A Survey of Henipavirus Tropism-Our Current Understanding from a Species/Organ and Cellular Level.

Henipaviruses are single-stranded RNA viruses that have been shown to be virulent in several species, including humans, pigs, horses, and rodents. Isolated nearly 30 years ago, these viruses have been shown to be of particular concern to public health, as at least two members (Nipah and Hendra viruses) are highly virulent, as well as zoonotic, and are thus classified as BSL4 pathogens. Although only 5 members of this genus have been isolated and characterized, metagenomics analysis using animal fluids and tissues has demonstrated the existence of other novel henipaviruses, suggesting a far greater degree of phylogenetic diversity than is currently known. Using a variety of molecular biology techniques, it has been shown that these viruses exhibit varying degrees of tropism on a species, organ/tissue, and cellular level. This review will attempt to provide a general overview of our current understanding of henipaviruses, with a particular emphasis on viral tropism.

An Overview of the Infectious Cycle of Bunyaviruses.

Bunyaviruses represent the largest group of RNA viruses and are the causative agent of a variety of febrile and hemorrhagic illnesses. Originally characterized as a single serotype in Africa, the number of described bunyaviruses now exceeds over 500, with its presence detected around the world. These predominantly tri-segmented, single-stranded RNA viruses are transmitted primarily through arthropod and rodent vectors and can infect a wide variety of animals and plants. Although encoding for a small number of proteins, these viruses can inflict potentially fatal disease outcomes and have even developed strategies to suppress the innate antiviral immune mechanisms of the infected host. This short review will attempt to provide an overall description of the order Bunyavirales, describing the mechanisms behind their infection, replication, and their evasion of the host immune response. Furthermore, the historical context of these viruses will be presented, starting from their original discovery almost 80 years ago to the most recent research pertaining to viral replication and host immune response.

Cloning Strategies for the Generation of Recombinant Capripoxvirus Through the Use of Screening and Selection Markers.

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is commonly used to generate capripoxvirus knockout viruses (KO), and is based on the targeting of a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses. An alternative step for the removal of both the EGFP and gpt cassettes and an optional selection step using CRISPR technology are also described.

A novel Schmallenberg virus subunit vaccine candidate protects IFNAR-/- mice against virulent SBV challenge.

Schmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR-/- mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.

Absence of CD59 in Guinea Pigs: Analysis of the Cavia porcellus Genome Suggests the Evolution of a CD59 Pseudogene.

CD59 is a membrane-bound regulatory protein that inhibits the assembly of the terminal membrane attack complex (C5b-9) of complement. From its original discovery in humans almost 30 years ago, CD59 has been characterized in a variety of species, from primates to early vertebrates, such as teleost fish. CD59 is ubiquitous in mammals; however, we have described circumstantial evidence suggesting that guinea pigs (Cavia porcellus) lack CD59, at least on erythrocytes. In this study, we have used a combination of phylogenetic analyses with syntenic alignment of mammalian CD59 genes to identify the only span of genomic DNA in C. porcellus that is homologous to a portion of mammalian CD59 and show that this segment of DNA is not transcribed. We describe a pseudogene sharing homology to exons 2 through 5 of human CD59 present in the C. porcellus genome. This pseudogene was flanked by C. porcellus homologs of two genes, FBXO3 and ORF91, a relationship and orientation that were consistent with other known mammalian CD59 genes. Analysis using RNA sequencing confirmed that this segment of chromosomal DNA was not transcribed. We conclude that guinea pigs lack an intact gene encoding CD59; to our knowledge, this is the first report of a mammalian species that does not express a functional CD59. The pseudogene we describe is likely the product of a genomic deletion event during its evolutionary divergence from other members of the rodent order.

DNA vaccination regimes against Schmallenberg virus infection in IFNAR-/- mice suggest two targets for immunization.

Schmallenberg virus (SBV) is an RNA virus of the Bunyaviridae family, genus Orthobunyavirus that infects wild and livestock species of ruminants. While inactivated and attenuated vaccines have been shown to prevent SBV infection, little is known about their mode of immunity; specifically, which components of the virus are responsible for inducing immunological responses in the host. As previous DNA vaccination experiments on other bunyaviruses have found that glycoproteins, as well as modified (i.e. ubiquitinated) nucleoproteins (N) can confer immunity against virulent viral challenge, constructs encoding for fragments of SBV glycoproteins GN and GC, as well as ubiquitinated and non-ubiquitinated N were cloned in mammalian expression vectors, and vaccinated intramuscularly in IFNAR-/- mice. Upon viral challenge with virulent SBV, disease progression was monitored. Both the ubiquitinated and non-ubiquitinated nucleoprotein candidates elicited high titers of antibodies against SBV, but only the non-ubiquitinated candidate induced statistically significant protection of the vaccinated mice from viral challenge. Another construct encoding for a putative ectodomain of glycoprotein GC (segment aa. 678-947) also reduced the SBV-viremia in mice after SBV challenge. When compared to other experimental groups, both the nucleoprotein and GC-ectodomain vaccinated groups displayed significantly reduced viremia, as well as exhibiting no clinical signs of SBV infection. These results show that both the nucleoprotein and the putative GC-ectodomain can serve as protective immunological targets against SBV infection, highlighting that viral glycoproteins, as well as nucleoproteins are potent targets in vaccination strategies against bunyaviruses.

Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses.

Seroprevalence of Sheep and Goat Pox, Peste Des Petits Ruminants and Rift Valley Fever in Saudi Arabia.

Sheep and goat pox, peste des petits ruminants and Rift Valley fever are important diseases of small ruminant livestock. Sheep and goat pox, along with peste des petits ruminants, are endemic throughout most of Africa, Asia and the Middle East. Whereas Rift Valley fever is endemic in Africa, outbreaks in the Middle East have been reported over the past decade, including the Arabian Peninsula. Saudi Arabia is a major importer of livestock, and understanding the prevalence of these viral infections would be useful for disease control. In this study, sera from sheep and goats were collected from 3 regions in Saudi Arabia. They were evaluated for antibodies specific to sheep and goat pox, peste des petits ruminants and Rift Valley fever by virus neutralization assays. To the best of our knowledge, this is the first study to evaluate the seroprevalence of these viruses in sheep and goats.

A lumpy skin disease virus deficient of an IL-10 gene homologue provides protective immunity against virulent capripoxvirus challenge in sheep and goats.

Sheep and goat pox continue to be important livestock diseases that pose a major threat to the livestock industry in many regions in Africa and Asia. Currently, several live attenuated vaccines are available and used in endemic countries to control these diseases. One of these is a partially attenuated strain of lumpy skin disease virus (LSDV), KS-1, which provides cross-protection against both sheep pox and goat pox. However, when used in highly stressed dairy cattle to protect against lumpy skin disease (LSD) the vaccine can cause clinical disease. In order to develop safer vaccines effective against all three diseases, a pathogenic strain of LSDV (Warmbaths [WB], South Africa) was attenuated by removing a putative virulence factor gene (IL-10-like) using gene knockout (KO) technology. This construct (LSDV WB005KO) was then evaluated as a vaccine for sheep and goats against virulent capripoxvirus challenge. Sheep and goats were vaccinated with the construct and the animals were observed for 21days. The vaccine appeared to be safe, and did not cause disease, although it induced minor inflammation at the injection site similar to that caused by other attenuated sheep and goat pox vaccines. In addition, no virus replication was detected in blood, oral or nasal swabs using real-time PCR following vaccination and low levels of neutralising antibodies were detected in both sheep and goats. Leukocytes isolated from vaccinated animals following vaccination elicited capripoxvirus-specific IFN-γ secretion, suggesting that immunity was also T-cell mediated. Following challenge with virulent capripoxvirus, vaccinated sheep and goats were found to be completely protected and exhibited no clinical disease. Furthermore, real-time PCR of blood samples at various time points suggested that viremia was absent in both groups of vaccinated animals, as opposed to capripoxvirus-related clinical disease and viremia observed in the unvaccinated animals. These findings suggest that this novel knockout strain of LSDV has potential as a vaccine to protect livestock against sheep pox and goat pox.

Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection.

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.

Capripoxvirus-vectored vaccines against livestock diseases in Africa.

Five different viral diseases of livestock, lumpy skin disease (LSD), sheep pox (SPP), goat pox (GTP), Rift Valley fever (RVF) and peste des petits ruminants (PPR), circulate in the same regions of Africa, imposing a major burden on economic activity and public health. While commercial vaccines against these viruses are available, the cost of implementing regular vaccination regimens against multiple diseases is prohibitive for most African farmers. A single, affordable multivalent vaccine that simultaneously protects against all 5 diseases would therefore be of significant benefit to the livestock sector in Africa. It could also serve as a platform for the development of new vaccines of significance to other developing countries around the world. In this paper, we present an overview of the economic importance of livestock in Africa, the pathogens responsible for RVF, PPR, SPP, GTP and LSD and the vaccination strategies currently used to combat them. We then review experience with the development of attenuated capripoxviruses as vaccines against LSD, SPP and GTP and of recombinant capripoxvirus-vectored vaccines against RVF and PPR. We conclude the article by presenting the rationale for a single, multivalent capripoxvirus-vectored vaccine that would protect against all 5 diseases of livestock, and describe the approach being taken by a consortium of Canadian and South African researchers to develop such a vaccine.

Bead-based suspension array for simultaneous detection of antibodies against the Rift Valley fever virus nucleocapsid and Gn glycoprotein.

A multiplex bead-based suspension array was developed that can be used for the simultaneous detection of antibodies against the surface glycoprotein Gn and the nucleocapsid protein N of Rift Valley fever virus (RVFV) in various animal species. The N protein and the purified ectodomain of the Gn protein were covalently linked to paramagnetic Luminex beads. The performance of the resulting multiplex immunoassay was evaluated by testing a comprehensive and well-characterized panel of sera from sheep, cattle and humans. The suitability of this multiplex immunoassay to differentiate infected from vaccinated animals (DIVA) was investigated by testing sera from lambs vaccinated with a paramyxovirus vaccine vector expressing the RVFV surface glycoproteins Gn and Gc. The results suggest that the bead-based suspension array can be used as a DIVA assay to accompany several recently developed experimental vaccines that are based on RVFV glycoproteins, and are devoid of the N protein.

A DNA vaccine encoding ubiquitinated Rift Valley fever virus nucleoprotein provides consistent immunity and protects IFNAR(-/-) mice upon lethal virus challenge.

Current vaccine candidates against Rift Valley fever virus (RVFV) incorporate the viral structural glycoproteins as antigens, since triggering antibody responses against them usually correlates with protection. Here, we have focused solely on the nucleoprotein of RVFV as a potential target for vaccine development. Previous studies in mouse models have already demonstrated that RVFV nucleoprotein can elicit partial protection when administered by means of a DNA vaccine or in recombinant, soluble, protein form. To determine whether this partially protective immune response could be augmented to a level comparable to DNA constructs encoding for RVFV glycoproteins, several targeting sequences were cloned adjacent to the RVFV nucleoprotein (N) gene. Immunization with a plasmid construct encoding for a ubiquitinated form of the viral nucleoprotein (pCMV-Ub-N) significantly increased the survival of IFNAR(-/-) mice following viral challenge to levels comparable with a recombinant DNA-vaccine encoding both RVFV glycoproteins. Mice immunized with pCMV-Ub-N also displayed higher levels of non-neutralizing anti-N antibodies and antigen-specific T-cell responses. This suggests a role for other cell mediated responses in protection against RVFV. These findings show the potential of RVFV N as a candidate antigen for vaccination, and present a new strategy in vaccine design against certain bunyaviruses, where glycoprotein variation may impede effective broad-based vaccination strategies.

Rift valley fever: recent insights into pathogenesis and prevention.

Rift Valley fever virus (RVFV) is a zoonotic pathogen that primarily affects ruminants but can also be lethal in humans. A negative-stranded RNA virus of the family Bunyaviridae, this pathogen is transmitted mainly via mosquito vectors. RVFV has shown the ability to inflict significant damage to livestock and is also a threat to public health. While outbreaks have traditionally occurred in sub-Saharan Africa, recent outbreaks in the Middle East have raised awareness of the potential of this virus to spread to Europe, Asia, and the Americas. Although the virus was initially characterized almost 80 years ago, the only vaccine approved for widespread veterinary use is an attenuated strain that has been associated with significant pathogenic side effects. However, increased understanding of the molecular biology of the virus over the last few years has led to recent advances in vaccine design and has enabled the development of more-potent prophylactic measures to combat infection. In this review, we discuss several aspects of RVFV, with particular emphasis on the molecular components of the virus and their respective roles in pathogenesis and an overview of current vaccine candidates. Progress in understanding the epidemiology of Rift Valley fever has also enabled prediction of potential outbreaks well in advance, thus providing another tool to combat the physical and economic impact of this disease.

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization.

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive ELISA and for antigen capture in a sandwich ELISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from Egyptian or South African lineages. These monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.

Protection against lethal Rift Valley fever virus (RVFV) infection in transgenic IFNAR(-/-) mice induced by different DNA vaccination regimens.

In this work, plasmid constructs encoding two different M segment ORFs, as well as the nucleoprotein N, have been used in different vaccination regimes to test protection against a RVFV-MP12 virus challenge in a transgenic mouse model with impaired interferon type I response (IFNAR(-/-)). We obtained dose dependent protection in animals immunized with a construct encoding both mature glycoproteins (pCMV-M4), whereas only partial protection in animals vaccinated with either N construct (pCMV-N) or a combination of both plasmids (pCMV-M4+pCMV-N). The protection elicited by the expression of the mature glycoproteins could be directly related to the induction of neutralizing antibodies against them. Interestingly, the combination of both vaccine constructs induced specific lymphoblast proliferation upon stimulation with a recombinant nucleoprotein.

The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar).

Fish embryos and hatchlings are exposed to pathogens long before maturation of their lymphoid organs. Little is known about defence mechanisms during the earliest stages of life, but innate mechanisms may be essential for survival. The complement system in fish is well developed and represents a major part of innate immunity. Complement factor 3 (C3) is central subsequent to activation of all pathways of the complement system, leading to inflammatory reactions, such as chemotaxis, opsonisation and lysis of pathogens. Hepatocytes represent the major source of C3, but modern molecular biological methods have confirmed that C3 is synthesised at multiple sites. Our main objective was to study the ontogeny of C3 in Atlantic salmon by mapping the commencement of synthesis and localisation of proteins. Eggs, embryos, hatchlings and adult fish were analysed for the presence of C3 mRNA and proteins. From immunohistochemical studies, C3 proteins were detected at several extrahepatic sites, such as the skeletal muscle, developing notochord and chondrocytes of the gill arch. Immunoblotting revealed presence of C3 proteins in the unfertilised egg, but C3 mRNA was only detected after fertilisation by real-time RT-PCR. Taken together, the results implicated the maternal transfer of C3 proteins as well as novel non-immunological functions during development.

B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities.

The present paradigm dictates that phagocytosis is accomplished mainly by 'professional' phagocytes (such as macrophages and monocytes), whereas B cells lack phagocytic capabilities. Here we demonstrate that B cells from teleost fish have potent in vitro and in vivo phagocytic activities. Particle uptake by B cells induced activation of 'downstream' degradative pathways, leading to 'phagolysosome' formation and intracellular killing of ingested microbes. Those results indicate a previously unknown function for B cells in the innate immunity of these primitive animals. A considerable proportion of Xenopus laevis B cells were also phagocytic. Our findings support the idea that B cells evolved from an ancestral phagocytic cell type and provide an evolutionary framework for understanding the close relationship between mammalian B lymphocytes and macrophages.

Maternal transfer of complement components C3-1, C3-3, C3-4, C4, C5, C7, Bf, and Df to offspring in rainbow trout (Oncorhynchus mykiss).

Defense mechanisms in developing fish are poorly known but before maturation of lymphoid organs and immunocompetence, innate mechanisms are essential. The complement system represents a major part of innate immunity. Our main objective was to map the presence of complement components early in fish development. Rainbow trout eggs, embryos, and hatchlings were assayed for the onset and duration of C3-1, C3-3, C3-4, C4, C5, C7, factor B, and factor D transcription using real-time reverse transcription-polymerase chain reaction. In general, complement transcript levels increased steadily from day 28 postfertilization to hatch, followed by a decrease during yolk-sac resorption. All the complement proteins studied were found in unfertilized eggs. There was no correlation between the transcript and protein levels throughout the study period. Complement proteins appeared in the liver, kidney, and intestine between day 7 and 35 but not until day 77 in the heart. This study is the first to address the ontogeny of several complement components and represents the first evidence that maternal transfer of complement components, other than C3, occurs in teleost fish.

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species.

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.