Establishing a molecular toolbox of lineage-specific real-time RT-PCR assays for the characterization of foot-and-mouth disease viruses circulating in Asia.

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

Elimination of Non-cytopathic Bovine Viral Diarrhea Virus From the LFBK-αvß6 Cell Line.

The LFBK-αvß6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvß6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvß6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 µM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvß6 cultures (renamed WRL-LFBK-αvß6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvß6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.

Evaluation of Cell Lines for the Isolation of Foot-and-Mouth Disease Virus and Other Viruses Causing Vesicular Disease.

The most sensitive cell culture system for the isolation of foot-and-mouth disease virus (FMDV) is primary bovine thyroid (BTY) cells. However, BTY cells are seldom used because of the challenges associated with sourcing thyroids from FMDV-negative calves (particularly in FMD endemic countries), and the costs and time required to regularly prepare batches of cells. Two continuous cell lines, a fetal goat tongue cell line (ZZ-R 127) and a fetal porcine kidney cell line (LFBK-αVß6), have been shown to be highly sensitive to FMDV. Here, we assessed the sensitivity of ZZ-R 127 and LFBK-αVß6 cells relative to primary BTY cells by titrating a range of FMDV original samples and isolates. Both the ZZ-R 127 and LFBK-αVß6 cells were susceptible to FMDV for >100 passages, and there were no significant differences in sensitivity relative to primary BTY cells. Notably, the LFBK-αVß6 cell line was highly sensitive to the O/CATHAY porcine-adapted FMDV strain. These results support the use of ZZ-R 127 and LFBK-αVß6 as sensitive alternatives to BTY cells for the isolation of FMDV, and highlight the use of LFBK-αVß6 cells as an additional tool for the isolation of porcinophilic viruses.

Molecular characterization of foot-and-mouth disease viruses circulating in Ethiopia between 2008 and 2019.

One of the constraints to controlling foot-and-mouth disease (FMD) in East Africa is the incomplete knowledge of the specific FMD virus (FMDV) strains circulating and the way in which these viruses move across countries in the region. This retrospective study focuses on Ethiopia, which has one of the largest FMD-susceptible livestock populations in Africa. Analyses of FMDV positive samples collected between 2008 and 2019 demonstrate that serotypes O (n = 175), A (n = 51) and SAT 2 (n = 33) were present in the country. Phylogenetic analysis of the VP1 sequences for these viruses showed that there were at least seven different FMD viral clades circulating during this period: O/EA-3, O/EA-4, A/AFRICA/G-I, A/AFRICA/G-IV, A/AFRICA/G-VII, SAT2/VII and SAT2/XIII. Although these results only represent a snapshot and might not reflect all FMDV lineages that were present, they highlight the importance of serotype O, as well as the complexity and co-existence of FMDV serotypes in Ethiopia and surrounding countries. These sequence data also support the idea that there are two FMDV ecosystems existing in East Africa. Data from retrospective studies, such as these presented here, will be beneficial for vaccine selection and vaccination campaigns to control FMDV within Ethiopia.

Inactivation of foot-and-mouth disease virus A/IRN/8/2015 with commercially available lysis buffers.

Laboratories working with foot-and-mouth disease virus (FMDV) must maintain a high level of biocontainment. However, if infectious virus is reliably inactivated during sample processing, molecular and serological testing can be performed at a lower level of containment. In this study, three commercial lysis buffers (AL, AVL, and MagMAX CORE) were tested in two laboratories for their ability to inactivate FMDV A/IRN/8/2015 in different sample matrices (cell culture supernatant, epithelial tissue suspension and milk). Residual infectivity after the addition of lysis buffer was evaluated by inoculating susceptible cell cultures. No cytopathic effect was observed for all three lysis buffers, indicating that the buffers are capable of reducing viral infectivity (estimated range 3.1 to >5.1 Log10). These results highlight the capacity of lysis buffers to decrease FMDV infectivity; however, additional validation experiments should be conducted, particularly if different sample matrices and/or lysis buffers are used.

Expression of APOBEC3 Lentiviral Restriction Factors in Cats.

Feline immunodeficiency virus (FIV) is a naturally occurring T-cell tropic lentiviral disease of felids with many similarities to HIV/AIDS in humans. Similar to primate lentiviral-host interactions, feline APOBEC3 (A3) has been shown to inhibit FIV infection in a host-specific manner and feline A3 degradation is mediated by FIV Vif. Further, infection of felids with non-native FIV strains results in restricted viral replication in both experimental and naturally occurring infections. However, the link between molecular A3-Vif interactions and A3 biological activity during FIV infection has not been well characterized. We thus examined expression of the feline A3 genes A3Z2, A3Z3 and A3Z2-Z3 during experimental infection of domestic cats with host-adapted domestic cat FIV (referred to as FIV) and non-adapted Puma concolor FIV (referred to as puma lentivirus, PLV). We determined A3 expression in different tissues and blood cells from uninfected, FIV-infected, PLV-infected and FIV/PLV co-infected cats; and in purified blood cell subpopulations from FIV-infected and uninfected cats. Additionally, we evaluated regulation of A3 expression by cytokines, mitogens, and FIV infection in cultured cells. In all feline cells and tissues studied, there was a striking difference in expression between the A3 genes which encode FIV inhibitors, with A3Z3 mRNA abundance exceeding that of A3Z2-Z3 by 300-fold or more. Interferon-alpha treatment of cat T cells resulted in upregulation of A3 expression, while treatment with interferon-gamma enhanced expression in cat cell lines. In cats, secondary lymphoid organs and peripheral blood mononuclear cells (PBMC) had the highest basal A3 expression levels and A3 genes were differentially expressed among blood T cells, B cells, and monocytes. Acute FIV and PLV infection of cats, and FIV infection of primary PBMC resulted in no detectable change in A3 expression with the exception of significantly elevated A3 expression in the thymus, the site of highest FIV replication. We conclude that cat A3 expression is regulated by cytokine treatment but, by and large, lentiviral infection did not appear to alter expression. Differences in A3 expression in different blood cell subsets did not appear to impact FIV viral replication kinetics within these cells. Furthermore, the relative abundance of A3Z3 mRNA compared to A3Z2-Z3 suggests that A3Z3 may be the major active anti-lentiviral APOBEC3 gene product in domestic cats.

Prior Puma Lentivirus Infection Modifies Early Immune Responses and Attenuates Feline Immunodeficiency Virus Infection in Cats.

We previously showed that cats that were infected with non-pathogenic Puma lentivirus (PLV) and then infected with pathogenic feline immunodeficiency virus (FIV) (co-infection with the host adapted/pathogenic virus) had delayed FIV proviral and RNA viral loads in blood, with viral set-points that were lower than cats infected solely with FIV. This difference was associated with global CD4⁺ T cell preservation, greater interferon gamma (IFN-γ) mRNA expression, and no cytotoxic T lymphocyte responses in co-infected cats relative to cats with a single FIV infection. In this study, we reinforced previous observations that prior exposure to an apathogenic lentivirus infection can diminish the effects of acute infection with a second, more virulent, viral exposure. In addition, we investigated whether the viral load differences that were observed between PLV/FIV and FIV infected cats were associated with different immunocyte phenotypes and cytokines. We found that the immune landscape at the time of FIV infection influences the infection outcome. The novel findings in this study advance our knowledge about early immune correlates and documents an immune state that is associated with PLV/FIV co-infection that has positive outcomes for lentiviral diseases.

Feline Immunodeficiency Virus Cross-Species Transmission: Implications for Emergence of New Lentiviral Infections.

Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of human immunodeficiency virus (HIV) emergence following human exposures to simian immunodeficiency virus (SIV), an understanding of processes that promote successful cross-species lentiviral transmissions is highly relevant. We previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) for a small number of animals in California and Florida. In this study, we investigate host-specific selection pressures, within-host viral fitness, and inter- versus intraspecies transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analyses of proviral and viral RNA levels demonstrate that PLVA fitness is severely restricted in mountain lions compared to that in bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but we did not detect selection among 20 PLVA isolates from bobcats. These findings support the hypothesis that PLVA is a bobcat-adapted virus which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals that intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following the initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to the emergence of HIV from nonhuman primates.IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which outcome will occur are complex, and the risk of new virus emergence is therefore difficult to predict. We used molecular techniques to evaluate the transmission, fitness, and adaptation of puma lentivirus A (PLVA) between bobcats and mountain lions in two geographic regions. Our findings illustrate that mountain lion exposure to PLVA is relatively common but does not routinely result in communicable infections in the new host. This is attributed to efficient species barriers that largely prevent lentiviral adaptation. However, the evolutionary capacity for lentiviruses to adapt to novel environments may ultimately overcome host restriction mechanisms over time and under certain ecological circumstances. This phenomenon provides a unique opportunity to examine cross-species transmission events leading to new lentiviral emergence.

Truncated Bovine Integrin Alpha-v/Beta-6 as a Universal Capture Ligand for FMD Diagnosis.

Foot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV) serotype-specific antibodies in small animals (rabbits and guinea pigs) that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvß6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvß6 and FMDV empty capsids in FMD diagnostic assays.

Domestic cat microsphere immunoassays: detection of antibodies during feline immunodeficiency virus infection.

Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9 and 28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases.

Microsphere immunoassay for the detection of cytokines in domestic cat (Felis catus) plasma: elevated IL-12/23 in acute feline immunodeficiency virus infections.

We recently described the development and validation of a highly sensitive and specific microsphere immunoassay capable of simultaneously quantifying three domestic cat cytokines in tissue culture supernatant. Here we describe the modification of this assay to measure interferon gamma (IFNγ), interleukin (IL)-10 and IL-12/IL-23 p40 (IL-12/23) in domestic cat plasma, report values obtained from plasma collected after feline immunodeficiency virus (FIV) exposure, and compare plasma concentrations to blood cell mRNA expression. The validated quantitation limits of this assay are 31-1000 pg/ml for IFNγ, 63-2000 pg/ml for IL-10, and 20-625 pg/ml for IL-12/23. Plasma cytokine levels from domestic cats infected with pathogenic and/or apathogenic FIV were determined at 3-4 and 7-8 weeks post-infection. IL-12/23 was elevated (p<0.05) during acute infection with both FIV strains in two similar studies, conducted five years apart in different feline cohorts (n=44 total animals). IL-12/23 concentrations ranged from 377 to 1904 pg/ml in naïve cats and 552 to 3460 pg/ml in infected cats. In contrast, the majority of plasma samples had IFNγ and IL-10 concentrations below the lowest standard tested. The inability to consistently detect levels of IFNγ and IL-10 in plasma, despite the fact that mRNA changes were detected, suggests that these cytokines may be secreted and/or cleared in a more highly regulated manner than IL-12/23, or perhaps exert local effects under tighter peripheral constraints and/or at a lower effective concentration.

Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines.

Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat (Felis catus) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 µl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.

Roles of human disturbance, precipitation, and a pathogen on the survival and reproductive probabilities of deer mice.

Climate change, human disturbance, and disease can have large impacts on the dynamics of a species by affecting the likelihood of survival and reproduction of individuals. We investigated the roles of precipitation, off-road vehicle (ORV) alteration of habitat, and infection with Sin Nombre virus on the survival and reproductive probabilities of deer mice (Peromyscus maniculatus). We used generalized linear mixed models to estimate the effects of these factors and their interactions by fitting capture-recapture data collected seasonally from 2002 to 2007 at 17 sites in the Great Basin Desert of central Utah, USA. During periods with high precipitation, we found no difference in survival and reproductive probabilities between seasons, but during drier periods, we found a reduction of overwinter survival and fall reproductive activity. Precipitation also interacted with disturbance to affect survival probabilities and female reproduction; in periods with low precipitation, deer mice on highly disturbed sites had extremely low survival probabilities and low reproductive probabilities of females compared to those of individuals from low-disturbance sites. However, high precipitation ameliorated the effect of disturbance on both parameters. Deer mice from sites with high impact of ORV disturbance also had low survival over summer. Additionally, male reproductive probabilities were diminished on highly disturbed sites in both seasons; in contrast, they were reduced only in the fall on low-disturbance sites. Density had an overall negative effect on survival and reproductive probabilities of deer mice. For females, the negative effect on reproductive activity was amplified in highly disturbed sites. We found no effect of hantavirus infection on survival probabilities of deer mice. Overall, this study revealed complexity in the determinants of deer mouse survival and reproduction given by the effects of a number of significant interactions among explanatory variables. Thus, factors that may not appear to have a strong effect when investigated alone can still be influential by modulating the effect of a different factor.

Seasonal variation in Sin Nombre virus infections in deer mice: preliminary results.

The proportion of deer mice (Peromyscus maniculatus) with recently acquired Sin Nombre virus (SNV) infections is an indicator of epizootic intensity and may be key in predicting outbreaks of hantavirus cardio-pulmonary syndrome in humans. We investigated whether incidence of recent infections was related to season, sex, reproductive status, or habitat disturbance. In May and September, 2006, we sampled 912 deer mice at six sites in Utah. We determined SNV antibody prevalence and estimated the number of recent infections with an avidity enzyme-linked immunosorbent assay. Antibody prevalence in adults (n = 735) was 22%, and putative maternal antibody prevalence in juveniles (n = 177) was 7%. Sampling period explained a significant amount of the variance in the probability of recent infections, which were two times more common in May versus September. Additionally, prevalence of high-avidity maternal antibodies (i.e., from dams with older infections) in juveniles did not correspond to the antibody avidity patterns in adult females. In May, no juveniles had high-avidity antibodies compared to adult females (49%); in September, avidity could not be measured in juveniles because none were seropositive, despite large sample sizes (n = 84) and an 11% seroprevalence in adult females. Based on the results, coupled with those from the literature, we speculate that the majority of new infections may occur predominantly in the spring and that SNV may impair reproductive output of females.