Elizabeth Rieder

The near complete coding genome sequences of 12 foot-and-mouth disease viruses of serotype O are reported here. These viruses were isolated from cattle in Uganda between 2014 and 2017. Eleven sequences belong to lineages I in the EA-2 topotype, while a single sequence belongs to the EA-1 topotype.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

Complete coding genome sequences of five foot-and-mouth disease virus (FMDV) serotype O strains that were isolated from the field between 2015 and 2016 showed five lineages within the EA-2 topotype circulating in four different regions (northern, western, eastern, and central) of Uganda. The genomic diversity may help in devising FMDV control strategies for Uganda.

Using georeferenced phylogenetic trees, phylogeography allows researchers to elucidate interactions between environmental heterogeneities and patterns of infectious disease spread. Concordant with the increasing availability of pathogen genetic sequence data, there is a growing need for tools to test epidemiological hypotheses in this field. In this study, we apply tools traditionally used in ecology to elucidate the epidemiology of foot-and-mouth disease virus (FMDV) in Uganda. We analyze FMDV serotype O genetic sequences and their corresponding spatiotemporal metadata from a cross-sectional study of cattle. We apply step selection function (SSF) models, typically used to study wildlife habitat selection, to viral phylogenies to show that FMDV is more likely to be found in areas of low rainfall. Next, we use a novel approach, a resource gradient function (RGF) model, to elucidate characteristics of viral source and sink areas. An RGF model applied to our data reveals that areas of high cattle density and areas near livestock markets may serve as sources of FMDV dissemination in Uganda, and areas of low rainfall serve as viral sinks that experience frequent reintroductions. Our results may help to inform risk-based FMDV control strategies in Uganda. More broadly, these tools advance the phylogenetic toolkit, as they may help to uncover patterns of spread of other organisms for which genetic sequences and corresponding spatiotemporal metadata exist.

Foot-and-mouth disease (FMD) is a viral infection of livestock of critical socioeconomic importance. Field studies from areas of endemic FMD suggest that animals can be simultaneously infected by more than one distinct variant of FMD virus (FMDV), potentially resulting in emergence of novel viral strains through recombination.

Inactivated, wild-type foot-and-mouth disease virus (FMDV) vaccines are currently used to control FMD around the world. These traditional FMD vaccines are produced using large quantities of infectious, virulent, wild-type FMD viruses, with the associated risk of virus escape from manufacturing facilities or incomplete inactivation during the vaccine formulation process. While higher quality vaccines produced from wild-type FMDV are processed to reduce non-structural antigens, there is still a risk that small amounts of non-structural proteins may be present in the final product. A novel, antigenically marked FMD-LL3B3D vaccine platform under development by Zoetis, Inc. and the USDA-ARS, consists of a highly attenuated virus platform containing negative antigenic markers in the conserved non-structural proteins 3D^pol and 3B that render resultant vaccines fully DIVA compatible. This vaccine platform allows for the easy exchange of capsid coding sequences to create serotype-specific vaccines. Here we demonstrate the efficacy of the inactivated FMD-LL3B3D-A₂₄ Cruzeiro vaccine in cattle against wild-type challenge with A₂₄ Cruzerio. A proprietary adjuvant system was used to formulate the vaccines that conferred effective protection at low doses while maintaining the DIVA compatibility. In contrast to wild-type FMDV, the recombinant FMD-LL3B3D mutant viruses have been shown to induce no clinical signs of FMD and no shedding of virus in cattle or pigs when inoculated as a live virus. The FMD-LL3B3D vaccine platform, currently undergoing development in the US, provides opportunities for safer vaccine production with full DIVA compatibility in support of global FMDV control and eradication initiatives.

In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3D ^pol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3D ^pol , T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3D ^pol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3D ^pol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Genetic Diversity of Circulating Foot and Mouth Disease Virus in Uganda Cross-Sectional Study During 2014–2017

Inactivated whole-virus vaccines are widely used for the control of foot-and-mouth disease (FMD). Their production requires the growth of large quantities of virulent FMD virus in biocontainment facilities, which is expensive and carries the risk of an inadvertent release of virus. Attenuated recombinant viruses lacking the leader protease coding region have been proposed as a safer alternative for the production of inactivated FMD vaccines (Uddowla et al., 2012, J Virol 86:11675-85). In addition to the leader deletion, the marker vaccine virus FMDV LL3B_PVKV3D_YR A₂₄ encodes amino acid substitutions in the viral proteins 3B and 3D that allow the differentiation of infected from vaccinated animals and has been previously shown to be effective in cattle and pigs. In the present study, two groups of six pigs each were inoculated with live FMDV LL3B_PVKV3D_YR A₂₄ virus either intradermally into the heel bulb (IDHB) or by intra-oropharyngeal (IOP) deposition. The animals were observed for 3 or 5 days after inoculation, respectively. Serum, oral and nasal swabs were collected daily and a thorough postmortem examination with tissue collection was performed at the end of the experiment. None of the animals had any signs of disease or virus shedding. Virus was reisolated from only one serum sample (IDHB group, sample taken on day 1) and one piece of heel bulb skin from the inoculation site of another animal (IDHB group, necropsy on day 3), confirming that FMDV LL3B_PVKV3D_YR A₂₄ is highly attenuated in pigs.

Here, we report the results of a cross‐sectional study designed to monitor the circulation and genetic diversity of foot and mouth disease virus (FMDV) in Uganda between 2014 and 2017. In this study, 13,614 sera and 2,068 oral‐pharyngeal fluid samples were collected from cattle and analysed to determine FMDV seroprevalence, circulating serotypes and their phylogenetic relationships. Circulation of FMDV was evidenced by the detection of antibodies against non‐structural proteins of FMDV or viral isolations in all districts sampled in Uganda. Sequence analysis revealed the presence of FMDV serotypes A, O, SAT 1 and SAT 2. FMDVs belonging to serotype O, isolated from 21 districts, were the most prevalent and were classified into six lineages within two East African topotypes, namely EA‐1 and EA‐2. Serotype A viruses belonging to the Africa G‐I topotype were isolated from two districts. SAT 1 viruses grouped within topotypes I and IV and SAT 2 viruses within topotypes VII, IV and X were isolated from six and four districts respectively. Phylogenetic analysis of SAT 1 and SAT 2 sequences from cattle clustered with historical sequences from African buffalo, indicating possible interspecies transmission at the wildlife‐livestock interface. In some cases, Uganda viruses also shared similarities to viral strains recovered from other regions in East Africa. This 3‐year study period provides knowledge about the geographical distribution of FMDV serotypes isolated in Uganda and insights into the genetic diversity of the multiple serotypes circulating in the country. Knowledge of circulating FMDV viruses will assist in antigenic matching studies to devise improved FMDV control strategies with vaccination and vaccine strain selection for Uganda.