Equine Rhinitis A Virus Mutants with Altered Acid Resistance Unveil a Key Role of VP3 and Intrasubunit Interactions in the Control of the pH Stability of the Aphthovirus Capsid.

Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and is genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intraprotomer interface between VP3 and VP2 or between VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV. IMPORTANCE: The viral capsid constitutes a sort of dynamic nanomachine that protects the viral genome against environmental assaults while accomplishing important functions such as receptor attachment for viral entry or genome release. We have explored the molecular determinants of aphthovirus capsid stability by isolating and characterizing a panel of equine rhinitis A virus mutants that differed on their acid sensitivity. All the mutations were located within a specific region of the capsid, the intraprotomer interface among capsid proteins, thus providing new insights into the regions that control the acid stability of aphthovirus capsid. These findings could positively contribute to the development of antiviral approaches targeting aphthovirus uncoating or the refinement of vaccine strategies based on capsid stabilization.

In vitro surrogate models to aid in the development of antivirals for the containment of foot-and-mouth disease outbreaks.

Foot-and-mouth disease virus (FMDV) is a highly pathogenic member of the genus Aphthovirus (family Picornaviridae) that is only to be manipulated in high-containment facilities, thus complicating research on and discovery of antiviral strategies against the virus. Bovine rhinitis B virus (BRBV) and equine rhinitis A virus (ERAV), phylogenetically most closely related to FMDV, were explored as surrogates for FMDV in antiviral studies. Although no efficient cell culture system has been reported so far for BRBV, we demonstrate that infection of primary bovine kidney cells resulted in an extensive but rather poorly-reproducible induction of cytopathic effect (CPE). Madin-Darby bovine kidney cells on the other hand supported viral replication in the absence of CPE. Antiviral tests were developed for ERAV in Vero A cells employing a viral RNA-reduction assay and CPE-reduction assay; the latter having a Z' factor of 0.83±0.07. The BRBV and ERAV models were next used to assess the anti-aphthovirus activity of two broad-spectrum antiviral agents 2'-C-methylcytidine (2CMC) and ribavirin, as well as of the enterovirus-specific inhibitor enviroxime. The effects of the three compounds in the CPE-reduction (ERAV) and viral RNA-reduction assays (BRBV and ERAV) were comparable. Akin to 2CMC, compound A, a recently-discovered non-nucleoside pan-serotype FMDV inhibitor, also inhibited the replication of both BRBV and ERAV, whereas enviroxime was devoid of activity. The BRBV and ERAV surrogate models reported here can be manipulated in BSL-2 laboratories and may facilitate studies to unravel the mechanism of action of novel FMDV inhibitors.

Antiviral effect of recombinant equine interferon-gamma on several equine viruses.

Recombinant equine interferon-gamma (reIFN-gamma) was prepared using a baculovirus expression system and its antiviral activity was investigated using several equine viruses. The reIFN-gamma suppressed the replication of all equine viruses used in the present experiment in horse cell cultures, but did not affect the growth of host cells at concentrations of less than 1000 u/ml. A strong antiviral effect was observed, especially against RNA viruses. Equine picornavirus, equine rhinovirus and equine arteritis virus could not be propagated at all in 100 u/ml reIFN-gamma when 100 TCID(50) of infective viruses was inoculated to cultivated horse cells. DNA viruses, equine herpesvirus types 1, 2, 3 and 4 and equine adenovirus, were less sensitive to reIFN-gamma but their growth became less than 1/100 in the cells treated with 100 u/ml reIFN-gamma compared to untreated cells. The antiviral effects were decreased in the cells of heterologous species and more than 1000 u/ml reIFN-gamma was required to induce an antiviral effect.

Sequence variation divides Equine rhinitis B virus into three distinct phylogenetic groups that correlate with serotype and acid stability.

Equine rhinitis B virus (ERBV), genus Erbovirus, family Picornaviridae, occurs as two serotypes, ERBV1 and ERBV2, and the few isolates previously tested were acid labile. Of 24 ERBV1 isolates tested in the studies reported here, 19 were acid labile and five were acid stable. The two available ERBV2 isolates, as expected, were acid labile. Nucleotide sequences of the P1 region encoding the capsid proteins VP1, VP2, VP3 and VP4 were determined for five acid-labile and three acid-stable ERBV1 isolates and one acid-labile ERBV2 isolate. The sequences were aligned with the published sequences of the prototype acid-labile ERBV1.1436/71 and the prototype ERBV2.313/75. The three acid-stable ERBV1 were closely related in a phylogenetic group that was distinct from the group of six acid-labile ERBV1, which were also closely related to each other. The two acid-labile ERBV2 formed a third distinct group. One acid-labile ERBV1 had a chimeric acid-labile/acid-stable ERBV1 P1 sequence, presumably because of a recombination event within VP2 and this was supported by SimPlot analysis. ERBV1 rabbit antiserum neutralized acid-stable and acid-labile ERBV1 isolates similarly. Accordingly, three distinct phylogenetic groups of erboviruses exist that are consistent with serotype and acid stability phenotypes.

Viral capsid mobility: a dynamic conduit for inactivation.

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

Inactivation of viruses by aziridines.

Ethyleneimine (EI) and N-acetylethyleneimine (AEI) have been shown to inactivate viruses belonging to most of the families described by the International Committee for the Taxonomy of Viruses. The mechanism by which they inactivate the viruses has not been established. In this paper, experiments with foot-and-mouth disease virus (FMDV) and poliovirus are described which indicate that the inactivating lesion is on the RNA.

Structural differences between foot-and-mouth disease and poliomyelitis viruses influence their inactivation by aziridines.

Inactivation of foot-and-mouth disease virus (FMDV) and poliovirus by ethyleneimine (EI) and N-acetylethyleneimine (AEI) has been studied at 25 degrees and at 37 degrees C and in different ionic conditions. FMDV is inactivated rapidly in 100 mM Tris pH 7.6 by each reagent at both temperatures. Poliovirus is also inactivated rapidly in 100 mM Tris by EI at both temperatures and by AEI at 37 degrees C. However, it is inactivated much more slowly by AEI at 25 degrees C; but if the virus is first incubated overnight at 2 degrees C with AEI before transferring to 25 degrees C inactivation then proceeds rapidly. Moreover, the rate of inactivation at 25 degrees C is markedly increased if the virus is suspended in 1 mM Tris. We had interpreted these differences as being due to the greater penetrability of poliovirus (i) in 100 mM Tris at 37 degrees C compared with 25 degrees C and (ii) at lower ionic strength. This interpretation has been confirmed by electron microscopy of FMDV and poliovirus particles stained with phosphotungstic acid. At the elevated temperature, poliovirus had an average diameter of 34+/-0. 21 nm and the stain outlined the nucleic acid core and the individual subunits, whereas at 25 degrees C it averaged 28+/-0.13 nm and the stain did not penetrate the particle. This study also showed that the particle diameter alters with changes in buffer concentration, being 28+/-0.13 nm in 100 mM Tris, 31+/-0.16 nm in 10 mM Tris and 34+/-0.21 nm in 1 mM Tris. The changes in poliovirus are reversible as addition of 1/10 volume of 1 M Tris to the virus in 1 mM Tris resulted in the return of the diameter to 28+/-0.13 nm. FMDV, on the other hand, was less sensitive to osmotic differences as its particle diameter only varied by 7% over the 100-fold change in buffer concentration compared with the 22% change observed for poliovirus.

Monoclonal antibodies to an Indian strain of type A foot-and-mouth disease virus.

A set of five neutralizing monoclonal antibodies (MAbs) to an Indian strain (IND17/77) of type A (subtype A22) foot-and-mouth disease (FMD) virus (FMDV) was used in the study. Four of the MAbs (27S, 37S, 85S, and 143S) identified a trypsin-sensitive (TS) epitope(s) and were specific for VP1, while the remaining MAb (145S) reacted with a trypsin-resistant (TR) epitope and was specific for VP3 in Western blot analysis. Both the epitopes (TS and TR) were conformation-independent in nature. Results obtained in MAb-competition enzyme-linked immunosorbent assay (ELISA), and profiling of the (MAb) neutralization-escape mutants in ELISA and cross-neutralization test revealed two overlapping TS epitopes (27S/37S and 85S/143S) on the virus. Variation at both these epitopes was observed in some field isolates of serotype A. Comparison of deduced amino acid sequence in the VP1 region (aa 140-213) between the parent virus and the mutants identified Gly148 and Arg153 as critical for the formation of both the TS epitopes. Substitution of R153 by Gly or Ser was observed in mutants with no reactivity for the MAbs 85S/143S. However, these mutants maintained partial reactivity with MAbs 27S/37S, and substitution of Gly148 by Glu eliminated both the epitopes. No amino acid substitution was observed in the VP1 region of aa 200-213. Efficient neutralization of the MAb neutralization escape mutants (MAb-resistant (MAR) mutants) by bovine vaccinate serum (BVS) indicated involvement of other epitopes on the virion surface in eliciting neutralizing antibodies following vaccination.

A universal virus inactivant for decontaminating blood and biopharmaceutical products.

Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. For biopharmaceutical products, removal can usually be achieved by a series of fractionation steps or by inactivation with a suitable reagent. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Viruses belonging to all the recognised families can be inactivated by imines. In this paper it is shown that the biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Also shown is that both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The RNA extracted from FMDV inactivated at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region. Surprisingly, it could be translated in the rabbit reticulocyte system although some of the products were different from those obtained with unmodified RNA.

Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L leaves.

Meliacine (MA), a peptide isolated from leaves of the high plant Melia azedarach L inhibited the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells. In this report, we establish that the MA-inhibitable process takes place within the first hour of the viral reproductive cycle. MA had no virucidal effect and did not affect adsorption and penetration of the virus in cells. In experiments with neutral red-labeled virus, it was found that MA significantly suppressed the development of photoresistance of the virus in infected cells. In untreated cultures nearly all virus which adsorbed to cells was uncoated within 1 h at 37 degrees C, whereas in treated cultures, even after 3 h only 3% of the virus was uncoated. Labeling of BHK-21 cells with acridine orange showed that MA affects the pH of intracellular acidic vesicles. Therefore, it is concluded that MA prevents the process of uncoating of FMDV in BHK-21 cells by inhibiting vacuolar acidification.

Evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate.

A variant of foot-and-mouth disease virus (FMDV) lacking the leader (L) coding region (A12-LLV2) was previously constructed and shown to be less virulent in cattle than its wild-type parent (A12-IC). In this study, cattle were tested for their clinical and immunological responses to subcutaneous inoculation with A12-LLV2 or A12-IC or to intramuscular vaccination with chemically inactivated A12-IC. Five weeks postinoculation animals were challenged by intradermal inoculation in the tongue with a virulent cattle-passaged virus. A12-LLV2-inoculated animals showed no clinical signs of disease and developed a neutralizing antibody response by 4 days postinoculation, whereas a companion control bovine did not seroconvert. After challenge, two of three inoculated animals did not develop lesions, but showed mild signs of infection. The third inoculated animal developed some lesions, but these were less severe than in the uninoculated control animal, which showed classical FMD. All animals inoculated with A12-IC developed a fever, two showed typical FMD lesions, and the companion control seroconverted, indicating that it had acquired infection by contact. The A12-IC-inoculated animals and the control were protected from challenge. Animals vaccinated with inactivated virus showed no clinical signs of disease and developed a neutralizing antibody response, and the control did not seroconvert. Upon challenge none of the vaccinated animals developed lesions, one developed a fever, and the control developed FMD. These experiments demonstrate the potential of a rationally designed live-attenuated FMDV vaccine.

Antiviral activity of crude extracts of Guarea guidona.

Crude extracts of leaves and fruits of Guarea guidona were tested for antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 micrograms/ml and 500 micrograms/ml. respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 micrograms/ml. Virucidal activity was not observed at 62.5 micrograms/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 micrograms/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.

Antiviral activity of crude extracts of Guarea guidona

Crude extracts of leaves and fruits of Guarea guidona were tested antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine Cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 mug/ml and 500 mug/ml, respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 mug/ml. Virucidal activity was not observed at 62.5 mug/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 mug/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.

Amitraz effects on foot-and-mouth disease virus in mammalian cells in vitro.

The toxicity of the acaricide amitraz and its effect on foot-and-mouth disease virus multiplication were evaluated in IB-RS-2 cells in vitro. A reduction of cell growth rate that was dependent on the dose and the length of treatment was observed in cells exposed to amitraz concentrations ranging from 20 to 50 micrograms/ml. Foot-and-mouth disease virus infectivity remained essentially unchanged in cells exposed to amitraz (20 micrograms/ml) 24 hr prior to virus infection or after the adsorption period. Viral RNA synthesis evaluated through [3H]uridine incorporation in cells treated for 24 hr prior to infection was not affected by amitraz.

Structure and immunogenicity of experimental foot-and-mouth disease and poliomyelitis vaccines.

The physico-chemical properties and immunogenicity of experimental vaccines against foot-and-mouth disease (FMD) and poliomyelitis, prepared by treatment of the viruses with N-acetylethyleneimine (AEI), formaldehyde or neutral red, have been studied. None of these reagents affects the rate of sedimentation of the particles or their reaction with antibody against the major immunogenic sites. FMD vaccines prepared by inactivation with AEI or neutral red, behaved like the untreated virus, in that they were disrupted on lowering the pH below 7. The RNA of the AEI-inactivated virus was degraded into slowly sedimenting molecules. Unlike AEI-inactivated virus, from which all the RNA could be extracted with phenol-SDS, the recovery from the neutral red inactivated virus was variable and was sometimes as low as 40%; this RNA gave a heterogenous profile in sucrose gradients. The capsid proteins in the AEI preparation migrated in SDS-PAGE to the same positions as those of untreated virus, but in the neutral red preparation there was evidence of cross-linking. In contrast, the formaldehyde-inactivated vaccine was stable below pH 7 and the RNA could not be released by extraction with phenol-SDS at pH 5, because the capsid proteins had become cross-linked and/or linked to the RNA. As with foot-and-mouth disease virus (FMDV), poliovirus which had been inactivated with formaldehyde did not release its RNA on extraction with phenol-SDS and the capsid proteins were also cross-linked. Surprisingly, although AEI cleaved the viral RNA slowly in situ, the virus was no longer infectious after 6 h. Neutral red did not reduce the infectivity of the virus. All of the preparations gave similar levels of neutralizing antibody after a single inoculation. The high levels obtained with the formaldehyde-inactivated vaccines have implications for the processing of fixed particles by the antigen-presenting cells.

Susceptibilidad de Picornavirus a un antiviral de origen vegetal (Meliacina) / Susceptibility of picornaviruses to an antiviral of vegetal origin (meliacine)

La planta superior Melia azederach L (MA) produce un polipéptido cíclico que puede aislarse de sus hojas y que tiene un amplio espectro antiviral contra virus con RNA de distintas familias. En este trabajo se estudió su efecto sobre dos Picornavirus: el virus fiebre aftosa (VFA) y el virus polio. Para ello se probaron las cepas A24, A87, C3 Resende, O1 Campos, O1 Caseros y O69 del VFA y los tipos 1, 2 y 3 del virus polio. Se encontró que las cepas muestran una susceptibilidad diferencial a una concentración no citotóxica del antiviral de 0,3 Ag/ml, siendo las cepas A24 y A87 las más susceptibles ya que resultaron inhibidas en un 90 por ciento. Para que ello ocurra el extracto de MA debe agregarse después de la adsorción viral y conservarse en el medio de cultivo hasta la cosecha de virus ya que con todas las cepas ensayadas el pretratamiento de las monocapas no resultó efectivo. Para determinar la influencia de la multiplicidad de infección (m.i.) en la diferente susceptibilidad observada se eligió la cepa O1 Campos del VFA y la tipo 3 de polio que resultaron ser poco sensibles cuando se usó una m.i. de 1, encontrándose una inhibición del 99 por ciento para ambos virus a una m.i. de 0,001. Estos resultados indican que los Picornavirus también son susceptibles a la inhibición por meliacina, que dicha inhibición varía con la cepa ensayada y que la aparente resistencia de estos virus estaría relacionada con la velocidad del ciclo de replicación, el que es superior al establecimiento del estado antiviral, cuando todas las células son infectadas simultáneamente (AU)

Susceptibilidad de Picornavirus a un antiviral de origen vegetal (Meliacina) / Susceptibility of picornaviruses to an antiviral of vegetal origin (meliacine)

La planta superior Melia azederach L (MA) produce un polipéptido cíclico que puede aislarse de sus hojas y que tiene un amplio espectro antiviral contra virus con RNA de distintas familias. En este trabajo se estudió su efecto sobre dos Picornavirus: el virus fiebre aftosa (VFA) y el virus polio. Para ello se probaron las cepas A24, A87, C3 Resende, O1 Campos, O1 Caseros y O69 del VFA y los tipos 1, 2 y 3 del virus polio. Se encontró que las cepas muestran una susceptibilidad diferencial a una concentración no citotóxica del antiviral de 0,3 µg/ml, siendo las cepas A24 y A87 las más susceptibles ya que resultaron inhibidas en un 90 por ciento. Para que ello ocurra el extracto de MA debe agregarse después de la adsorción viral y conservarse en el medio de cultivo hasta la cosecha de virus ya que con todas las cepas ensayadas el pretratamiento de las monocapas no resultó efectivo. Para determinar la influencia de la multiplicidad de infección (m.i.) en la diferente susceptibilidad observada se eligió la cepa O1 Campos del VFA y la tipo 3 de polio que resultaron ser poco sensibles cuando se usó una m.i. de 1, encontrándose una inhibición del 99 por ciento para ambos virus a una m.i. de 0,001. Estos resultados indican que los Picornavirus también son susceptibles a la inhibición por meliacina, que dicha inhibición varía con la cepa ensayada y que la aparente resistencia de estos virus estaría relacionada con la velocidad del ciclo de replicación, el que es superior al establecimiento del estado antiviral, cuando todas las células son infectadas simultáneamente

Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.

RNA molecules containing the 3' terminal region of foot-and-mouth disease virus (FMDV) RNA in both antisense and sense orientations were able to inhibit viral FMDV translation and infective particle formation in BHK-21 cells following comicroinjection or cotransfection with infectious viral RNA. Antisense, but not sense, transcripts from the 5' noncoding region including the proximal element of the internal ribosome entry site and the two functional initiation AUGs were also inhibitory, both in in vitro translation and in vivo in comicroinjected or cotransfected BHK-21 cells. This effect was not observed with nonrelated RNA transcripts from lambda phage. The inhibitions found were permanent, sequence specific, and dose dependent; an inverse correlation between the length of the transcript and the extent of the antiviral effect was seen. In all cases, the extent of inhibition increased when viral RNAs and transcripts were allowed to reanneal before transfection, concomitant with a decrease in the doses required. The antiviral effect was specific for FMDV, since transcripts failed to inhibit infective particle formation by other picornavirus, such as encephalomyocarditis virus. These results indicate that the ability of RNA transcripts to inhibit viral multiplication depends on their efficient hybridization with target regions on the viral genome. Furthermore, cells transfected with the 5'1as transcript, which is complementary to the 5' noncoding region, showed a significant reduction of plaque-forming ability during the course of a natural infection. RNA 5'1as was able to inhibit FMDV RNA translation in vitro, suggesting that the inhibitions observed are mediated by a blockage of the viral translation initiation. Conversely, hybridization of short sequences of both sense and antisense transcripts from the 3' end induces distortion of predicted highly ordered structural motifs, which could be required for the synthesis of negative-stranded viral RNA, and correlates with inhibition of viral propagation.