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Development and Validation of a Mucosal Antibody (IgA) Test to Identify Persistent Infection with Foot-and-Mouth Disease Virus.

ABSTRACT: It is well known that approximately 50% of cattle infected with foot-and-mouth disease (FMD) virus (FMDV) may become asymptomatic carrier (persistently infected) animals. Although transmission of FMDV from carrier cattle to naïve cattle has not been demonstrated experimentally, circumstantial evidence from field studies has linked FMDV-carrier cattle to cause subsequent outbreaks. Therefore, the asymptomatic carrier state complicates the control and eradication of FMD. Current serological diagnosis using tests for antibodies to the viral non-structural proteins (NSP-ELISA) are not sensitive enough to detect all carrier animals, if persistently infected after vaccination and do not distinguish between carriers and non-carriers. The specificity of the NSP ELISA may also be reduced after vaccination, in particular after multiple vaccination. FMDV-specific mucosal antibodies (IgA) are not produced in vaccinated cattle but are elevated transiently during the acute phase of infection and can be detected at a high level in cattle persistently infected with FMDV, irrespective of their vaccination status. Therefore, detection of IgA by ELISA may be considered a diagnostic alternative to RT-PCR for assessing FMDV persistent infection in ruminants in both vaccinated and unvaccinated infected populations. This study reports on the development and validation of a new mucosal IgA ELISA for the detection of carrier animals using nasal, saliva, and oro-pharyngeal fluid (OPF) samples. The diagnostic performance of the IgA ELISA using nasal samples from experimentally vaccinated and infected cattle demonstrated a high level of specificity (99%) and an improved level of sensitivity (76.5%). Furthermore, the detection of carrier animals reached 96.9% when parallel testing of samples was carried out using both the IgA-ELISA and NSP-ELISA.

SUBMITTER: Biswal JK

PROVIDER: S-EPMC8147266 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In foot-and-mouth disease (FMD)-endemic countries, vaccination is commonly used to control the disease, whilst in FMD-free countries, vaccination is considered as an option, in addition to culling the infected and in contact animals. FMD vaccines are mainly comprised of inactivated virions and stimulate protective antibodies to virus structural proteins. In contrast, infection with FMD virus leads to virus replication and additional antibody responses to viral nonstructural proteins (NSP). Therefore, antibodies against NSPs are used to differentiate infection in vaccinated animals (DIVA), in order to estimate the prevalence of infection or its absence. Another advantage of NSP antibody tests is that they detect FMD infection in the field, irrespective of the serotypes of virus in circulation. In cattle, the NSP tests that target the 3ABC polyprotein provides the highest sensitivity, detecting up to 90% of vaccinated animals that become carriers after exposure to infection, with a specificity of around 99%. Due to insufficient diagnostic sensitivity and specificity, detection of a low level of infection is difficult at the population level with a high degree of confidence. The low level of non-specific responses can be overcome by retesting samples scored positive using a second confirmatory test, which should have at least comparable sensitivity to the first test. In this study, six in-house tests were developed incorporating different NSP antigens, and validated using bovine sera from naïve animals, field cases and experimentally vaccinated and/or infected animals. In addition, two (short and long incubation) new commercial NSP tests based on 3ABC competitive blocking ELISAs (ID Screen® FMD NSP Competition, IDvet, France) were validated in this study. The two commercial ELISAs had very similar sensitivities and specificities that were not improved by lengthening the incubation period. Several of the new in-house tests had performance characteristics that were nearly as good as the commercial ELISAs. Finally, the in-house tests were evaluated for use as confirmatory tests following screening with the PrioCHECK® and ID Screen® FMDV NS commercial kits, to assess the diagnostic performance produced by a multiple testing strategy. The in-house tests could be used in series (to confirm) or in parallel (to augment) with the PrioCHECK® and IDvet® FMDV NS commercial kits, in order to improve either the specificity or sensitivity of the overall test system, although this comes at the cost of a reduction in the counterpart (sensitivity/specificity) parameter.

Project description:African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centers of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centers, probang samples (oropharyngeal scrapings), and tonsil swabs to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1 coding region of the SAT1 virus inoculum clustered around 2 subpopulations differing by 22 single-nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions, depending on the day postinfection (dpi). There was a significant difference between the populations of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral populations in samples from individual animals, germinal centers, and epithelial tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to postmortem showed less within-sample variability of VP1 than that of tissue sample sequences analyzed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralize the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study, there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo.IMPORTANCE Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven-hoofed animals and is recognized as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV, and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low-frequency sequence variants over time, there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.

Project description:Foot-and-mouth disease virus (FMDV) could cause acute infection in host cells, or they could coexist with host cells to generate persistent infection. In persistent infection, the virus could survive for a long time in the host and could be transmitted between different host cells. In the case of FMDV-persistent infection cell line, there is a remarkable significant cellular heterogeneity in the FMDV-persistent infection cell line due to differences of viral load in the individual cells within the cell line. However, the mechanisms of FMDV-persistent infection are not well understood. It is now generally accepted that multiple factors contribute to the coevolution of viruses and cells during the course of persistent infection. The outcome would influence the development of persistent FMDV infection conjointly, reaching a state of equilibrium ultimately. Therefore, in order to elucidate the mechanism of cellular heterogeneity in FMDV-persistent infection cell line, single-cell sequencing was performed on BHK-Op, and pseudotime trajectory plot was draw through cell cluster. Based on the cell clusters, we predicted the development and progression of the FMDV-persistent infection. It could be well explained by the fact that, in BHK-Op cells, there are a fraction of infected cells and a fraction of virus-exposed but uninfected bystander cells. By further comparing the transcripts in cell clusters, we found that these genes were involved in changes in ribosome biogenesis, cell cycle, and intracellular signaling including the interferon signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway. Through comprehensive cross-tabulation analysis of differential expressed genes in various cluster of cells, we identified a high association of Fos, a downstream transcription factor of the MAPK/extracellular signal-regulated kinase (ERK) signaling pathway, with viral replication during the formation of FMDV-persistent infection. Through the further study of Fos, we found that downregulation of Fos facilitates viral clearance during FMDV-persistent infection. Upregulation of c-Raf, which is the upstream of the MAPK/ERK signaling pathway, could promote FMDV replication through downregulation of Fos. Our research is the first to provide insight into the mechanism of the formation FMDV-persistent infection through single-cell sequencing using persistent infection cell line. Pseudotime trajectory analysis was the first time to apply for FMDV-persistent infection cell line. Our work highlights the detailed overview of the evolution of FMDV-persistent infection. We also analyzed the differential expressed genes in the replication or elimination of FMDV within the host. We found that the MAPK/ERK signaling pathway and its downstream transcription factor Fos play an important role in FMDV-persistent infection.

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Development and Validation of a Mucosal Antibody (IgA) Test to Identify Persistent Infection with Foot-and-Mouth Disease Virus.

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