Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis.

Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease characterized by virus persistence and recurrent arthritis. These studies demonstrate in vitro neutralization of CAEV infectivity by serum from goats infected with CAEV. Serum neutralizing activity was not detectable until 10 to 36 months postinfection, and titers were relatively low (less than or equal to 1:8). Serum neutralization was caused by antibody and was virus specific. Antigenic variants of CAEV were isolated from cell-free joint fluid of arthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent development of antigenic variants may promote CAEV persistence in vivo and provide a stimulus for recurrent arthritis.     

Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of expression of cytokines in macrophages. This finding suggests that CAEV may modulate the accessory functions of infected macrophages and the antiviral immune response in vivo.     

Presence of caprine arthritis-encephalitis virus (CAEV) infected cells in flushing media following oviductal-stage embryo collection

To improve the knowledge on the risk of transmission of the caprine arthritis-encephalitis virus (CAEV) during embryo manipulations, we conducted a double-nested polymerase chain reaction (PCR) for CAEV proviral-DNA on flushing media recovered from the oviducts 48 h after the beginning of estrus and on blood from 89 donor does. Sixty-four does had negative blood and flushing media by PCR. Among the 25 CAEV infected goats (blood PCR positive), 11 were PCR flushing media positive (P < 0.01). Cell lysate from flushing media samples that were PCR positive were serially diluted 10 times at 1:100. Starting with the second 1:100 dilution all the cell lysate samples were PCR negative. The mean number of embryos recovered was not significantly different between goats with flushing media PCR positive and goats with flushing media PCR negative (6.0 +/- 5.4 versus 7.8 +/- 4.4, respectively; mean +/- S.D.) nor between goats with blood PCR positive and goats with blood PCR negative (7.0 +/- 5.0 versus 5.9 +/- 5.3; mean +/- S.D.). The presence of CAEV infected cells in oviductal flushing media from infected donor does was indicated for the first time during this study. The absence of flushing media PCR positive for goat blood PCR negative seemed to allow the use of the blood PCR test to confidently predict the absence of CAEV provirus in the oviductal fluid.     

African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg(2+), the preferred cation, and has an apparent K(m) for dUTP of 1 microM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vDeltaE165R) has been obtained. Growth kinetics showed that vDeltaE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.     

Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.

Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.     

Combined eradication strategy for CAE in a dairy goat farm in Japan

We conducted an eradication program from 2002 to 2006 against caprine arthritis-encephalitis (CAE) virus (CAEV) in an important farm that maintained goat breeds and had a high prevalence of CAEV infection. The program did not involve the slaughter and replacement of entire flocks, but rather the prevention of both vertical and horizontal transmission. The program consisted of (1) removal of kids immediately after birth, (2) segregation of each generation, and (3) culling of positive goats in periodical tests. All goats born before 2002 were regarded as infected and grouped into herd A. Kids born during the program were divided into several herds on the basis of CAEV infection risk, raised with calf milk replacer, and periodically tested by polymerase chain reaction (PCR) and the agar gel immunodiffusion (AGID) test. A total of 205 kids were produced from 137 parents in herd A, 92 of which were distinctly infected. Only 11 of the 205 kids were infected with CAEV and were culled. The remaining 194 kids and all other kids born from other herds were negative by PCR and AGID testing throughout the program. The milk yield of primiparous does was significantly increased after the eradication program. These findings indicate that the combine use of isolated and milk-deprived rearing and periodical detection testing are effective in establishing a CAEV-free flock from an infected flock.     

Presence of caprine arthritis-encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does

Transmission of caprine arthritis-encephalitis virus (CAEV) is not completely understood and the vertical route of infection from the goat to the embryo or to the fetus needs to be investigated. This route of infection involves the presence of CAEV in the genital tract tissues. Prior studies have detected CAEV-infected cells in genital secretions and in flushing media recovered during embryo collection from infected goats. To specify the origin of these cells, we conducted a double-nested polymerase chain reaction (PCR) test on embryo flushing media and on mammary gland, mammary lymph node, synovial membrane, pelvic lymph node, uterus and oviduct tissues from 25 CAEV-infected (blood PCR positive) embryo donor goats for the presence of CAEV proviral DNA.The presence of proviral DNA was found in 22 of 25 mammary gland samples, 14 of 25 uterus samples, and in 16 of 25 oviduct samples. Nineteen of 25 goats had at least one positive genital tract sample. Flushing media from 11 goats were PCR positive. All goats with positive-flushing media were oviduct positive. Of this group of does, except for 1 of the 11, infection of flushing media correlated with infection of almost all the other tissues examined. The frequency of positive tissues for flushing media-positive goats (61/66; 92%) was significantly higher than that for flushing media-negative goats (50/84; 60%) (P<0.01).This study demonstrated the presence of CAEV-infected cells in the goat genital tract. The presence of CAEV-infected cells in the uterus and oviducts suggests potential for vertical transmission of CAEV from doe to embryo or fetus.     

Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses.

The ovine-caprine lentiviruses share nucleotide homology and serological properties in their gag-pol genes and gene products but constitute two distinct biological groups represented by ovine visna virus of Icelandic origin and by caprine arthritis-encephalitis and ovine progressive pneumonia viruses of U.S. origin. Two members of each group, visna 1514 and its antigenic variant LV1-1 in the first group and CAEV/CO and S93, a field isolate virus from a local arthritic sheep, in the second group, were examined in the present study in competitive-binding studies in fibroblast and macrophage cell cultures. The cultures were preinoculated with each of the four viruses and then reinoculated with either 1514 virus or CAEV/CO, labeled with [35S]methionine. Both 1514 and CAEV/CO caused homologous interference. LV1-1 and S93 viruses shared the interference patterns of 1514 and CAEV/CO, respectively. 1514 and LV1-1 did not interfere with binding of CAEV/CO. Similarly, CAEV/CO and S93 did not interfere with binding of 1514. Remarkably, certain combinations, such as S93 plus 1514, resulted in enhanced binding of the second virus. Other experiments showed that the enhancement in binding extended to enhancement in replication of the second virus. These latter data suggested that individual cells supported replication of both viruses. Further testing of this phenomenon showed that goats could be doubly infected with two noninterfering viruses, 1514 and CAEV/CO. The ability of noninterfering related lentiviruses to infect the same cell and also the same host animal may be important in the natural history of these viruses in providing ideal conditions for the development of new recombinant viruses.     

Antibody reactivity to the immunodominant epitopes of the caprine arthritis-encephalitis virus gp38 transmembrane protein associates with the development of arthritis.

High titers of antibodies to caprine arthritis-encephalitis virus (CAEV) envelope (Env) glycoproteins are found in infected goats developing a progressive arthritis. In order to identify linear B epitopes of the CAEV Env, which may be involved in the immunopathology of arthritis, we constructed a lambda gt11 Env expression library. By combining library screening with sera from naturally infected Swiss goats with an enzyme immunoassay with overlapping peptides (pepscan), four group-specific epitopes could be precisely defined in the transmembrane envelope proteins: TM1 to TM4, including a conserved structure (TM3) that corresponds to the immunodominant epitope of human immunodeficiency virus type 1 and other lentiviruses. A panel of 190 CAEV naturally infected goat serum samples, obtained from animals with defined clinical status, was tested for reactivity to synthetic peptides corresponding to the TM epitopes in an enzyme-linked immunosorbent assay. Antibody reactivity to two epitopes was highly associated (TM3, P = 0.002, and TM4, P < 0.001) with the presence of clinically detectable arthritis. Such an association is absent for anti-Gag antibody. Antibodies to the immunodominant structures of the TM glycoprotein could thus have an important role in the immunopathogenic process leading to disease.     

Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection

During an infection with herpes simplex virus, activity of cellular dUTPase decreases as a function of time, post-infection, while virus-encoded dUTPase activity increases. Prelabeling of cells with 35S-methionine and immunoprecipitation analysis, using monoclonal antibodies, indicates that cellular dUTPase protein levels remain the same (with respect to levels in uninfected cells) throughout the infection period. New synthesis of cellular dUTPase does not occur in infected cells as determined by 35S-methionine labeling during infection. Further characterization of the cellular dUTPase, in uninfected cells, reveals that the protein is post-translationally phosphorylated at serine residues. Pulse labeling of virus-infected cells with 32P-orthophosphate reveals that the phosphorylation rate of the cellular dUTPase protein decreases significantly as a function of time post-infection. In an effort to establish that phosphate turnover was occurring on the cellular dUTPase protein, cells were prelabeled with 32P-orthophosphate and then infected with HSV in the absence of label. Evidence from this experiment indicates that the phosphate moiety is removed from the cellular dUTPase protein during the infection. A series of viable virus mutants was generated by insertional inactivation of the HSV dUTPase gene. These mutants do not express viral dUTPase activity and HSV dUTPase protein is not detected by western blot analysis. However, in contrast to the wild-type situation, these mutant virus retain significant cellular dUTPase activity throughout infection. Interestingly, phosphorylation of cellular dUTPase protein is now readily detectable in each of the mutant virus-infected cells. These studies indicate that cellular dUTPase activity is diminished in wild-type HSV-infected cells by a process of dephosphorylation. It also appears that in mutant HSV, lacking the virus dUTPase, the mechanism of dephosphorylation and thus inactivation of cellular dUTPase is not functional. The end result is that the mutant virus can now rely on the cellular activity for its survival.     

The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.     

Lack of risk of transmission of caprine arthritis-encephalitis virus (CAEV) after an appropriate embryo transfer procedure

The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.     

The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies

Equine infectious anemia virus (EIAV) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes, tat, rev, and S2. Although serological analyses demonstrate the expression of the S2 protein in persistently infected horses, the role of this viral gene remains undefined. We recently reported that the S2 gene is not essential for EIAV replication in primary equine macrophages, as EIAV mutants lacking the S2 gene replicate to levels similar to those of the parental virus (F. Li, B. A. Puffer, and R. C. Montelaro, J. Virol. 72:8344-8348, 1998). We now describe in vivo studies that examine the evolution and role of the S2 gene in ponies experimentally infected with EIAV. The results of these studies reveal for the first time that the S2 gene is highly conserved during persistent infection and that deletion of the S2 gene reduces viral virulence and virus replication levels compared to those of the parental virus containing a functional S2 gene. These data indicate that the EIAV S2 gene is in fact an important determinant of viral replication and pathogenic properties in vivo, despite the evident lack of S2 influence on viral replication levels in vitro. Thus, these observations suggest in vivo functions of EIAV S2 that are not adequately reflected in simple infections of cultured cells, including natural target macrophages.     

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 10⁷ spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 μl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 10⁵ TCID₅₀/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.     

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.     

Host Range of Small-Ruminant Lentivirus Cytopathic Variants Determined with a Selectable Caprine Arthritis- Encephalitis Virus Pseudotype System

The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.