Juan M. Pacheco

Early events in the pathogenesis of foot-and-mouth disease in cattle after controlled aerosol exposure.

The goal of this study was to identify the primary sites of replication of foot-and-mouth disease virus (FMDV) in cattle subsequent to aerogenous inoculation. A novel aerosol inoculation method was developed to simulate natural, airborne transmission and thereby allow the identification of early replication sites. Virus distribution after aerosol inoculation was compared at 24h post inoculation with simple nasal instillation. Aerosol inoculation of FMDV consistently resulted in virus detection by real-time reverse transcriptase-polymerase chain reaction and viral isolation in the soft palate, pharynx, and lungs. Viral antigen was also detected in each of these tissues by immunohistochemistry. Aerosol exposure resulted in typical clinical signs of FMD when animals were kept alive long enough to develop disease. This aerosol infection method is highly reproducible regarding inoculum dose and volume, and allowed the detailed study of early events in FMDV-infected cattle. Extensive postmortem sampling and trimodal virus detection system allows a more precise determination of FMDV localization than previously reported.

Emergence in Asia of Foot-and-Mouth Disease Viruses with Altered Host Range: Characterization of Alterations in the 3A Protein

ABSTRACT In 1997, an epizootic in Taiwan, Province of China, was caused by a type O foot-and-mouth disease virus which infected pigs but not cattle. The virus had an altered 3A protein, which harbored a 10-amino-acid deletion and a series of substitutions. Here we show that this deletion is present in the earliest type O virus examined from the region (from 1970), whereas substitutions surrounding the deletion accumulated over the last 29 years. Analyses of the growth of these viruses in bovine cells suggest that changes in the genome in addition to the deletion, per se, are responsible for the porcinophilic properties of current Asian viruses in this lineage.

Role of Nonstructural Proteins 3A and 3B in Host Range and Pathogenicity of Foot-and-Mouth Disease Virus

ABSTRACT The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese strain of FMDV (O/TAW/97) to cause disease in bovines. Recently, an Asian virus with a second 3A deletion (amino acids 133 to 143) has also been detected (N. J. Knowles et al., J. Virol. 75:1551-1556, 2001). Genetically engineered viruses harboring the amino acids 93 to 102 or 133 to 143 grew well in porcine cells but replicated poorly in bovine cells, whereas a genetically engineered derivative of the O/TAW/97 virus expressing a full-length 3A (strain A12) grew well in both cell types. Interestingly, a virus with a deletion spanning amino acid 93 to 144 also grew well in porcine cells and caused disease in swine. Further, genetically engineered viruses containing only a single copy of VPg were readily recovered with the full-length 3A, the deleted 3A (amino acids 93 to 102), or the “super” deleted forms of 3A (missing amino acids 93 to 144). All of the single-VPg viruses were attenuated in porcine cells and replicated poorly in bovine cells. The single-VPg viruses produced a mild disease in swine, indicating that the VPg copy number is an important determinant of host range and virulence. The association of VPg copy number with increased virulence in vivo may help to explain why all naturally occurring FMDVs have retained three copies of VPg.

The Early Pathogenesis of Foot-and-Mouth Disease in Cattle After Aerosol Inoculation Identification of the Nasopharynx as the Primary Site of Infection

To characterize the early events of foot-and-mouth disease virus (FMDV) infection in cattle subsequent to simulated natural exposure, 16 steers were aerosol inoculated with FMDV and euthanized at various times. Samples were collected from each steer antemortem (serum, nasal swabs, and oral swabs) and postmortem (up to 40 tissues per animal) and screened for FMDV by virus isolation and for FMDV RNA by real-time reverse transcription polymerase chain reaction. Tissues that tested positive for FMDV or viral RNA were examined by immunohistochemistry and multichannel immunofluorescence microscopy. In previremic steers, FMDV was most consistently localized to nasopharyngeal tissues, thereby indicating this region as the most important site of primary viral replication. The earliest site of microscopic localization of FMDV antigens was the lymphoid follicle-associated epithelium of the pharyngeal mucosa-associated lymphoid tissue of the nasopharynx at 6 hours postaerosolization. At early time points after aerosol inoculation, viral antigens colocalized with cytokeratin-positive pharyngeal epithelial cells; intraepithelial FMDV-negative, MHCII/CD11c-double-positive dendritic cells were present in close proximity to FMDV-positive cells. Onset of viremia coincided with marked increase of viral loads in pulmonary tissues and with substantial decrease of viral detection in nasopharyngeal tissues. These data indicate that subsequent to aerogenous exposure to FMDV, the temporally defined critical pathogenesis events involve (1) primary replication in epithelial cells of the pharyngeal mucosa-associated lymphoid tissue crypts and (2) subsequent widespread replication in pneumocytes in the lungs, which coincides with (3) the establishment of sustained viremia.

Detection of foot-and-mouth disease virus infected cattle using infrared thermography.

Abstract In this study, infrared thermography (IRT) was assessed as a means of detecting foot-and-mouth disease virus (FMDV)-infected cattle before and after the development of clinical signs. Preliminary IRT imaging demonstrated that foot temperatures increased in FMDV-infected animals. The maximum foot temperatures of healthy (n =53), directly inoculated (DI) (n =12), contact (CT) (n =6), and vaccine trial (VT) (n =21) cattle were measured over the course of FMD infection. A cut-off value was established at 34.4°C (sensitivity=61.1%, specificity=87.7%) with the aim of detecting FMDV-infected animals both before and after clinical signs were observed. Seven of 12 (58%) DI and 3/6 (50%) CT animals showed maximum foot temperatures exceeding the 34.4°C cut-off before the development of foot vesicles. In contrast, only 5/21 (24%) VT animals displayed pre-clinical foot temperatures above this cut-off possibly indicating partial vaccine protection of this group. These results show IRT as a promising screening technology to quickly identify potentially infected animals for confirmatory diagnostic testing during FMD outbreaks. Further evaluation of this technology is needed to determine the value of IRT in detecting animals with mild clinical signs or sub-clinical infections.

Evaluation of Genetically Engineered Derivatives of a Chinese Strain of Foot-and-Mouth Disease Virus Reveals a Novel Cell-Binding Site Which Functions in Cell Culture and in Animals

ABSTRACT Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS). After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A12 infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.

Continuous porcine kidney cell line constitutively expressing bovine αVβ6 integrin with increased susceptibility to foot and mouth disease virus

ABSTRACT Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the αVβ6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the αV and β6 bovine integrin subunits. This stable cell line (LFBK-αVβ6) showed β6 expression and enhanced susceptibility to FMDV infection for ≥100 cell passages. LFBK-αVβ6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-αVβ6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.

Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication

Abstract Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus within the Picornaviridae family. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. The origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. By using confocal microscopy we showed in FMDV-infected cells co-localization of non-structural viral proteins 2B, 2C and 3A with LC3 (an autophagosome marker) and viral structural protein VP1 with Atg5 (autophagy-related protein), and LC3 with LAMP-1. Importantly, treatment of FMDV-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering RNAs, decreased viral replication. Altogether, these studies strongly suggest that autophagy may play an important role during the replication of FMDV.

Bovine Type III Interferon Significantly Delays and Reduces the Severity of Foot-and-Mouth Disease in Cattle

ABSTRACT Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.

A Safe Foot-and-Mouth Disease Vaccine Platform with Two Negative Markers for Differentiating Infected from Vaccinated Animals

ABSTRACT Vaccination of domestic animals with chemically inactivated foot-and-mouth disease virus (FMDV) is widely practiced to control FMD. Currently, FMD vaccine manufacturing requires the growth of large volumes of virulent FMDV in biocontainment-level facilities. Here, two marker FMDV vaccine candidates (A24LL3DYR and A24LL3BPVKV3DYR) featuring the deletion of the leader coding region (Lpro) and one of the 3B proteins were constructed and evaluated. These vaccine candidates also contain either one or two sets of mutations to create negative antigenic markers in the 3D polymerase (3Dpol) and 3B nonstructural proteins. Two mutations in 3Dpol, H27Y and N31R, as well as RQKP9-12→PVKV substitutions, in 3B2 abolish reactivity with monoclonal antibodies targeting the respective sequences in 3Dpol and 3B. Infectious cDNA clones encoding the marker viruses also contain unique restriction endonuclease sites flanking the capsid-coding region that allow for easy derivation of custom designed vaccine candidates. In contrast to the parental A24WT virus, single A24LL3DYR and double A24LL3BPVKV3DYR mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A24LL3DYR virus in the heel bulbs showed no clinical signs of disease, no fever, and no FMD transmission to in-contact animals. Immunization of cattle with chemically inactivated A24LL3DYR and A24LL3BPVKV3DYR vaccines provided 100% protection from challenge with parental wild-type virus. These attenuated, antigenically marked viruses provide a safe alternative to virulent strains for FMD vaccine manufacturing. In addition, a competitive enzyme-linked immunosorbent assay targeted to the negative markers provides a suitable companion test for differentiating infected from vaccinated animals.