Modelling Mutation in Equine Infectious Anemia Virus Infection Suggests a Path to Viral Clearance with Repeated Vaccination

Equine Infectious Anemia Virus (EIAV) is a lentivirus similar to HIV that infects horses. Clinical and experimental studies demonstrating immune control of EIAV infection hold promise for efforts to produce an HIV vaccine. Antibody infusions have been shown to block both wild-type and mutant virus infection, but the mutant sometimes escapes. Using these data, we develop a mathematical model that describes the interactions between antibodies and both wild-type and mutant virus populations, in the context of continual virus mutation. The aim of this work is to determine whether repeated vaccinations through antibody infusions can reduce both the wild-type and mutant strains of the virus below one viral particle, and if so, to examine the vaccination period and number of infusions that ensure eradication. The antibody infusions are modelled using impulsive differential equations, a technique that offers insight into repeated vaccination by approximating the time-to-peak by an instantaneous change. We use impulsive theory to determine the maximal vaccination intervals that would be required to reduce the wild-type and mutant virus levels below one particle per horse. We show that seven boosts of the antibody vaccine are sufficient to eradicate both the wild-type and the mutant strains. In the case of a mutant virus infection that is given infusions of antibodies targeting wild-type virus (i.e., simulation of a heterologous infection), seven infusions were likewise sufficient to eradicate infection, based upon the data set. However, if the period between infusions was sufficiently increased, both the wild-type and mutant virus would eventually persist in the form of a periodic orbit. These results suggest a route forward to design antibody-based vaccine strategies to control viruses subject to mutant escape.

Truncation of the cytoplasmic tail of equine infectious anemia virus increases virion production by improving Env cleavage and plasma membrane localization

Envelope glycoproteins (Envs) of lentiviruses harbor unusually long cytoplasmic tails (CTs). Natural CT truncations always occur in vitro and are accompanied by attenuated virulence, but their effects on viral replication have not been fully elucidated. The Env in equine infectious anemia virus (EIAV) harbors the longest CT in the lentiviral family, and a truncated CT was observed in a live attenuated vaccine. This study demonstrates that CT truncation significantly increased EIAV production, as determined by comparing the virion yields from EIAV infectious clones in the presence or absence of the CT. A significant increase in a cleaved product from the CT-truncated Env precursor, but not the full-length Env, was observed. We further confirmed that the presence of the CT inhibited the cleavage of the Env precursor and found that a functional domain located at the C-terminus was responsible for this function. Moreover, CT-truncated Env was mainly localized at the plasma membrane (PM), while full-length Env was mainly localized in the cytoplasm. The CT-truncation caused a dramatic reduction in the endocytosis of Env. These results suggest that the CT can modulate the processing and trafficking of EIAV Env and thus regulate EIAV replication. Importance The mature lentivirus envelope glycoprotein (Env) is composed of a surface unit (SU) and a transmembrane unit (TM), which are cleaved products of the Env precursor. After mature Env is heterodimerically formed from the cleavage of the Env precursor, it is trafficked to the plasma membrane (PM) for incorporation and virion assembly. Env harbors a long cytoplasmic tail (CT), which has been increasingly found to play multiple roles in the Env biological cycle. Here, we revealed for the first time that the CT of equine infectious anemia virus (EIAV) Env inhibits cleavage of the Env precursor. Simultaneously, the CT promoted Env endocytosis, resulting in weakened Env localization at the PM. We also validated that the CT could significantly decrease EIAV production. These findings suggest that the CT regulates the processing and trafficking of EIAV Env to balance virion production.

Low Transmission Rates of Equine Infectious Anemia Virus (EIAV) in Foals Born To Seropositive Feral Mares Inhabiting the Amazon Delta Region Despite Climatic Conditions Supporting High Insect Vector Populations

Background Marajó Island within in the Amazon River Delta supports numerous bands of feral equids including the genetically distinct Marajoara horses. Roughly 40% of the equids on the island are infected with the Equine infectious anemia virus (EIAV). In the absence of iatrogenic transmission, spread of this lentivirus is mediated mainly by hematophagous insects whose year-round prevalence on the island is supported by favorable climatic conditions. The euthanasia of all infected equids within the population is not a feasible strategy when the prevalence of the disease is high or in highly specialized or rare breeds of equid such as the Marajoara horse. Preservation of these animals is complicated by high rates of seropositivity with the potential for vertical transmission or insect mediated transmission following parturition of foal. Therefore, the aim of this study was to evaluate EIAV vertical and post-partum insect-mediated transmission rates among foals born to seropositive feral mares until natural weaning. Serum samples of foals born to seropositive feral mares from Soure municipality, within Marajó Island, were collected to investigate their serological status, using an indirect ELISApgp45 with positive samples being tested in the classical agar gel immunodiffusion (AGID) assay to confirm the results. Results Twenty-eight foals were sampled and their serological status was monitored over a 2-year period. Depending on the birth date, some of them were sampled up to six times. All foals remained with their respective mares until fully weaned, approximately 10 months of age, and just 2 of 28 foals (7.14%) in the study group became seropositive against EIAV. Conclusion The results showed that in most cases it is possible to obtain negative foals born to and eventually weaned by EIA positive mares, even in equatorial regions where substantial rainfall and high temperatures favor the proliferation of insect vectors.

Identification and genetic characterization of equine infectious anemia virus in Western Balkans

Background Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse. Results the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain. Conclusions This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5′-LTR/ tat segment of PCR product revealed 83–93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/ tat region represents an important target for the detection of non-clinical horses.

Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.