Short time window for transmissibility of African swine fever virus from a contaminated environment.

Since the introduction of African swine fever virus (ASFV) into the Baltic states and Poland in 2014, the disease has continued to spread within these regions. In 2017, the virus spread further west and the first cases of disease were reported in the Czech Republic and Romania, in wild boar and domestic pigs, respectively. To control further spread, knowledge of different modes of transmission, including indirect transmission via a contaminated environment, is crucial. Up until now, such an indirect mode of transmission has not been demonstrated. In this study, transmission via an environment contaminated with excretions from ASFV-infected pigs was investigated. Following euthanasia of pigs that were infected with an isolate of ASFV from Poland (POL/2015/Podlaskie/Lindholm), healthy pigs were introduced into the pens, in which the ASFV-infected pigs had been housed. Introduction was performed at 1, 3, 5 or 7 days, following euthanasia of the infected pig groups. Pigs, that were introduced into the contaminated environment after 1 day, developed clinical disease within 1 week, and both ASFV DNA and infectious virus were isolated from their blood. However, pigs introduced into the contaminated pens after 3, 5 or 7 days did not develop any signs of ASFV infection and no viral DNA was detected in blood samples obtained from these pigs within the following 3 weeks. Thus, it was shown that exposure of pigs to an environment contaminated with ASFV can result in infection. However, the time window for transmissibility of ASFV seems very limited, and, within our experimental system, there appears to be a rapid decrease in the infectivity of ASFV in the environment.

Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain.

Outbreaks of porcine epidemic diarrhoea (PED) were reported across Europe during the 1980s and 1990s, but only sporadic outbreaks occurred in recent years. PED virus (PEDV) spread for the first time into the USA in 2013 and has caused severe economic losses. Retrospectively, it was found that two different strains of PEDV have been introduced into the United States, both are closely related to strains circulating in China where a new wave of the disease occurred from 2010 onwards. Since autumn 2014, new outbreaks of PED have occurred in Europe. In this study, weaned piglets were inoculated with an early European isolate (Br1/87) or faecal/intestinal suspensions derived from pigs infected with a recent European strain of PEDV (from Germany) or a US strain of PEDV. No evidence for infection resulted from inoculation of pigs with the German sample that contained high levels of PEDV RNA; there were no clinical signs, excretion of viral RNA or anti-PEDV antibody production. In contrast, all the pigs in the other two groups showed evidence of infection. Mild clinical signs of disease, mainly diarrhoea, occurred in piglets inoculated with the Br1/87 and US PEDV strains. PEDV RNA was detected throughout the intestine in euthanized animals at 4 days post-inoculation. In addition, within these animals, low levels of viral RNA were detected in lungs and livers with higher levels in spleens. Seroconversion against PEDV occurred in all surviving infected animals within 10 days. PEDV RNA excretion occurred for at least 2 weeks. The US PEDV RNA was detected at low levels in serum samples on multiple days. It is apparent that current diagnostic systems can detect infection by the different virus strains.

Characterization of a Novel Chimeric Swine Enteric Coronavirus from Diseased Pigs in Central Eastern Europe in 2016.

During a severe outbreak of diarrhoea and vomiting in a pig herd in Central Eastern Europe, faecal samples were tested positive for porcine epidemic diarrhoea virus (PEDV) and negative for transmissible gastroenteritis virus (TGEV) using a commercial RT-qPCR assay that can detect both of these coronaviruses. However, further analyses, using other TGEV- and PEDV-specific RT-qPCR assays, provided results inconsistent with infection by either of these viruses. Sequencing of an amplicon (ca. 1.6 kb), generated by an RT-PCR specific for the PEDV S-gene, indicated a very close similarity (ca. 99% identity) to recently described chimeric viruses termed swine enteric coronaviruses (SeCoVs). These viruses (with an RNA genome of ca. 28 kb) were first identified in Italy in samples from 2009 but have not been detected there since 2012. A closely related virus was detected in archived samples in Germany from 2012, but has not been detected subsequently. Building on the initial sequence data, further amplicons were generated and over 9 kb of sequence corresponding to the 3'-terminus of the new SeCoV genome was determined. Sequence comparisons showed that the three known SeCoVs are ≥98% identical across this region and contain the S-gene and 3a sequences from PEDV within a backbone of TGEV, but the viruses are clearly distinct from each other. It is demonstrated, for the first time, that pigs from within the SeCoV-infected herd seroconverted against PEDV but tested negative in a TGEV-specific ELISA that detects antibodies against the S protein. These results indicate that SeCoV is continuing to circulate in Europe and suggest it can cause a disease that is very similar to PED. Specific detection of the chimeric SeCoVs either requires development of a new diagnostic RT-qPCR assay or the combined use of assays targeting the PEDV S-gene and another part of the TGEV genome.

Assessing the potential spread and maintenance of foot-and-mouth disease virus infection in wild ungulates: general principles and application to a specific scenario in Thrace.

Foot-and-mouth disease (FMD), due to infection with serotype O virus, occurred in wild boar and within eleven outbreaks in domestic livestock in the south-east of Bulgaria, Thrace region, in 2011. Hence, the issue of the potential for the spread and maintenance of FMD virus (FMDV) infection in a population of wild ungulates became important. This assessment focused on the spread and maintenance of FMDV infection within a hypothetical wild boar and deer population in an environment, which is characterized by a climate transitional between Mediterranean and continental and variable wildlife population densities. The assessment was based on three aspects: (i) a systematic review of the literature focusing on experimental infection studies to identify the parameters describing the duration of FMDV infection in deer and wild boar, as well as observational studies assessing the occurrence of FMDV infection in wild deer and wild boar populations, (ii) prevalence survey data of wild boar and deer in Bulgaria and Turkey and (iii) an epidemiological model, simulating the host-to-host spread of FMDV infections. It is concluded, based on all three aspects, that the wildlife population in Thrace, and so wildlife populations in similar ecological settings, are probably not able to maintain FMD in the long term in the absence of FMDV infection in the domestic host population. However, limited spread of FMDV infection in time and space in the wildlife populations can occur. If there is a continued cross-over of FMDV between domestic and wildlife populations or a higher population density, virus circulation may be prolonged.

Challenges for Serology-Based Characterization of Foot-and-Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011.

Control of foot-and-mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK® FMDV NS ELISA, solid-phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti-NSP positive cattle sera. However, 35% of the anti-NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa-2 (EA-2) topotype, each differing from the currently used vaccine strain (EA-1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT-SPBEs, perform vaccine matching and implement improved regional FMD control.

Analysis of Recent Serotype O Foot-and-Mouth Disease Viruses from Livestock in Kenya: Evidence of Four Independently Evolving Lineages.

Foot-and-mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these viruses were determined to ascertain whether these were independent outbreaks or one single strain spreading throughout the country. The sequences of the complete VP1-coding region were analysed from viruses sampled within different areas of Kenya during 2010 and 2011. The results indicated that the 2010 to 2011 outbreaks in Kenya were caused by four independent strains. By comparison with earlier type O isolates from Eastern Africa, it was apparent that the outbreaks were caused by viruses from three different lineages of topotype EA-2 and a fourth virus strain belonging to topotype EA-4. The topotypes EA-1 and EA-3 were not detected from these outbreaks. Implications of these results for FMD control in Eastern Africa are discussed.

Rapid spread of Schmallenberg virus-infected biting midges (Culicoides spp.) across Denmark in 2012.

Detection of Schmallenberg virus RNA, using real-time RT-PCR, in biting midges (Culicoides spp.) caught at 48 locations in 2011 and four well-separated farms during 2012 in Denmark, revealed a remarkably rapid spread of virus-infected midges across the country. During 2012, some 213 pools of obsoletus group midges (10 specimens per pool) were examined, and of these, 35 of the 174 parous pools were Schmallenberg virus RNA positive and 11 of them were positive in the heads. Culicoides species-specific PCRs identified both C. obsoletus and C. dewulfi as vectors of Schmallenberg virus.

Foot-and-mouth disease virus serotype O phylodynamics: genetic variability associated with epidemiological factors in Pakistan.

One of the most challenging aspects of foot-and-mouth disease (FMD) control is the high genetic variability of the FMD virus (FMDV). In endemic settings such as the Indian subcontinent, this variability has resulted in the emergence of pandemic strains that have spread widely and caused devastating outbreaks in disease-free areas. In countries trying to control and eradicate FMD using vaccination strategies, the constantly evolving and wide diversity of field FMDV strains is an obstacle for identifying vaccine strains that are successful in conferring protection against infection with field viruses. Consequently, quantitative knowledge on the factors that are associated with variability of the FMDV is prerequisite for preventing and controlling FMD in the Indian subcontinent. A hierarchical linear model was used to assess the association between time, space, host species and the genetic variability of serotype O FMDV using viruses collected in Pakistan from 2005 to 2011. Significant (P<0.05) amino acid and nucleotide variations were associated with spatial distance, but not with differences in host species, which is consistent with the frequent multi-species infection of this serotype O FMDV. Results from this study will contribute to the understanding of FMDV variability and to the design of FMD control strategies in Pakistan. Viruses sequenced here also provide the earliest reported isolate from the Pan Asia II(ANT-10) sublineage, which has caused several outbreaks in the Middle East and spread into Europe (Bulgaria) and Africa (Libya).

Detection and genetic characterization of foot-and-mouth disease viruses in samples from clinically healthy animals in endemic settings.

A total of 1501 oral swab samples from Pakistan, Afghanistan and Tajikistan were collected from clinically healthy animals between July 2008 and August 2009 and assayed for the presence of foot-and-mouth disease virus (FMDV) RNA. The oral swab samples from two (of four) live animal markets in Pakistan (n = 245), one (of three) live animal market in Afghanistan (n=61) and both the live animal markets in Tajikistan (n=120) all tested negative. However, 2 of 129 (∼2%) samples from Gondal and 11 of 123 (9%) from Chichawatni markets in Pakistan were positive for FMDV RNA. Similarly, 12 of 81 (15%) samples from Kabul and 10 of 20 (50%) from Badakhshan in Afghanistan were found to be positive. Serotypes A and O of FMDV were identified within these samples. Oral swab samples were also collected from dairy colonies in Harbanspura, Lahore (n=232) and Nagori, Karachi (n=136), but all tested negative for FMDV. In the Landhi dairy colony, Pakistan, a cohort of 179 apparently healthy animals was studied. On their arrival within the colony, thirty-nine (22%) of these animals were found positive for FMDV RNA (serotype A was identified), while 130 (72.6%) had antibodies to FMDV non-structural proteins. Thus, newly introduced animals may be a significant source of the disease in the colony. Only two animals from the cohort were detected as becoming positive for FMDV RNA during a follow-up period of 4months; however, only 10 animals remained negative for anti-NSP antibodies during this period.

Serotype identification and VP1 coding sequence analysis of foot-and-mouth disease viruses from outbreaks in eastern and northern Uganda in 2008/9.

In April 2008, foot-and-mouth disease (FMD) outbreaks were reported in Kamuli district of the eastern region of Uganda. Soon after lifting the quarantines in this area, further FMD outbreaks were reported in northern Uganda, which spread to more than 10 districts. The aim of this study was to identify the serotype and compare the variable protein (VP)1 coding sequences of the viruses responsible for FMD outbreaks during 2008 and 2009, to trace the transmission pathways of the disease in Uganda. Probang and epithelial swab samples were collected from cattle with clinical signs of FMD in the two regions, and the presence of FMDV RNA in these samples was determined using a standard diagnostic RT-PCR assay. From the total of 27 positive samples, the VP1 coding region was amplified and sequenced. Each of these sequences showed >99% identity to each other, and just five distinct sequences were identified. BLAST searches and phylogenetic analysis of the complete variable protein (VP)1 coding sequences revealed that they belonged to serotype O, topotype EA-2. The close similarity between the virus sequences suggested introduction from a single source. We therefore conclude that FMD in the northern region of Uganda was most likely introduced from the outbreak in the eastern region across Lake Kyoga through movement of live animals. This has significant implications for the effectiveness of the current FMD control measures.

Modulation of cytokine mRNA expression in pharyngeal epithelial samples obtained from cattle infected with foot-and-mouth disease virus.

A novel technique of endoscopical collection of small tissue samples was used to obtain sequential tissue samples from the dorsal soft palate (DSP) of individual cattle infected with foot-and-mouth disease virus (FMDV) at different phases of the infection. Levels of mRNA encoding interferon (IFN)-α and IFN-ß as well as tumour necrosis factor (TNF)-α were measured in these samples by quantitative reverse transcriptase polymerase chain reaction. Expression of IFN-ß mRNA was significantly down-regulated in the biopsy samples harvested during the acute phase of infection, while there was no statistically significant effect on the expression of IFN-α mRNA compared with baseline levels. In contrast, the mRNA encoding TNF-α was significantly up-regulated in samples collected during both acute and late (>28 days post infection) phases of infection. There were also significantly higher levels of TNF-α mRNA expressed in samples derived from animals that were identified subsequently as persistently infected FMDV-carriers. It was concluded that there was a significant difference in the host-response in the DSP of calves that were identified as persistently infected, subclinical carriers of FMDV.

Low diversity of foot-and-mouth disease serotype C virus in Kenya: evidence for probable vaccine strain re-introductions in the field.

Most viruses are maintained by complex processes of evolution that enable them to survive but also complicate efforts to achieve their control. In this paper, we study patterns of evolution in foot-and-mouth disease (FMD) serotype C virus isolates from Kenya, one of the few places in the world where serotype C has been endemic and is suspected to remain. The nucleotide sequences encoding the capsid protein VP1 from eight isolates collected between 1967 and 2004 were analysed for patterns of sequence divergence and evolution. Very low nucleotide diversity (π = 0·0025) and remarkably little change (only five segregating sites and three amino-acid changes) were observed in these isolates collected over a period of almost 40 years. We interpret these results as being suggestive of re-introductions of the vaccine strain into the field. The implications of these results for the maintenance of serotype C FMD virus and the use of vaccination as a control measure in Kenya are discussed.

Co-circulation of two extremely divergent serotype SAT 2 lineages in Kenya highlights challenges to foot-and-mouth disease control.

Amongst the SAT serotypes of foot-and-mouth disease virus (FMDV), the SAT 2 serotype is the most widely distributed throughout sub-Saharan Africa. Kenyan serotype SAT 2 viruses have been reported to display the highest genetic diversity for the serotype globally. This complicates diagnosis and control, and it is essential that patterns of virus circulation are known in order to overcome these difficulties. This study was undertaken to establish patterns of evolution of FMDV serotype SAT 2 in Kenya using complete VP1 coding sequences in a dataset of 65 sequences from Africa, collected over a period of 50 years. Two highly divergent lineages were observed to co-circulate, and occasional trans-boundary spread was inferred, emphasizing the value of constant monitoring and characterization of field strains for improved diagnosis and appropriate vaccine application as well as the need for regional approaches to control.

Bluetongue in Denmark during 2008.

Following the first ever case of bluetongue in Denmark during late 2007, further outbreaks were observed in Denmark during 2008, despite vaccination against bluetongue virus (BTV) serotype 8 (BTV-8) in the southern part of the country. In total, 15 separate outbreaks of infection were identified, mostly as a result of clinical suspicions but also because of surveillance of bulk milk samples. These outbreaks led to extensions of the original vaccination zone planned for 2008. Blood samples from clinical suspects were analysed using ELISA and real-time RT-PCR assays for the presence of anti-BTV antibodies and viral RNA, respectively. A newly infected calf from the primary outbreak in 2008 was studied for a period of three months, during which time it seroconverted to BTV, but the presence of viral RNA in its blood was maintained throughout this time. Each outbreak was caused by BTV-8, as determined by a serotype-specific real-time RT-PCR assay. Furthermore, the nucleotide sequence of a portion of segment 2 of the viral RNA (encoding the outer capsid protein VP2) from the samples analysed was identical to the BTV-8 segment 2 that circulated in the Netherlands during 2006.

Molecular characterization of SAT 2 foot-and-mouth disease virus from post-outbreak slaughtered animals: implications for disease control in Uganda.

In Uganda, limiting the extent of foot-and-mouth disease (FMD) spread during outbreaks involves short-term measures such as ring vaccination and restrictions of the movement of livestock and their products to and from the affected areas. In this study, the presence of FMD virus RNA was investigated in cattle samples 3 months after FMD quarantine measures had been lifted following an outbreak in 2004. Oropharyngeal tissue samples were obtained from 12 cattle slaughtered in a small town abattoir in Kiboga. FMD virus RNA was detected by diagnostic RT-PCR in nine of the 12 tissue samples. Part of the coding region for the capsid protein VP1 was amplified and sequenced. All samples were identified as belonging to the SAT 2 serotype. The implications for FMD control of both virus introduction into Uganda and the presence of carrier animals following outbreaks are discussed.

Translation and replication of FMDV RNA.

Foot-and-mouth disease virus (FMDV) RNA is infectious. After delivery of the RNA (about 8.3 kb) into the cytoplasm of a cell, the RNA must initially be translated to produce the viral proteins required for RNA replication and for the packaging of the RNA into new virions. Subsequently there has to be a switch in the function of the RNA; translation has to be stopped to permit RNA replication. The signals required for the control of the different roles of viral RNA must be included within the viral RNA sequence. Many cellular proteins interact with the viral RNA and probably also with the virus-encoded proteins. The functions of different RNA elements within the viral RNA and the various virus-encoded proteins in determining the efficiency of virus replication are discussed. Unique aspects of FMDV RNA translation and replication are emphasised.

Cleavage of translation initiation factor 4AI (eIF4AI) but not eIF4AII by foot-and-mouth disease virus 3C protease: identification of the eIF4AI cleavage site.

The translation initiation factor eIF4A is cleaved within mammalian cells infected by foot-and-mouth disease virus (FMDV). The FMDV 3C protease cleaves eIF4AI (between residues E143 and V144), but not the closely related eIF4AII. Modification of eIF4AI, to produce a sequence identical to eIF4AII around the cleavage site, blocked proteolysis. Alignment of mammalian eIF4AI onto the three-dimensional structure of yeast eIF4A located the scissile bond within an exposed, flexible portion of the molecule. The N- and C-terminal cleavage products of eIF4AI generated by FMDV 3C dissociate. Cleavage of eIF4AI by FMDV 3C is thus expected to inactivate it.

An attenuating mutation in the 2A protease of swine vesicular disease virus, a picornavirus, regulates cap- and internal ribosome entry site-dependent protein synthesis.

Virulent and avirulent strains of swine vesicular disease virus (SVDV), a picornavirus, have been characterized previously. The major determinants for attenuation have been mapped to specific residues in the 1D-2A-coding region. The properties of the 2A proteases from the virulent and avirulent strains of SVDV have now been examined. Both proteases efficiently cleaved the 1D/2A junction in vitro and in vivo. However, the 2A protease of the avirulent strain of SVDV was much less effective than the virulent-virus 2A protease at inducing cleavage of translation initiation factor eIF4GI within transfected cells. Hence the virulent-virus 2A protease is much more effective at inhibiting cap-dependent protein synthesis. Furthermore, the virulent-virus 2A protease strongly stimulated the internal ribosome entry sites (IRESs) from coxsackievirus B4 and from SVDV, while the avirulent-virus 2A protease was significantly less active in these assays. Thus, the different properties of the 2A proteases from the virulent and avirulent strains of SVDV in regulating protein synthesis initiation reflect the distinct pathogenic properties of the viruses from which they are derived. A single amino acid substitution, adjacent to His21 of the catalytic triad, is sufficient to confer the characteristics of the virulent-strain 2A protease on the avirulent-strain protease. It is concluded that the efficiency of picornavirus protein synthesis, controlled directly by the IRES or indirectly by the 2A protease, can determine virus virulence.

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems.

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.

Eukaryotic initiation factors 4A (eIF4A) and 4G (eIF4G) mutually interact in a 1:1 ratio in vivo.

mRNA translation in eukaryotic cells involves a set of proteins termed translation initiation factors (eIFs), several of which are involved in the binding of ribosomes to mRNA. These include eIF4G, a modular scaffolding protein, and eIF4A, an RNA helicase, of which two closely related forms are known in mammals, eIF4A(I) and eIF4A(II). In mammals, eIF4G possesses two independent sites for binding eIF4A, whereas in other eukaryotes (e.g. yeast) only one site appears to be present, thus raising the issue of the stoichiometry of eIF4G.eIF4A complexes in different eukaryotes. We show that in human embryonic kidney cells eIF4G is associated with eIF4A(I) or eIF4A(II) but not with both simultaneously, suggesting a stoichiometry of 1:1 rather than 1:2. To confirm this, eIF4A(I) or eIF4A(II) was expressed in a tagged form in these cells, and complexes with eIF4G were again isolated. Complexes containing tagged eIF4A(I) or eIF4A(II) contained no endogenous eIF4A, supporting the notion that eIF4G binds only one molecule of eIF4A. Each binding site in eIF4G can bind either eIF4A(I) or eIF4A(II). The data imply that the second binding site in mammalian eIF4A does not bind an additional eIF4A molecule and that initiation factor complexes in different eukaryotes contain one eIF4A per eIF4G.