Antiviral activity of K22 against members of the order Nidovirales.

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Optimization and application of a DNA-launched infectious clone of equine arteritis virus.

Reverse genetics is one of the most powerful tools in modern virology. Equine arteritis virus (EAV) is the prototype member of the Equartevirus. In this study, a new reverse genetics system for the recovery of equine arteritis virus from a cDNA plasmid, which contains viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences in both terminals of the viral genome, was developed by optimization of the promoter and terminator regions. Cellular RNA polymerase II drove the transcription of the viral genome. The results showed that the rescued virus (ic-EAV) shared similar morphological and growth characteristics with the wild-type (WT) virus, and could be distinguished from the WT virus via an engineered BspEI restriction site in the nsp3 gene. By using the reverse genetics method established in this study, a G-to-C silent mutation at site 12642 resulted in a significant change in the plaque size of the rescued virus. Moreover, an eGFP-labeled EAV was constructed by introducing the eGFP gene into the infectious clone of EAV, which facilitated the observation of the infection of EAV in target cells. Hence, the newly reverse genetics method of EAV established in this study can be easily manipulated and would be helpful for studying the pathogenic mechanism of EAV.

Intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in RK13 cells infected with equine arteritis virus.

The modulation of the expression of caspases by viruses influences the cell survival of different cell types. Equine arteritis virus (EAV) induces apoptosis of BHK21 and Vero cell lines, but it is not known whether EAV induces apoptosis in RK13 cells, a common cell line routinely used in EAV diagnosis and research. In this study, we determined that caspase-3 expression was triggered after infection of RK13 cells with EAV in a time- and dose-dependent manner. We also detected caspase-8 and caspase-9 activation, indicating the stimulation of both extrinsic and intrinsic apoptosis pathways. Finally, we found caspase-12 activation, an indicator of endoplasmic reticulum stress-induced apoptosis. The variability observed in the apoptotic response in the different cell lines demonstrates that apoptosis depends on the distinctive sensitivity of each cell line used for investigation.

Development and characterization of an infectious cDNA clone of the modified live virus vaccine strain of equine arteritis virus.

A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals.

Curing of HeLa cells persistently infected with equine arteritis virus by a peptide-conjugated morpholino oligomer.

A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region of the EAV genome. We found that persistently infected HeLa cells passaged three times in the presence of 5-10 microM EAV-specific PPMO produced no detectable virus. The PPMO-cured HeLa cells were free of infectious virus, viral antigen and EAV RNA as measured by plaque assay, indirect immunofluorescence assay and RT-PCR, respectively. Furthermore, when re-challenged with EAV at several passages after discontinuation of PPMO treatments, PPMO-cured HeLa cells were found to be refractory to re-infection and to the re-establishment of viral persistence. While these findings demonstrate that PPMO can be used to eliminate persistent EAV infection in cell culture, the efficacy of PPMO against EAV in vivo remains to be addressed.

Persistent equine arteritis virus infection in HeLa cells.

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53-->Cys and Val55-->Ala), GP2 (Leu15-->Ser, Trp31-->Arg, Val87-->Leu, and Ala112-->Thr), GP3 (Ser115-->Gly and Leu135-->Pro), and GP4 (Tyr4-->His and Ile109-->Phe) proteins or with a single point mutation in the GP5 protein (Pro98-->Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.

An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene.

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP-nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.

Mutagenesis analysis of the nsp4 main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing.

Nonstructural protein 4 (nsp4; 204 amino acids) is the chymotrypsin-like serine main proteinase of the arterivirus Equine arteritis virus (order Nidovirales), which controls the maturation of the replicase complex. nsp4 includes a unique C-terminal domain (CTD) connected to the catalytic two-beta-barrel structure by the poorly conserved residues 155 and 156. This dipeptide might be part of a hinge region (HR) that facilitates interdomain movements and thereby regulates (in time and space) autoprocessing of replicase polyproteins pp1a and pp1ab at eight sites that are conserved in arteriviruses. To test this hypothesis, we characterized nsp4 proteinase mutants carrying either point mutations in the putative HR domain or a large deletion in the CTD. When tested in a reverse genetics system, three groups of mutants were recognized (wild-type-like, debilitated, and dead), which was in line with the expected impact of mutations on HR flexibility. When tested in a transient expression system, the effects of the mutations on the production and turnover of replicase proteins varied widely. They were cleavage product specific and revealed a pronounced modulating effect of moieties derived from the nsp1-3 region of pp1a. Mutations that were lethal affected the efficiency of polyprotein autoprocessing most strongly. These mutants may be impaired in the accumulation of nsp5-7 and/or suffer from delayed or otherwise perturbed processing at the nsp5/6 and nsp6/7 junctions. On average, the production of nsp7-8 seems to be the most resistant to debilitating nsp4 mutations. Our results further prove that the CTD is essential for a vital nsp4 property other than catalysis.

Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle.

The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.

Study on the epidemiology of equine arteritis virus infection with different diagnostic techniques by investigating 96 cases of equine abortion in Hungary.

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.

Evidence that use of an inactivated equine herpesvirus vaccine induces serum cytotoxicity affecting the equine arteritis virus neutralisation test.

Several laboratories worldwide have recently experienced problems related to serum cytotoxicity with the equine arteritis virus (EAV) neutralisation test (VN) when using Office International des Epizooties (OIE) reference laboratory prescribed rabbit kidney (RK-13) indicator cells. Cytotoxicity can be mistaken for viral cytopathic effect and has led to increasing difficulties in test interpretation, consequently causing disruption to both equine breeding and disease surveillance. Results from experimental and field-derived data suggest that this serum cytotoxicity is associated with use of a tissue-culture-derived equine herpesvirus vaccine, probably manifested through a vaccine-induced anti-cellular antibody response directed against RK-13 cells. Two alternative EAV VN methods were shown to significantly reduce the effects of cytotoxicity (from 73 to <5% prevalence) among vaccinated horses but did not completely eliminate the problem. Use of ELISA-based tests, which are not affected by serum cytotoxicity but which are not currently recognised as international standards, should be evaluated as a useful backup in screening equine sera for EAV VN antibodies.

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells.

OBJECTIVE: To compare growth characteristics of strains of equine arteritis virus (EAV) of differing virulence to horses in rabbit kidney (RK)-13 cells and equine endothelial cells (EECs) cultured from the pulmonary artery of a foal. SAMPLE POPULATION: 13 strains of EAV, including 11 field isolates of differing virulence to horses; the highly virulent, horse-adapted Bucyrus strain; and the modified-live virus (MLV) vaccine derived from it. PROCEDURE: The growth characteristics of the 13 strains were compared in EECs and RK-13 cells. Viral nucleoprotein expression, cytopathogenicity, and plaque size were compared to determine whether growth characteristics of the 13 strains were predictive of their virulence to horses. RESULTS: Cytopathogenicity, viral nucleoprotein expression, and plaque size induced by all 13 viruses were similar in RK-13 cells, whereas virulent strains of EAV caused significantly larger plaques in EECs than did the avirulent strains of EAV. Paradoxically, the highly attenuated MLV vaccine and 1 field isolate of EAV caused plaques in EECs that were larger than those caused by any of the other viruses, and sequence analysis confirmed the field isolate of EAV to be indistinguishable from the MLV vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: With the notable exception of the MLV vaccine, growth of the various strains of EAV in EECs was predictive of their individual virulence to horses. Thus, EECs provide a relevant and useful model to further characterize determinants of virulence and attenuation amongst strains of EAV.

Growth characteristics of a highly virulent, a moderately virulent, and an avirulent strain of equine arteritis virus in primary equine endothelial cells are predictive of their virulence to horses.

Equine viral arteritis (EVA) is an endotheliotropic viral disease of horses caused by equine arteritis virus (EAV). Although there is only one serotype of EAV, there is marked variation in the virulence of different strains of the virus. The replication and cytopathogenicity of three well-characterized strains of EAV of different virulence to horses were compared in rabbit kidney (RK-13) and primary equine pulmonary artery endothelial cells (ECs). Viral protein expression, plaque size, and cytopathogenicity of all three viruses were similar in RK-13 cells, whereas two virulent strains of EAV were readily distinguished from an avirulent strain by their plaque morphology and cytopathogenicity in primary equine ECs. Furthermore, EAV nucleocapsid protein was detected by flow cytometric analysis significantly later in ECs infected with the avirulent than those infected with the virulent strains of EAV. Primary equine ECs provide a convenient and relevant model for in vitro characterization of the pathogenesis of EVA and the virulence determinants of EAV.

Induction of apoptosis by equine arteritis virus infection.

Equine arteritis virus (EAV) is the etiological agent of equine viral arteritis, a contagious viral disease of equids. EAV is the prototype virus of the arteriviruses, a group of small enveloped viruses with positive single-stranded RNA genomes. Because apoptosis or programmed cell death is believed to play an important role in the biogenesis of several cytopathogenic viruses, we examined whether EAV was able to induce cell apoptosis in vitro. To do this, Vero cells were infected with EAV at a multiplicity of infection of 0.1 tissue culture infectious dose (TCID50) per cell, and analyzed at various time intervals for the appearance of apoptotic signs. Fragmentation of chromosomal DNA into nucleosomal oligomers and caspase activation were observed in the infected cells at the time (e.g. 24h postinfection) where a noticeable cytopathic effect was observed. The kinetics of the DNA fragmentation correlated with that of the production of progeny virus, so that viral multiplication was not interrupted by the apoptotic cell damage. All these data provide evidence that EAV is able to induce apoptotic cell death in vitro.

Effects of chlorine, iodine, and quaternary ammonium compound disinfectants on several exotic disease viruses.

The effects of three representative disinfectants, chlorine (sodium hypochlorite), iodine (potassium tetraglicine triiodide), and quaternary ammonium compound (didecyldimethylammonium chloride), on several exotic disease viruses were examined. The viruses used were four enveloped viruses (vesicular stomatitis virus, African swine fever virus, equine viral arteritis virus, and porcine reproductive and respiratory syndrome virus) and two non-enveloped viruses (swine vesicular disease virus (SVDV) and African horse sickness virus (AHSV)). Chlorine was effective against all viruses except SVDV at concentrations of 0.03% to 0.0075%, and a dose response was observed. Iodine was very effective against all viruses at concentrations of 0.015% to 0.0075%, but a dose response was not observed. Quaternary ammonium compound was very effective in low concentration of 0.003% against four enveloped viruses and AHSV, but it was only effective against SVDV with 0.05% NaOH. Electron microscopic observation revealed the probable mechanism of each disinfectant. Chlorine caused complete degeneration of the viral particles and also destroyed the nucleic acid of the viruses. Iodine destroyed mainly the inner components including nucleic acid of the viruses. Quaternary ammonium compound induced detachment of the envelope of the enveloped viruses and formation of micelle in non-enveloped viruses. According to these results, chlorine and iodine disinfectants were quite effective against most of the viruses used at adequately high concentration. The effective concentration of quaternary ammonium compound was the lowest among the disinfectants examined.

Effect of heparin on infection of cells by equine arteritis virus.

The number of plaques formed by equine arteritis virus (EAV) and Aujesky's disease virus (ADV) was reduced to 14% and 5% of the untreated control (100%), respectively, by 10 U/ml of heparin, but could not be reduced below to 13 and 4%, respectively, by use of concentration up to 100 U/ml. An inhibitory effect of heparin, at concentration up to 100 U/ml, was not observed on parainfluenza virus 3 (PIV-3). Heparinase treatment of RK13 cells reduced the number of EAV-, as well as ADV-induced plaques. On the other hand, the number of PIV-3 induced plaques did not decrease after treatment of RK13 cells with heparinase.

Ultrastructure and immuno-cytochemistry of BHK-21 cells infected with a modified Bucyrus strain of equine arteritis virus.

Morphogenesis of a modified Bucyrus strain of equine arteritis virus (EAV) in BHK-21 cells was studied. Bacillary tubules were first detected in the cytoplasm 8 h after infection, and mature virions 79 to 122 nm in diameter, 101 nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (RER) at 12 h or later. They had isometrical cores and morphological subunits in the outer layer. Budding occurred from the RER and the outer nuclear membrane, but not from the cell surface. Structural linkage was detected between the tubule and the virus core. Aberrant strands were occasionally demonstrated within the nucleus 12 h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus.

Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.