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We have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (EIAV). The clones were recovered from fetal equine kidney (FEK) cells infected with a virulent, cell culture-adapted virus stock (designated PV) and have been characterized at a molecular level. Each clone has unique envelope and long terminal repeat (LTR) sequences. We further investigated LTR sequence variation in the PV stock using PCR amplification to obtain additional LTR clones from infected FEK cells and from peripheral blood mononuclear ceils (PBMCs) from animals experimentally infected with PV.

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.

Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinicalEquine infectious anemia virus (EIAV) is unique among lentiviruses in that the clinical course of infection in equids results initially in a rapid and dynamic series of clearly demarcated cycles of disease and associated viremia that begin by 3 weeks postinfection and continue at irregular intervals separated by weeks or months (reviewed in reference 25). Disease cycles last 3 to 5 days and are characterized by fever, diarrhea, lethargy, edema, anemia, and thrombocytopenia. This stage of disease, defined as chronic equine infectious anemia (EIA), typically lasts about 8 to 12 months postinfection, with the frequency and severity of clinical episodes decreasing with time. In contrast to the progressive degenerative disease associated with most lentiviral infections, horses infected with EIAV typically make a transition during the first year postinfection from chronic EIA to an inapparent infection in which clinical symptoms are absent and viremia is usually undetectable for the remainder of the animal's life span of up to about 20 years. Thus, the EIAV systems provides a novel model in which to examine the dynamics of lentivirus replication during clearly defined cycles of disease and during long-term asymptomatic infections.Several lines of evidence indicate that the control of EIAV replication and disease in long-term inapparent carriers is mediated by virus-specific host immune responses that evolve during the first year postinfection to achieve an enduring effective suppression of virus replication. For example, experimental infection of foals with severe combined immunodeficiency results in a progressive infection leading to death, demonstrating the necessity of the host immune system in accomplishing the temporal control of virus replication associated with infection of immunocompetent horses (29). In addition, it has been shown that severe stress or treatment of long-term inapparent carriers with immunosuppressive drugs can cause recrudescence of viremia and disease, even after decades of clinical quiescence (16,48). Finally, it has been demonstrated that transfer of whole blood from long-term inapparent carriers to naive horses results in EIAV infection and disease in the recipient horses (12). Taken together, these observations demonstrate the lack of attenuation of the infect-* Corresponding author. Mailing address:

As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive for EIAV replication, the mutant virus replicated as well as the parental virus. In primary cultures of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infection decreased, the difference between the parental and mutant viruses increased, such that at the lowest multiplicity of infection tested, there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shetland ponies that were infected with the parental and mutant viruses showed transient virus RNA levels in plasma approximately 5 to 10 days postinfection. The peak virus levels in plasma (as measured by a quantitative reverse transcriptase PCR assay) were 10-to 100-fold lower in the mutant virus-infected animals than in the animals infected with the parental virus. However, ponies infected with the mutant virus mounted similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for the efficient replication of the virus in macrophages in vitro and in vivo and suggests that variations in the DU sequence could markedly affect the biological and pathogenic properties of EIAV.

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.

Results suggested that administration of this MLV vaccine to ponies with exercise-induced immunosuppression was safe and that administration of a single dose to ponies provided clinical protection 3 months later.