered to mammals via the respiratory route. We recently showed that intramuscular vaccination with NDV-based Rift Valley fever virus (RVFV) vaccines provides complete protection in mice and induces neutralizing antibodies in sheep and cattle, the main target species of RVFV. Here, we discuss the use of NDV as a vaccine vector for applications in mammalian livestock with an emphasis on the vaccination route. We also report the results of novel experiments that underscore our notion that vaccination via a parenteral route is more effective than immunization via the respiratory route.

Interventions against West Nile virus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus: where are we?

ARBO-ZOONET is an international network financed by the European Commission's seventh framework program. The major goal of this initiative is capacity building for the control of emerging viral vector-borne zoonotic diseases, with a clear focus on West Nile virus, Rift Valley fever virus, and Crimean-Congo hemorrhagic fever virus. To evaluate the status quo of control measures against these viruses, an ARBO-ZOONET meeting was held in Istanbul, Turkey, from 19 to 20 November 2009. The symposium consisted of three themes: (1) vaccines: new and existing ones; (2) antivirals: existing and new developments; and (3) antivector vaccines. In addition, a satellite workshop was held on epidemiology and diagnosis. The meeting brought together foremost international experts on the subjects from both within and without the ARBO-ZOONET consortium. This report highlights selected results from these presentations and major conclusions that emanated from the discussions held.

Challenges for porcine reproductive and respiratory syndrome virus (PRRSV) vaccinology.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat for the pig industry. Vaccines have been developed, but these failed to provide sustainable disease control, in particular against genetically unrelated strains. Here we give an overview of current knowledge and gaps in our knowledge that may be relevant for the development of a future generation of more effective vaccines. PRRSV replicates in cells of the monocyte/macrophage lineage, induces apoptosis and necrosis, interferes with the induction of a proinflammatory response, only slowly induces a specific antiviral response, and may cause persistent infections. The virus appears to use several evasion strategies to circumvent both innate and acquired immunity, including interference with antigen presentation, antibody-mediated enhancement, reduced cell surface expression of viral proteins, and shielding of neutralizing epitopes. In particular the downregulation of type I interferon-alpha production appears to interfere with the induction of acquired immunity. Current vaccines are ineffective because they suffer both from the immune evasion strategies of the virus and the antigenic heterogeneity of field strains. Future vaccines therefore must "uncouple" the immune evasion and apoptogenic/necrotic properties of the virus from its immunogenic properties, and they should induce a broad immune response covering the plasticity of its major antigenic sites. Alternatively, the composition of the vaccine should be changed regularly to reflect presently and locally circulating strains. Preferably new vaccines should also allow discriminating infected from vaccinated pigs to support a virus elimination strategy. Challenges in vaccine development are the incompletely known mechanisms of immune evasion and immunity, lack of knowledge of viral sequences that are responsible for the pathogenic and immunosuppressive properties of the virus, lack of knowledge of the forces that drive antigenic heterogeneity and its consequences for immunogenicity, and a viral genome that is relatively intolerant for subtle changes at functional sites.

In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes.

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3.9 kb (M39). Expression of the reporter gene beta-glucuronidase (GUS) (2-8 microg per 10(6) cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1-2 microg per 10(6) cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development.