Detection of African Swine Fever Virus and Blood Meals of Porcine Origin in Hematophagous Insects Collected Adjacent to a High-Biosecurity Pig Farm in Lithuania; A Smoking Gun?

A seasonal trend of African swine fever (ASF) outbreaks in domestic pig farms has been observed in affected regions of Eastern Europe. Most outbreaks have been observed during the warmer summer months, coinciding with the seasonal activity pattern of blood-feeding insects. These insects may offer a route for introduction of the ASF virus (ASFV) into domestic pig herds. In this study, insects (hematophagous flies) collected outside the buildings of a domestic pig farm, without ASFV-infected pigs, were analyzed for the presence of the virus. Using qPCR, ASFV DNA was detected in six insect pools; in four of these pools, DNA from suid blood was also identified. This detection coincided with ASFV being reported in the wild boar population within a 10 km radius of the pig farm. These findings show that blood from ASFV-infected suids was present within hematophagous flies on the premises of a pig farm without infected animals and support the hypothesis that blood-feeding insects can potentially transport the virus from wild boars into domestic pig farms.

Unbiased Virus Detection in a Danish Zoo Using a Portable Metagenomic Sequencing System.

Metagenomic next-generation sequencing (mNGS) is receiving increased attention for the detection of new viruses and infections occurring at the human-animal interface. The ability to actively transport and relocate this technology enables in situ virus identification, which could reduce response time and enhance disease management. In a previous study, we developed a straightforward mNGS procedure that greatly enhances the detection of RNA and DNA viruses in human clinical samples. In this study, we improved the mNGS protocol with transportable battery-driven equipment for the portable, non-targeted detection of RNA and DNA viruses in animals from a large zoological facility, to simulate a field setting for point-of-incidence virus detection. From the resulting metagenomic data, we detected 13 vertebrate viruses from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including avian leukosis virus in domestic chickens (Gallus gallus), enzootic nasal tumour virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA (CRESS DNA) viruses in several mammal species. More significantly, we demonstrate that the mNGS method is able to detect potentially lethal animal viruses, such as elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the newly described human-associated gemykibivirus 2, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for the first time.

The N-terminal region (VP4) of the foot-and-mouth disease capsid precursor (P1-2A) is not required during its synthesis to allow subsequent processing by the 3C protease.

The capsid precursor (P1-2A) of foot-and-mouth disease virus is processed by the 3C protease (3Cpro) to VP0, VP3 and VP1 plus 2A. During capsid assembly, the VP0 is cleaved to VP4 plus VP2. Single amino acid changes in a conserved motif (YCPRP) near the C-terminus of VP1 can block processing of the capsid precursor by the 3Cpro, although the cleavage sites are located hundreds of amino acids distant from this motif, presumably due to misfolding. In contrast, we show here that the absence of the VP4 sequence during the synthesis of the capsid precursor does not affect its subsequent processing. Cleavage of this truncated precursor by 3Cpro at the VP3/VP1 and VP2/VP3 junctions occurred efficiently. Thus, in contrast to the presence of the YCPRP motif in VP1, there are no critical motifs near the N-terminus of the precursor, within VP4, required for correct cleavage by 3Cpro.

Improvements in metagenomic virus detection by simple pretreatment methods.

Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified; this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.

Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark.

Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera-i.e., Pedacovirus, Nyctacovirus, and Myotacovirus-that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates-including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus-present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

Picornaviruses: A View from 3A.

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells.

Border disease virus (BDV) envelope glycoprotein E2 is required for entry into cells and is a determinant of host tropism for sheep and pig cells. Here, we describe adaptive changes in the BDV E2 protein that modify virus replication in pig cells. To achieve this, two BDV isolates, initially collected from a pig and a sheep on the same farm, were passaged in primary sheep and pig cells in parallel with a rescued variant of the pig virus derived from a cloned full-length BDV cDNA. The pig isolate and the rescued virus shared the same amino acid sequence, but the sheep isolate differed at ten residues, including two substitutions in E2 (K771E and Y925H). During serial passage in cells, the viruses displayed clear selectivity for growth in sheep cells; only the cDNA-derived virus adapted to grow in pig cells. Sequencing revealed an amino acid substitution (Q739R) in the E2 domain DA of this rescued virus. Adaptation at the same residue (Q739K/Q739R) was also observed after passaging of the pig isolate in sheep cells. Use of reverse genetics confirmed that changing residue Q739 to R or K (each positively charged) was sufficient to achieve adaptation to pig cells. Furthermore, this change in host tropism was suppressed if Q739R was combined with K771E. Another substitution (Q728R), conferring an additional positive charge, acquired during passaging, restored the growth of the Q739R/K771E variant. Overall, this study provided evidence that specific, positively charged, residues in the E2 domain DA are crucial for pig-cell tropism of BDV.

Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants.

Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity.IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

Foot-and-mouth disease virus: Prospects for using knowledge of virus biology to improve control of this continuing global threat.

Understanding of the biology of foot-and-mouth disease virus (FMDV) has grown considerably since the nucleotide sequence of the viral RNA was determined. The ability to manipulate the intact genome and also to express specific parts of the genome individually has enabled detailed analyses of viral components, both RNA and protein. Such studies have identified the requirements for specific functional elements for virus replication and pathogenicity. Furthermore, information about the functions of individual virus proteins has enabled the rational design of cDNA cassettes to express non-infectious empty capsid particles that can induce protective immunity in the natural host animals and thus represent new vaccine candidates. Similarly, attempts to block specific virus activities using antiviral agents have also been performed. However, currently, only the well-established, chemically inactivated FMDV vaccines are commercially available and suitable for use to combat this important disease of livestock animals. These vaccines, despite certain shortcomings, have been used very successfully (e.g. in Europe) to control the disease but it still remains endemic in much of Africa, southern Asia and the Middle East. Hence there remains a significant risk of reintroduction of the disease into highly susceptible animal populations with enormous economic consequences.

Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites.

Many picornaviruses cause important diseases in humans and other animals including poliovirus, rhinoviruses (causing the common cold) and foot-and-mouth disease virus (FMDV). These small, non-enveloped viruses comprise a positive-stranded RNA genome (ca. 7-9 kb) enclosed within a protein shell composed of 60 copies of three or four different capsid proteins. For the aphthoviruses (e.g. FMDV) and cardioviruses, the capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to generate VP0, VP3 and VP1 plus 2A. For enteroviruses, e.g. poliovirus, the capsid precursor is P1 alone, which is cleaved by the 3CD protease to generate just VP0, VP3 and VP1. The sequences required for correct processing of the FMDV capsid protein precursor in mammalian cells were analyzed. Truncation of the P1-2A precursor from its C-terminus showed that loss of the 2A peptide (18 residues long) and 27 residues from the C-terminus of VP1 (211 residues long) resulted in a precursor that cannot be processed by 3Cpro although it still contained two unmodified internal cleavage sites (VP0/VP3 and VP3/VP1 junctions). Furthermore, introduction of small deletions within P1-2A identified residues 185-190 within VP1 as being required for 3Cpro-mediated processing and for optimal accumulation of the precursor. Within this C-terminal region of VP1, five of these residues (YCPRP), are very highly conserved in all FMDVs and are also conserved amongst other picornaviruses. Mutant FMDV P1-2A precursors with single amino acid substitutions within this motif were highly resistant to cleavage at internal junctions. Such substitutions also abrogated virus infectivity. These results can explain earlier observations that loss of the C-terminus (including the conserved motif) from the poliovirus capsid precursor conferred resistance to processing. Thus, this motif seems essential for maintaining the correct structure of picornavirus capsid precursors prior to processing and subsequent capsid assembly; it may represent a site that interacts with cellular chaperones.

Cleavages at the three junctions within the foot-and-mouth disease virus capsid precursor (P1-2A) by the 3C protease are mutually independent.

The foot-and-mouth disease virus capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to VP0, VP3, VP1 and 2A. The P1-2A precursor (wt or mutant) was expressed alone or with 3Cpro and processing of P1-2A was determined. The VP2 K217R and VP3 I2P substitutions (near the VP0/VP3 junction) strongly reduced the processing at this junction by 3Cpro while the substitution VP2 K217E blocked cleavage. At the VP3/VP1 junction, the substitutions VP3 Q2221P and VP1 T1P each severely inhibited processing at this site. Blocking cleavage at either junction did not prevent processing elsewhere in P1-2A. These modifications were also introduced into full-length FMDV RNA; only wt and the VP2 K217R mutant were viable. Uncleaved VP0-VP3 and the processed products were observed within cells infected with the mutant virus. The VP0-VP3 was not incorporated into empty capsids or virus particles. The three junctions within P1-2A are processed by 3Cpro independently.

Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E14, S15, and N16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P17, G18, or P19, which displayed little or no "cleavage" activity in vitro, were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E14, S15, N16, and P19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV.

Selection of functional 2A sequences within foot-and-mouth disease virus; requirements for the NPGP motif with a distinct codon bias.

Foot-and-mouth disease virus (FMDV) has a positive-sense ssRNA genome including a single, large, open reading frame. Splitting of the encoded polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues long), which induces a nonproteolytic, cotranslational "cleavage" at its own C terminus. A conserved feature among variants of 2A is the C-terminal motif N16P17G18/P19, where P19 is the first residue of 2B. It has been shown previously that certain amino acid substitutions can be tolerated at residues E14, S15, and N16 within the 2A sequence of infectious FMDVs, but no variants at residues P17, G18, or P19 have been identified. In this study, using highly degenerate primers, we analyzed if any other residues can be present at each position of the NPG/P motif within infectious FMDV. No alternative forms of this motif were found to be encoded by rescued FMDVs after two, three, or four passages. However, surprisingly, a clear codon preference for the wt nucleotide sequence encoding the NPGP motif within these viruses was observed. Indeed, the codons selected to code for P17 and P19 within this motif were distinct; thus the synonymous codons are not equivalent.

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Sequence adaptations affecting cleavage of the VP1/2A junction by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited cleavage of this junction and produced 'self-tagged' virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus [Gullberg et al. (2013). J Virol 87: , 11591-11603]. It was shown here that introduction of this E83K change alone into a serotype O virus resulted in the rapid accumulation of a second site substitution within the 2A sequence (L2P), which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained the uncleaved VP1-2A protein. The 2A L2P substitution resulted in the VP1/2A junction being highly resistant to cleavage by the 3C protease, hence it may be a preferred route for 'tagging' virus particles.

Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells.

The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3C(pro)) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3C(pro) can be toxic for cells. The expression level of 3C(pro) activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia-virus-based transient expression system, P1-2A (from serotypes O and A) and 3C(pro) were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3C(pro) expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3C(pro) with reduced catalytic activity or using two different mutant IRES elements (the wt GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin αvß6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.

Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells.

The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3C(pro)) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3C(pro) can be expected to be produced at equivalent concentrations. However, using transient-expression assays, within mammalian cells, it is possible to modify the relative amounts of the substrate and protease. It has now been shown that optimal production of the processed capsid proteins from P1-2A is achieved with reduced levels of 3C(pro) expression, relative to the P1-2A, compared with that achieved with a single P1-2A-3C polyprotein. Expression of the FMDV 3C(pro) is poorly tolerated by mammalian cells and higher levels of the 3C(pro) greatly inhibit protein expression. In addition, it is demonstrated that both the intact P1-2A precursor and the processed capsid proteins can be efficiently detected by FMDV antigen detection assays. Furthermore, the P1-2A and the processed forms each bind to the integrin αvß6, the major FMDV receptor. These results contribute to the development of systems which efficiently express the components of empty capsid particles and may represent the basis for safer production of diagnostic reagents and improved vaccines against foot-and-mouth disease.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity.

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: investigation of possible sites of virus replication and persistence.

Foot-and-mouth disease (FMD) is a highly contagious viral infection of significant financial importance to the export and trade of agricultural products. The occurrence of persistently infected "carriers" of FMD-virus (FMDV) in ruminant species adds further complications to disease control. There have been significant discrepancies in reports regarding the pathogenesis of FMDV infection in cattle with specific emphasis on the anatomical sites involved in early and persistent virus replication. In this study, collection of small biopsy samples from the dorsal soft palate (DSP) of live animals was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through measurements of the levels of FMDV RNA in the DSP as well as mandibular and retropharyngeal lymph nodes beyond 28 days after infection. Results indicated only low levels of FMDV RNA present in samples of pharyngeal epithelia during both early and persistent phases of infection with significantly higher levels of virus detected in pharyngeal excretions. It is concluded that the targeted area for sampling within the DSP does not harbour significant levels of virus replication during acute or persistent FMDV infection in cattle. Furthermore, the DSP and the mandibular and retropharyngeal lymph nodes cannot be concluded to be principal sites for persistence of FMDV.