Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT)

The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated [1]. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission.     

Experimental risk assessment of bovine viral diarrhea virus transmission via in vitro embryo production using somatic cell nucleus transfer

The objective of this study was to perform a comprehensive risk assessment on infectious disease transmission in the system of in vitro embryo production via somatic cell nucleus transfer (SCNT) technology using bovine viral diarrhea virus (BVDV) as a model. The risks of BVDV transmission in each step of the SCNT embryo production procedure, from donor cells to preimplantation SCNT embryo culture, were carefully examined using a sensitive real-time polymerase chain reaction assay. The identified primary source of BVDV transmission in SCNT embryo production was donor cell infection, most likely caused by contaminated fetal bovine serum in the culture medium. The risk of disease transmission through contaminated oocytes from an abattoir was relatively low, and it can be greatly minimized by cumulus cell removal and adequate oocyte washing procedures. Of the 200 cumulus-oocyte complexes (COCs) and more than 1500 cumulus cell-free oocyte (CFO) samples collected from multiple sources over a course of 7 months, only 2.5% of the COCs were BVDV positive, and all of the CFOs (100%) were BVDV negative. To evaluate the risk of BVDV introduction during in vitro SCNT embryo culture, 324 SCNT embryos were produced from 18 different cell lines using oocytes from 26 different batches collected over a course of 9 months. The embryos were cultured in vitro for 7 days and then tested for BVDV. All of the 324 SCNT embryos (100%) were negative, indicating that the embryo culture system is virtually risk-free for BVDV transmission. Based on these results, a standard operational protocol (SOP) for SCNT embryo production was proposed to greatly minimize the risk of BVDV transmission through the SCNT embryo production system. This SOP could be a starting point to produce a SCNT system that is virtually risk-free for disease transmission in general.     

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.     

Replication of Equine Infectious Anemia Virus in Engineered Mouse NIH 3T3 Cells

We employed the equine lentivirus equine infectious anemia virus (EIAV) to investigate the cellular restrictions for lentivirus replication in murine NIH 3T3 cells. The results of these studies demonstrate that NIH 3T3 cells expressing the EIAV receptor ELR1 and equine cyclin T1 supported productive replication of EIAV and produced infectious virions at levels similar to those found in a reference permissive equine cell line. The studies presented here demonstrate, for the first time, differential levels of restriction for EIAV and human immunodeficiency virus type 1 (HIV-1) replication in murine cells and suggest that these differences can be exploited to reveal critical virus-cell interactions required for HIV-1 assembly and budding of lentivirus particles.     

Risk assessment of porcine reproductive and respiratory syndrome virus (PRRSV) transmission via somatic cell nuclear transfer (SCNT) embryo production using oocytes from commercial abattoirs

Somatic cell nuclear transfer (SCNT) technology has become a powerful tool for reproductive biology to preserve and propagate valuable genetics for livestock. Embryo production through SCNT involves enucleation of the oocyte and insertion of a somatic donor cell into the oocyte. These procedures lead to a few small openings on the zona pellucida that may elevate risk of viral infection for the produced SCNT embryos. The oocytes used for SCNT are mainly obtained from abattoirs where viral contamination is almost inevitable. Therefore, a systematic evaluation of risk of disease transmission through SCNT embryo production is necessary prior large scale implementation of this technology in the livestock industry. The objective of the current study was to evaluate the risk of disease transmission via SCNT embryo production and transfer by testing for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) throughout the process of SCNT embryo production. The presence of PRRSV in each step of SCNT embryo production, from donor cells to pre-implantation SCNT embryo culture, was carefully examined using a real-time PCR assay with a sensitivity of five copies per-reaction. All 114 donor cell lines derived from pig skin tissue over a period of 7 years in our facility tested negative for PRRSV. Out of the 68 pooled follicular fluid samples collected from 736 ovaries, only four (5.9%) were positive indicating a small amount of viral molecule present in the oocyte donor population. All 801 Day 7 SCNT embryos produced in four separate trials and over 11,571 washed oocytes obtained in 67 batches over 10 months tested negative. These oocytes were collected from multiple abattoirs processing animals from areas with high density of pig population and correspond to a donor population of over 5828 individuals. These results indicate that the oocytes from abattoirs were free of PRRSV infection and therefore could be safely used for in vitro embryo production. Additionally, the established SCNT embryo production system, including donor cell testing, oocytes decontamination, and pathogen free embryo reconstruction and culturing, bears no risk of PRRSV transmission.     

Major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes from horses with equine infectious anemia virus recognize Env and Gag/PR proteins.

Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. To begin dissection of the immune mechanisms involved in EIAV control, peripheral blood mononuclear cells (PBMC) from infected horses were evaluated for CTL to EIAV-infected cells. By using noninfected and EIAV-infected autologous equine kidney (EK) cells in 51Cr-release assays, EIAV-specific cytotoxic activity was detected in unstimulated PBMC from three infected horses. The EIAV-specific cytotoxic activity was major histocompatibility complex (MHC) restricted, as determined by assaying EIAV-infected heterologous EK targets, and was mediated by CD8+ T lymphocytes, as determined by depleting these cells by a panning procedure with an anti-CD8 monoclonal antibody. MHC-restricted CD8+ CTL in unstimulated PBMC from infected horses caused significant specific lysis of autologous EK cells infected with recombinant vaccinia viruses expressing EIAV genes, either env or gag plus 5' pol. The EIAV-specific MHC-restricted CD8+ CTL were detected in two EIAV-infected horses within a few days after plasma viremia occurred and were present after viremia was terminated. The detection of these immune effector cells in EIAV-infected horses permits further studies to determine their in vivo role.     

Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.     

Structure of Equine Infectious Anemia Virus Matrix Protein

The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.     

Mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.     

马传染性贫血病毒囊膜基因的研究进展

马传染性贫血病毒是反转录病毒科慢病毒属的成员之一 ,不仅与人免疫缺陷病毒具有序列同源性 ,而且与其血清具有交叉反应。马传染性贫血驴白细胞弱毒疫苗是迄今为止唯一研究成功的慢病毒疫苗。在马传贫病毒囊膜基因的研究中有助于弄清其抗原变异、持续感染和疫苗免疫机理 ,为艾滋病疫苗的研究提供借鉴。对囊膜基因的结构、变异及其在机体免疫应答中的作用进行了讨论。     

An equine infectious anemia virus variant superinfects cells through novel receptor interactions

Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAV_vMA-1c, can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAV_vMA-1c superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAV_vMA-1c overcomes this restriction. We found that the cellular receptor used by EIAV, equine lentivirus receptor 1 (ELR1), remains on the surface of cells chronically infected with EIAV, suggesting that wild-type EIAV interferes with superinfection by masking ELR1. The addition of soluble wild-type SU protein to the medium during infection blocked infection by wild-type strains of virus, implicating SU as the viral protein responsible for interfering with virion entry into previously infected cells. Additionally, interference of wild-type EIAV binding to ELR1 by the addition of either anti-ELR1 antibodies or the ELR1 ectodomain prevented entry of the wild-type strains of EIAV into two permissive cell populations. Many of these same interference treatments prevented EIAV_vMA-1c infection of endothelial cells but only modestly affected the ability of EIAV_vMA-1c to enter and kill previously infected ED cells. These findings indicate that EIAV_vMA-1c retains the ability to use ELR1 for entry and suggest that this virus can interact with an additional, unidentified receptor to superinfect ED cells.     

Protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency

Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro. Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.