Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins.

Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically Differentiate the Infected from the Vaccinated Animals (DIVA). We developed an indirect ELISA using HeV-G expressed in Leishmania tarentolae and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from naïve, vaccinated and infected horses that either originated from a Hendra-virus free region, or had been pre-tested in validated diagnostic tests. Our data confirm the reliability of this approach, as HeV-N-specific antibodies were only detected in sera from infected horses, while HeV-G-specific antibodies were detected in infected and vaccinated horses with a high level of specificity and sensitivity. Given the excellent correlation of data obtained for German and Australian HeV-negative horses, we assume that this test can be applied for the testing of horse serum samples from a variety of geographical regions.

Identification of African swine fever virus-like elements in the soft tick genome provides insights into the virus' evolution.

BACKGROUND: African swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus' evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host's genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery. RESULTS: For the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks. CONCLUSION: Here, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector 'arms race', a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV.

Productive replication of peste des petits ruminants virus Nigeria 75/1 vaccine strain in vero cells correlates with inefficiency of maturation of the viral fusion protein.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus, in the family Paramyxoviridae expresses two membrane glycoproteins, the fusion (F) and haemagglutinin (H) glycoproteins which mediate virus-to-cell fusion and cell-to-cell fusion leading to the induction of syncytia in PPRV infected cells. In the context of the characterization of the virulent lineage IV strain PPRV Kurdistan 2011, isolated from wild goats from the Kurdistan region in Iraq, we observed that both PPRV Kurdistan 2011 and the PPRV Nigeria 75/1 vaccine strain led to induction of large syncytia in Vero-dogSLAM cells within 48 h whereas both failed to induce detectable cell-cell fusion events in two Vero cell lines of differing passage histories. We were unable to detect syncytium formation in transiently transfected cells expressing PPRV F or H alone whereas co-expression of F and H induced large syncytia - in Vero-dogSLAM cells only. In VeroMontpellier cells expressing PPRV F and H, fused cells were rarely detectable indicating that PPRV mediated cell fusion activity is impaired in this cell line. Surprisingly, on Vero-dogSLAM cells the vaccine strain grew to titers of 105.25 TCID50/ml, whereas infectious virus yield was about 200-fold higher on VeroMontpellier and Vero-76 cells. In contrast, the virulent Kurdistan 2011 strain grew to a maximum titer of 107.0 TCID50/ml on Vero-dogSLAM cells and only 104.5 TCID50/ml on normal Vero cells. This was as expected since Vero cells lacking the SLAM receptor for PPRV are regarded as not so permissive for infection. To elucidate the divergent productive replication behaviour of PPRV Nigeria 75/1 vaccine strain on Vero vs Vero-dogSLAM cells, we examined whether intracellular transport and/or maturation of the viral envelope glycoproteins F and H might be implicated with this phenomenon. The results indicate that F in contrast to the H glycoprotein matures inefficiently during intracellular transport in VeroMontpellier cells, thus leading to an absence of detectable syncytia formation. However, in the case of the PPRV Nigeria 75/1 vaccine strain this did not impair efficient virus assembly and release.

The intracellular proteome of African swine fever virus.

African swine fever (ASF) is a viral disease that affects members of the Suidae family such as African bush pigs, warthogs, but also domestic pigs, and wild boar. It is transmitted by direct contact of naïve with infected animals, by soft ticks of the Ornithodoros genus, or indirectly by movement of infected animals, improper disposal of contaminated animal products or other sources related to human activity. The recent spread of ASF into Eastern and Central European countries is currently threatening the European pig industry. The situation is aggravated as to-date no efficient vaccine is available. African swine fever virus (ASFV) is a large enveloped ds DNA-virus encoding at least 150 open reading frames. Many of the deduced gene products have not been described, less functionally characterized. We have analysed ASFV protein expression in three susceptible mammalian cell lines representing a susceptible host (wild boar) and two non-susceptible species (human and green monkey) by mass spectrometry and provide first evidence for the expression of 23 so far uncharacterized ASFV ORFs. Expression levels of several newly identified ASFV proteins were remarkably high indicating importance in the viral replication cycle. Moreover, expression profiles of ASFV proteins in the three cell lines differed markedly.

Efficient transgene insertion in a pseudorabies virus vector by CRISPR/Cas9 and marker rescue-enforced recombination.

For development of vectored vaccines against porcine pathogens the genome of the pseudorabies virus vaccine strain Bartha (PrV-Ba) was previously cloned as an infectious bacterial artificial chromosome (BAC), containing the bacterial replicon and a reporter gene cassette encoding enhanced green fluorescent protein (EGFP) at the nonessential glycoprotein G locus. To facilitate substitution of this insertion, this BAC was now modified by deletion of the adjacent promoter and initiation codon of the essential glycoprotein D (gD) gene of PrV-Ba. Furthermore, rabbit kidney (RK13) cells stably expressing Cas9 nuclease and an EGFP gene-specific guide RNA were prepared to induce site specific cleavage of the BAC DNA. After co-transfection of these cells with the modified BAC and recombination plasmids containing expression cassettes for new transgenes flanked by PrV DNA sequences including the intact 5'-end of the gD gene, >95% of the recombinants exhibited the desired gene substitutions, while no EGFP-expressing progeny virus was detectable. This approach was used for insertion and expression of the open reading frames E199L, CP204L (p30) and KP177R (p22) of African swine fever virus. The studies revealed that codon adaptation significantly enhanced expression of E199L, and that the chimeric CAG promoter increased transgene expression compared to cytomegalovirus immediate-early promoters.

Genetic fusion of peste des petits ruminants virus haemagglutinin and fusion protein domains to the amino terminal subunit of glycoprotein B of bovine herpesvirus 1 interferes with transport and function of gB for BHV-1 infectious replication.

Peste des petits ruminants is an emerging, often fatal viral disease of domestic and wild small ruminants caused by peste des petits ruminants virus. The haemagglutinin and the fusion protein are viral envelope glycoproteins and essential for the infection process and both induce a protective immune response in infected or vaccinated animals. Attempts to generate pseudotyped bovine herpesvirus 1 recombinants firstly by integration of expression cassettes for PPRV-H and PPRV-F into the herpesviral genome or secondly to generate pseudotyped BHV-1 by genetically fusing relevant parts of both PPRV glycoproteins to the amino-terminal subunit of glycoprotein B, approaches that had been successful for heterologous viral membrane glycoproteins in the past, failed repeatedly. We therefore analyzed at which intracellular stage generation of viable BHV-1 hybrid-gB recombinants might be inhibited. Results obtained from transient protein expression experiments revealed that, dependent on the fusion protein, transport of the hybrid glycoproteins beyond the endoplasmic reticulum is impeded. Thus, expression of heterologous glycoproteins using BHV-1 interferes more than expected from published experience with BHV-1 gB transport and consequently with virus replication.

Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L).

African swine fever is a devastating viral disease of domestic and wild pigs against which no vaccine or therapy is available. Therefore, we applied the CRISPR (clustered regularly interspaced short palindromic repeats) - Cas9 nuclease system to target the double-stranded DNA genome of African swine fever virus (ASFV). To this end, a permissive wild boar lung (WSL) cell line was modified by stable transfection with a plasmid encoding Cas9 and a guide RNA targeting codons 71 to 78 of the phosphoprotein p30 gene (CP204L) of ASFV. Due to targeted Cas9 cleavage of the virus genome, plaque formation of ASFV was completely abrogated and virus yields were reduced by four orders of magnitude. The specificity of these effects could be demonstrated by using a natural ASFV isolate and escape mutants possessing nucleotide exchanges within the target sequence, which were not inhibited in the Cas9-expressing cell line. Growth of the cell line was not affected by transgene expression which, as well as virus inhibition, proved to be stable over at least 50 passages. Thus, CRISPR-Cas9 mediated targeting of the ASFV p30 gene is a valid strategy to convey resistance against ASF infection, which may also be applied in its natural animal host.

BacMam Platform for Vaccine Antigen Delivery.

Recombinant baculo viruses based on Autographa californica multiple nuclear polyhedrosis virus carrying vertebrate cell active expression cassettes, so-called BacMam viruses, are increasingly used as gene delivery vectors for vaccination of animals against pathogens. Different approaches for generation of BacMams exist and a variety of transfer vectors to improve target protein expression in vivo have been constructed. Here we describe a use of transfer vector which contains an insect cell-restricted expression cassette for the green fluorescent protein and thus enables easy monitoring of BacMam virus rescue, fast plaque purification of recombinants and their convenient titer determination and which has been proven to be efficacious for gene delivery in vaccination/challenge experiments.

Related strains of African swine fever virus with different virulence: genome comparison and analysis.

Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.

ANP32B is a nuclear target of henipavirus M proteins.

Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.

A novel bromodeoxyuridine-resistant wild boar lung cell line facilitates generation of African swine fever virus recombinants.

Manipulation of African swine fever virus (ASFV) genomes, in particular those from field strains, is still a challenge. We have shown recently that generation of a green-fluorescent-protein-expressing, thymidine-kinase-negative (TK-) mutant of the low-pathogenic African swine fever virus field strain NHV was supported by a TK- Vero cell line. Since NHV, like other ASFV field strains, does not replicate well in Vero cells, a bromodeoxyuridine (BrdU)- resistant cell line derived from wild boar lung (WSL) cells, named WSL-Bu, was selected. WSL cells were used because they are suitable for productive replication of NHV and other ASFV field strains. Here, we show that WSL-Bu cells enable positive selection of both TK- and TK+ ASFV recombinants, which allows for novel strategies for construction of ASFV mutants. We further demonstrate for a low-pathogenic ASFV strain that TK expression is required for infectious replication in macrophages infected at low multiplicity and that vaccinia TK fully complements ASFV TK in this respect.

High-level protein expression following single and dual gene cloning of infectious bronchitis virus N and S genes using baculovirus systems.

Baculovirus is an efficient system for the gene expression that can be used for gene transfer to both insect and different vertebrate hosts. The nucleocapsid gene (N) of the infectious bronchitis virus was cloned in a baculovirus expression system for insect cell expression. Dual expression vectors containing IBV N and spike (S) proteins of the avian infectious bronchitis virus were engineered under the control of human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in both vertebrate and insect cells. Transduction of the N gene in the insect Sf9 cells revealed a high level of protein expression. The expressed protein, used in ELISA, effectively detected chicken anti-IBV antibodies with high specificity. Transduction of mammalian and avian cells with BacMam viruses revealed that dual expression cassettes yielded high levels of protein from both transcription units.

Infection of differentiated airway epithelial cells from caprine lungs by viruses of the bovine respiratory disease complex.

Bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3) and bovine herpesvirus type 1 (BHV-1) are important pathogens associated with the bovine respiratory disease complex (BRDC). Non-bovine ruminants such as goats may also be infected and serve as a virus reservoir to be considered in the development of control strategies. To evaluate the susceptibility of caprine airway epithelial cells to infection by viruses of BRDC, we established a culture system for differentiated caprine epithelial cells. For this purpose, we generated precision-cut lung slices (PCLS), in which cells are retained in their original structural configuration and remain viable for more than a week. The three bovine viruses were found to preferentially infect different cell types. Ciliated epithelial cells were the major target cells of BPIV3, whereas BHV-1 preferred basal cells. Cells infected by BRSV were detected in submucosal cell layers. This spectrum of susceptible cells is the same as that reported recently for infected bovine PCLS. While infection of caprine cells by BRSV and BPIV3 was as efficient as that reported for bovine cells, infection of caprine cells by BHV-1 required a tenfold higher dose of infectious virus as compared to infection of bovine airway cells. These results support the notion that non-bovine ruminants may serve as a reservoir for viruses of BRDC and introduce a culture system to analyze virus infection of differentiated airway epithelial cells from the caprine lung.

Three viruses of the bovine respiratory disease complex apply different strategies to initiate infection.

Bovine respiratory disease complex (BRDC) is the major cause of serious respiratory tract infections in calves. The disease is multifactorial, with either stress or reduced immunity allowing several pathogens to emerge. We investigated the susceptibility of bovine airway epithelial cells (BAEC) to infection by the three major viruses associated with the BRDC: bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BHV-1) and bovine parainfluenza virus type 3 (BPIV3). For this purpose, two culture systems for well-differentiated BAEC were used: the air-liquid interface (ALI) system, where filter-grown BAEC differentiate into a pseudostratified respiratory epithelium and precision-cut lung slices (PCLS) where BAEC are maintained in the original tissue organisation. Comparative infection studies demonstrated that entry and release of BPIV3 occurred specifically via the apical membrane with ciliated cells being the major target cells. By contrast, airway epithelial cells were largely resistant to infection by BHV-1. When the epithelial barrier was abolished by opening tight junctions or by injuring the cell monolayer, BHV-1 infected mainly basal cells. Respiratory epithelial cells were also refractory to infection by BRSV. However, this virus infected neither differentiated epithelial cells nor basal cells when the integrity of the epithelial barrier was destroyed. In contrast to cells of the airway epithelium, subepithelial cells were susceptible to infection by BRSV. Altogether, these results indicate that the three viruses of the same disease complex follow different strategies to interact with the airway epithelium. Possible entry mechanisms are discussed.

BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus.

Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

Virus species polemics: 14 senior virologists oppose a proposed change to the ICTV definition of virus species.

The Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) has recently decided to modify the current definition of virus species (Code of Virus Classification and Nomenclature Rule 3.21) and will soon ask the full ICTV membership (189 voting members) to ratify the proposed controversial change. In this discussion paper, 14 senior virologists, including six Life members of the ICTV, compare the present and proposed new definition and recommend that the existing definition of virus species should be retained. Since the pros and cons of the proposal posted on the ICTV website are not widely consulted, the arguments are summarized here in order to reach a wider audience.

Novel approach for the generation of recombinant African swine fever virus from a field isolate using GFP expression and 5-bromo-2'-deoxyuridine selection.

Generation of African swine fever virus (ASFV) recombinants has so far relied mainly on the manipulation of virus strains which had been adapted to growth in cell culture, since field isolates do not usually replicate efficiently in established cell lines. Using wild boar lung cells (WSL) which allow for propagation of ASFV field isolates, a novel approach for the generation of recombinant ASFV directly from field isolates was developed which includes the integration into the viral thymidine kinase (TK) locus of an ASFV p72-promoter driven expression cassette for enhanced green fluorescent protein (EGFP) embedded in a 16 kbp mini F-plasmid into the genome of the ASFV field strain NHV. This procedure enabled the monitoring of recombinant virus replication by EGFP autofluorescence. Selection for the TK-negative (TK(-)) phenotype of the recombinants on TK(-) Vero (VeroTK(-)) cells in the presence of 5-bromo-2'-deoxyuridine (BrdU) led to efficient isolation of recombinant virus due to the elimination of TK(+) wild type virus by BrdU-phosporylation in infected VeroTK(-) cells. The recombinant NHV-dTK-GFP produced titres of both cell-associated and secreted viral progeny in WSL cells similar to parental NHV indicating that insertion of large heterologous sequences into the viral TK locus and EGFP expression do not impair viral replication in these cells. In summary, a novel method has been developed for generation of ASFV recombinants directly from field isolates, providing an efficacious method for further manipulations of wild-type virus genomes.

Potential of adenovirus and baculovirus vectors for the delivery of shRNA against morbilliviruses.

Morbilliviruses are important pathogens of humans, ruminants, carnivores and marine mammals. Although good vaccines inducing long-term immunity are available, recurrent outbreaks of measles, canine distemper and peste des petits ruminants (PPR) are observed. In control strategies, antivirals thus could be useful to confine virus spread and application of interfering RNAs is a promising approach, provided they can be delivered efficiently into the host cells. We have constructed recombinant adenovirus and baculovirus vectors expressing short hairpin RNAs (shRNAs) against the PPR virus (PPRV) and compared them in vitro. It was found that both recombinant viruses inhibited PPRV replication with the baculovirus vector, which inhibited generation of infectious progeny by more than 2 log10 and the nucleoprotein expression of PPRV by 73%, being the more efficient. The results show that baculoviral shRNA-expressing vectors have the potential for therapeutic use against morbillivirus infections.

A proposal to change existing virus species names to non-Latinized binomials.

A proposal has been posted on the ICTV website (2011.001aG.N.v1.binomial_sp_names) to replace virus species names by non-Latinized binomial names consisting of the current italicized species name with the terminal word "virus" replaced by the italicized and non-capitalized genus name to which the species belongs. If implemented, the current italicized species name Measles virus, for instance, would become Measles morbillivirus while the current virus name measles virus and its abbreviation MeV would remain unchanged. The rationale for the proposed change is presented.