B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses.

Insights into the molecular and biological features of the dUTPase-related gene of bovine immunodeficiency virus.

This study was stimulated by our previous research of the dUTPase-related protein from bovine immunodeficiency virus (BIV) (Voronin et al., 2014). Despite the lack of detectable enzymatic BIV dUTPase activity (both of the recombinant protein and in virions), mutating the dUTPase gene was deleterious to viral production. However, cDNA synthesis and integration were apparently unaffected. Consequently, we have studied here two important issues. First, we showed that in cDNA produced by the dUTPase-mutated virions, the incidence of mutations was not higher than that found in wild-type BIV-infected cells. Second, single mutations, introduced in preserved dUTPase residues Asp48 and Asn57 (in the putative dUTPase active site or close to it), have led to abortive BIV infections (except for the conservative Asp48Glu mutation). Therefore, we postulate that the BIV dUTPase-related protein has a critical role in retroviral replication at steps that take place after viral cDNA synthesis and integration.

Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production.

The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of antiviral strategies.IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag although HIV-1 Vif proteins bind Pr55Gag less efficiently than those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of antiviral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

Role of cullin-elonginB-elonginC E3 complex in bovine immunodeficiency virus and maedi-visna virus Vif-mediated degradation of host A3Z2-Z3 proteins.

BACKGROUND: All lentiviruses except equine infectious anemia virus (EIVA) antagonize antiviral family APOBEC3 (A3) proteins of the host through viral Vif proteins. The mechanism by which Vif of human, simian or feline immunodeficiency viruses (HIV/SIV/FIV) suppresses the corresponding host A3s has been studied extensively. RESULTS: Here, we determined that bovine immunodeficiency virus (BIV) and maedi-visna virus (MVV) Vif proteins utilize the Cullin (Cul)-ElonginB (EloB)-ElonginC (EloC) complex (BIV Vif recruits Cul2, while MVV Vif recruits Cul5) to degrade Bos taurus (bt)A3Z2-Z3 and Ovis aries (oa)A3Z2-Z3, respectively, via a proteasome-dependent but a CBF-ß-independent pathway. Mutation of the BC box in BIV and MVV Vif, C-terminal hydrophilic replacement of btEloC and oaEloC and dominant-negative mutants of btCul2 and oaCul5 could disrupt the activity of BIV and MVV Vif, respectively. While the membrane-permeable zinc chelator TPEN could block BIV Vif-mediated degradation of btA3Z2-Z3, it had minimal effects on oaA3Z2-Z3 degradation induced by MVV Vif, indicating that Zn is important for the activity of BIV Vif but not MVV Vif. Furthermore, we identified a previously unreported zinc binding loop [C-x1-C-x1-H-x19-C] in the BIV Vif upstream BC box which is critical for its degradation activity. CONCLUSIONS: A novel zinc binding loop was identified in the BIV Vif protein that is important for the E3 ubiquination activity, suggesting that the degradation of btA3Z2-Z3 by BIV and that of oaA3Z2-Z3 by MVV Vif may need host factors other than CBF-ß.

The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein.

BACKGROUND: Deoxyuridine 5'-triphosphate nucleotide-hydrolases (dUTPases) are essential for maintaining low intra-cellular dUTP/dTTP ratios. Therefore, many viruses encode this enzyme to prevent dUTP incorporation into their genomes instead of dTTP. Among the lentiviruses, the non-primate viruses express dUTPases. In bovine immunodeficiency virus (BIV), the putative dUTPase protein is only 74 residues-long, compared to ~130 residues in other lentiviruses. RESULTS: In this study, the recombinant BIV dUTPase, as well as infectious wild-type (WT) BIV virions, were shown to lack any detectable dUTPase activity. Controls of recombinant dUTPase from equine infectious anemia virus (EIAV) or of EIAV virions showed substantial dUTPase activities. To assess the importance of the dUTPase to BIV replication, we have generated virions of WT BIV or BIV with mutations in the dUTPase gene. The two mutant viral dUTPases were the double mutant D48E/N57S (in the putative enzyme active site and its vicinity) and a deletion of 36 residues. In dividing Cf2Th cells and under conditions where the WT virus was infectious and generated progeny virions, both mutant viruses were defective, as no progeny viruses were generated. Analyses of the integrated viral cDNA showed that cells infected with the mutant virions carry in their genomic DNA levels of integrated BIV DNA that are comparable to those in WT BIV-infected cells. CONCLUSIONS: The herby presented results show that the two BIV mutants with the modified dUTPase gene could infect cells, as viral cDNA was synthesized and integrated into the host cell DNA. However, no virions were generated by cells infected by these mutants. The most likely explanation is that either the integrated cDNA of the mutants is defective (due to potential multiple mutations, introduced during reverse-transcription) or that the original dUTPase mutations have led to severe blocks in viral replication at steps post integration. These results emphasize the importance of the dUTPase-related sequence to BIV replication, despite the lack of any detectable catalytic activity.

Cellular requirements for bovine immunodeficiency virus Vif-mediated inactivation of bovine APOBEC3 proteins.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) viral infectivity factor (Vif) form a CRL5 E3 ubiquitin ligase complex to suppress virus restriction by host APOBEC3 (A3) proteins. The primate lentiviral Vif complex is composed of the unique cofactor core binding factor ß (CBF-ß) and canonical ligase components Cullin 5 (CUL5), Elongin B/C (ELOB/C), and RBX2. However, the mechanism by which the Vif protein of the related lentivirus bovine immunodeficiency virus (BIV) overcomes its host A3 proteins is less clear. In this study, we show that BIV Vif interacts with Cullin 2 (CUL2), ELOB/C, and RBX1, but not with CBF-ß or CUL5, to form a CRL2 E3 ubiquitin ligase and degrade the restrictive bovine A3 proteins (A3Z2Z3 and A3Z3). RNA interference-mediated knockdown of ELOB or CUL2 inhibited BIV Vif-mediated degradation of these A3 proteins, whereas knockdown of CUL5 or CBF-ß did not. BIV Vif with mutations in the BC box (Vif SLQ-AAA) or putative VHL box (Vif YI-AA), which cannot interact with ELOB/C or CUL2, respectively, lost the ability to counteract bovine A3 proteins. Moreover, CUL2 and UBE2M dominant negative mutants competitively inhibited the BIV Vif-mediated degradation mechanism. Thus, although the general strategy for inhibiting A3 proteins is conserved between HIV-1/SIV and BIV, the precise mechanisms can differ substantially, with only the HIV-1/SIV Vif proteins requiring CBF-ß as a cofactor, HIV-1/SIV Vif using CUL5-RBX2, and BIV Vif using CUL2-RBX1. IMPORTANCE: Primate lentivirus HIV-1 and SIV Vif proteins form a ubiquitin ligase complex to target host antiviral APOBEC3 proteins for degradation. However, the mechanism by which the nonprimate lentivirus BIV Vif inhibits bovine APOBEC3 proteins is unclear. In the present study, we determined the mechanism for BIV Vif-mediated degradation of bovine APOBEC3 proteins and found that it differs from the mechanism of HIV-1/SIV Vif by being CBF-ß independent and requiring different ubiquitin ligase scaffolding proteins (CUL2-RBX1 instead of CUL5-RBX2). BIV Vif is the only known retroviral protein that can interact with CUL2. This information broadens our understanding of the distinct mechanisms by which the Vif proteins of different lentiviruses facilitate viral infection. This novel mechanism for assembly of the BIV Vif-APOBEC3 ubiquitin ligase complex advances our understanding of viral hijacking of host E3 ubiquitin ligases and illustrates the evolutionary flexibility of lentiviruses.

Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1.

The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to block HIV-1 and SIV(mac) infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine-aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIV(mac). We demonstrated that the HD domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active purified SAMHD1 protein from insect cells. Interestingly, we showed that double-stranded RNA inhibits the enzymatic activity of SAMHD1 in vitro suggesting the possibility that RNA from a pathogen might modulate the enzymatic activity of SAMHD1 in cells. By contrast, we found that the SAM domain is dispensable for retroviral restriction, oligomerization and RNA binding. Finally we tested the ability of SAMHD1 to block the infection of retroviruses other than HIV-1 and SIV(mac). These results showed that SAMHD1 blocks infection of HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Equine infectious anemia virus (EIAV), N-tropic murine leukemia virus (N-MLV), and B-tropic murine leukemia virus (B-MLV).

Bovine ISG15: an antiviral and inducible protein in BIV infected fetal bovine lung cells.

Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15's expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells.

Bovine immunodeficiency virus produces a transient viraemic phase soon after infection in Bos javanicus.

Infection of Bali cattle (Bos javanicus) in Indonesia with a non-pathogenic bovine lentivirus similar to Bovine immunodeficiency virus (BIV) is suspected but efforts to detect the virus have been unsuccessful. To define the kinetics of BIV infection in Bali cattle, 13 were infected with the R-29 strain of BIV and monitored for 60 days. No clinical effects were detected. Proviral DNA was detected in peripheral blood mononuclear cells from 4 to 60 days with peak titres 20 days post-infection (dpi). There was a transient viraemia from 4 to 14 dpi with a maximum titre of 1x10(4)genome copies/ml plasma. An antibody response to the transmembrane (TM) glycoprotein commenced 12 dpi but an antibody response to the capsid (CA) protein was detected in one animal only and not until 34 dpi. The results indicated that detection of BIV in infected Bali cattle would have a greater chance of success soon after infection and prior to the onset of a CA antibody response.

Comparative functional analysis of Jembrana disease virus Tat protein on lentivirus long terminal repeat promoters: evidence for flexibility at its N-terminus.

BACKGROUND: Jembrana disease virus (JDV) encodes a potent regulatory protein Tat that strongly stimulates viral expression by transactivating the long terminal repeat (LTR) promoter. JDV Tat (jTat) promotes the transcription from its own LTR as well as non-cognate LTRs, by recruiting host transcription factors and facilitating transcriptional elongation. Here, we compared the sequence requirements of jTat for transactivation of JDV, bovine immunodeficiency virus (BIV) and human immunodeficiency virus (HIV) LTRs. RESULTS: In this study, we identified the minimal protein sequence for LTR activation using jTat truncation mutants. We found that jTat N-terminal residues were indispensable for transactivating the HIV LTR. In contrast, transactivation of BIV and JDV LTRs depended largely on an arginine-rich motif and some flanking residues. Competitive inhibition assay and knockdown analysis showed that P-TEFb was required for jTat-mediated LTR transactivation, and a mammalian two-hybrid assay revealed the robust interaction of jTat with cyclin T1. In addition, HIV LTR transactivation was largely affected by fusion protein at the jTat N-terminus despite the fact that the cyclin T1-binding affinity was not altered. Furthermore, the jTat N-terminal sequence enabled HIV Tat to transactivate BIV and JDV LTRs, suggesting the flexibility at the jTat N-terminus. CONCLUSION: This study showed the distinct sequence requirements of jTat for HIV, BIV and JDV LTR activation. Residues responsible for interaction with cyclin T1 and transactivation response element are the key determinants for transactivation of its cognate LTR. N-terminal residues in jTat may compensate for transactivation of the HIV LTR, based on the flexibility.

The non-toxic A subunit of Shiga toxin type 1 prevents replication of bovine immunodeficiency virus in infected cells.

Shiga toxins are ribosome-inactivating proteins many of which are antiviral. Shiga toxin-producing Escherichia coli (STEC) may be pathogenic to humans, but are carried without ill effects by ruminants. We hypothesize that STEC have antiviral activity in ruminants, and showed previously that the non-toxic subunit A of Shiga toxin 1 (StxA1) acts selectively on cells infected with bovine leukemia virus, without harming normal cells, and that the numbers of intestinal STEC are inversely correlated with viral load in bovine leukemia virus-infected sheep. The purpose of the present study was to characterize StxA1 activity against a second bovine retrovirus, bovine immunodeficiency virus (BIV). Flow cytometry showed that StxA1 treatment induced apoptosis in BIV-infected cells but not in uninfected cells and immunoblot analysis showed that StxA1 curtailed synthesis of Gag p26 protein. A systematic electron microscopy description of BIV infection in fetal bovine lung fibroblasts showed an orderly sequence of changes in cell membrane, endoplasmic reticulum, Golgi, nucleus, and mitochondria, and suggested that the infected cells produce the virus within multivesicular bodies (MVBs). StxA1 interfered with all manifestations of BIV-induced transformation of infected cells into BIV-producing units. BIV-infected cells provided a suitable experimental system for investigation of the mechanism of Stx-antiviral activity.

Differential sensitivity of viruses to the antiviral activity of Shiga toxin 1 A subunit.

The non-toxic enzymic A subunit of Shiga toxin 1 (StxA1) reduces expression and replication of the bovine retroviruses, bovine leukemia virus and bovine immunodeficiency virus (BIV). Here, the impact of StxA1 on representative positive and negative stranded RNA viruses was compared. BIV and equine infectious anemia virus were sensitive to picomolar concentrations of StxA1 while poliovirus, rhinovirus, and vesicular stomatitis virus were only marginally sensitive to nanomolar concentrations of toxin. Thus, the length of the reproductive cycle and/or other factors, but not viral encapsulation may play a role in determining sensitivity to StxA1. The effects of StxA1 at concentrations from 0.01 to 10 microg/ml on the most sensitive virus (BIV-infected cultures of fetal bovine lung cells) were analyzed by electron microscopy 48 h post challenge. Cells treated with 0.1 microg StxA1/ml or higher toxin concentrations were similar in appearance and showed progressively fewer viral factories with increasing toxin concentration. However, cells treated with 0.01 microg/ml StxA1 had a radically different appearance, exhibiting smooth cell membranes and high vacuolization. These results showed that complex retroviruses were more sensitive to StxA1 than single-stranded RNA viruses and that StxA1 interfered with retroviral replication in a concentration-dependent manner.

Characterization of BIV Env core: implication for mechanism of BIV-mediated cell fusion.

Entry of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), requires folding of two heptad repeat regions (HR1 and HR2) of gp41 into a trimer-of-hairpins, which subsequently brings virus and cell membrane into fusion. This motif is a generalized feature of viral fusion proteins and has been exploited in generating antiviral fusion agents. In the present paper, we report structural characters of Env protein from another lentivirus, bovine immunodeficiency virus (BIV), which contributes to a good animal model of HIV. BIV HR1 and HR2 regions are predicted by two different programs and expressed separately or conjointly in Escherichia coli. Biochemical and biophysical analyses show that the predicted HRs of BIV Env can form a stable trimer-of-hairpins or six-helix bundle just like that formed by feline immunodeficiency virus Env. Cell fusion assay demonstrates that the HR2 peptide of BIV can efficiently inhibit the virus-mediated cell fusion.

In vivo transcription of bovine leukemia virus and bovine immunodeficiency-like virus.

Cellular tropism and transcription of bovine leukemia virus (BLV) and bovine immunodeficiency-like virus (BIV) were investigated using peripheral blood mononuclear cells (PBMC) collected from a cow infected with both viruses. Each PBMC subset, purified by magnetic cell sorting, was subjected to PCR and RT-PCR for detection of their integrated proviruses and transcript mRNAs. Both BLV and BIV genomes were detected by nested PCR in CD3(+), CD4(+), CD8(+) and gammadelta T cells, B cells and monocytes. However, BLV tax transcription was only detected in B cells, and only B cells also formed BLV syncytia in CC81 cells. On the other hand, BIV transcript was detected in each subpopulation of PBMC. These results indicated that BLV can infect T cells and monocytes as well as B cells, but can be expressed by transcription only in B cells. In contrast, BIV can express its transcripts in all infected cells.

Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus.

The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.

Construction and characterization of a chimeric virus (BIV/HIV-1) carrying the bovine immunodeficiency virus gag-pol gene.

HIV-1(HXB2) 5'LTR region, most of BIV(R29) gag-pol segment and HIV-1(HXB2) pol IN-3'LTR region were respectively amplified. A chimeric clone, designated as pHBIV(3753), was constructed by cloning three fragments sequentially into pUC18. MT4 cells were transfected with pHBIV3753. The replication and expressions of the chimeric virus (HBIV3753) were monitored by RT activity and IFA. The results firstly demonstrated that it is possible to generate a new type of the BIV/HIV-1 chimeric virus containing BIV gag-pol gene.

Experimental collection and transfer of embryos from bovine immunodeficiency virus (BIV) infected cattle.

Three experiments were conducted to determine whether the lentivirus, bovine immunodeficiency virus (BIV) is likely to be transmitted via embryo transfer. In the first experiment, embryos collected from BIV-negative heifers were exposed in vitro to BIV for 24 h, washed and then tested for the presence of the provirus. In the second experiment, embryos obtained from BIV-negative heifers were transferred to the uterine horns of BIV-infected heifers; 24 h later these embryos were recovered and tested for the presence of BIV. In the third experiment, embryos were collected from heifers experimentally infected with BIV and then transferred to BIV-negative recipients. In all three experiments, (BIV) proviral DNA was not detected by PCR in association with any oocytes, embryos, follicular fluid, oviductal or uterine washes. Twelve single embryos collected from BIV experimentally infected donors were transferred to BIV-negative recipients resulting in the birth of 7 calves all of which were also negative for BIV; the recipients remained BIV-negative throughout the experiment. In conclusion, this study demonstrates that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are unlikely to transmit this agent either to the recipients or the resulting offspring.

Lectins from bulbs of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae).

The isolation of three lectins with similar N-terminal amino acid sequences from the bulbs of the Chinese daffodil Narcissus tazetta was achieved. The isolation protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on mannose-agarose, and fast protein liquid chromatography-gel filtration on Superose 12. The lectins were all adsorbed on mannose-agarose and demonstrated a single band with a molecular weight of 13 kDa in SDS-polyacrylamide gel electrophoresis and a single 26 kDa peak in gel filtration, indicating that they were mannose-binding, dimeric proteins. The lectins differed in hemagglutinating activity, with the magnitude of the activity correlating with the ionic strength of the buffer required to elute the lectin from the DEAE-cellulose column. The bulb lectin did not exert potent cytotoxicity against cancer cell lines or fetal bovine lung cells but inhibited syncytium formation in, and reinstated viability of, fetal bovine lung cells infected with bovine immunodeficiency virus.

Viral contamination of embryos cryopreserved in liquid nitrogen.

Despite the worldwide application of embryo-freezing technology as the means of preserving germplasm of mammalian species, there is no information available on the possible transmission of infectious agents to cryopreserved embryos via contaminated liquid nitrogen (LN). Recently, it has been reported that new methods of cryopreservation which employ ultrarapid freezing or vitrification require direct contact between the freezing medium containing oocytes or embryos and liquid phase nitrogen (LPN). As models for human and animal viral pathogens three bovine viruses, bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV), and bovine immunodeficiency virus (BIV), were employed to study the potential for their transmission by experimentally contaminated LN to embryos frozen and stored in open freezing containers. Bovine embryos in a mixture of 20% ethylene glycol, 20% ME(2)SO, and 0.6% sucrose were vitrified in either unsealed standard 0.25 ml or modified open pulled straws or in plastic cryovials and then plunged into contaminated LPN. After 3-5 weeks of storage in LN, embryos were thawed and sequentially washed and only those with intact ZP were pooled together and tested in batches of three for viral contamination. From this pool of 83 batches, 13 of 61 (21.3%) batches exposed to BVDV and BHV-1 tested positive for viral association while all 22 batches exposed to BIV in unsealed containers tested negative. All control embryos vitrified in sealed cryovials and straws were free from viral contamination.

The antibody response of cattle infected with bovine immunodeficiency virus to peptides of the viral transmembrane protein.

The development of the antibody response to peptides of the transmembrane glycoprotein of bovine immunodeficiency virus (BIV) was followed over a period of 50 weeks in six cattle experimentally infected with the BIV(FL112) isolate. Antibody was detected by an enzyme immunoassay using either a linear or a cyclized peptide with structural features common to an immunodominant region of other lentiviruses. The assay was specific for BIV, detecting antibody in bovine sera to BIV(FL112) or BIV(R29) but not to six other common viruses of cattle. Antibody was present in the sera of all cattle inoculated with BIV(FL112) within 4 weeks of infection, peaked between 10 and 30 weeks and persisted in most cattle during the 50 weeks of observation. These features indicate that this assay may be useful in identifying cattle infected with other strains of BIV in the field.