Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents

Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (He Ne ) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 10(6) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/ml) followed by He Ne or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.     

Viremia and virus shedding in elk infected with type 1 and virulent type 2 bovine viral diarrhea virus

In order to determine whether elk (Cervus elaphus) could be infected with and shed bovine viral diarrhea virus (BVDV) and to determine whether BVDV could cause disease in elk, two groups of five yearling elk each and two control cattle were experimentally inoculated intranasally with type 1 Singer strain or a virulent type 2 isolate of BVDV, strain 24515. Virulence of the type 2 isolate was confirmed by inoculation of a control bovine cow which developed diarrhea, dehydration, severe thrombocytopenia, hemorrhages, and enteritis with intestinal necrosis. None of the elk inoculated with type 1 or type 2 BVDV developed clinical signs of illness. However, all elk became infected as demonstrated by viremia, nasal shedding, and/or seroconversion. One uninoculated, in-contact elk contracted type 1 BVDV and seroconverted. Thus, although BVDV does not appear capable of producing disease in nonpregnant elk, the species is susceptible to infection and can shed and transmit BVDV.     

E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7). It has been shown that cleavage between E2 and p7 is incomplete, resulting in proteins E2-p7, E2, and p7. We found no precursor-product relationship between E2-p7 and E2, which indicates a stable nature of E2-p7. To study the function of the E2-p7 region of the polyprotein, mutations were introduced into an infectious cDNA of bovine viral diarrhea virus (BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletion of p7. To prevent synthesis of E2-p7, a translational stop codon was introduced after the last codon of the E2 gene and an internal ribosome entry site element followed by a signal peptide coding sequence was inserted upstream of the p7 gene. Transfection of RNA transcribed from the bicistronic construct led to the release of infectious virus particles. Thus, synthesis of E2-p7 is not essential for the generation of infectious virions. Cell lines constitutively expressing BVDV p7 and/or E2 were generated for complementation studies. Transfection of BVDV RNAs with point mutations or a deletion in the E2-p7 region into the complementing cell lines led to the generation of infectious virions. According to our studies, p7 as well as E2 can be complemented in trans.     

The effect of infection with bovine viral diarrhea virus on the fertility of Swiss dairy cattle

Bovine viral diarrhea virus is a major cattle pathogen with a worldwide distribution. Animals may be infected with BVD virus transiently or persistently. Transient infection leads to protective immunity. Persistent infection is unique because it is associated with an immunotolerance that is specific to the infecting strain of BVD virus. Persistent infection results from viral invasion of fetuses between the second and fourth month of development. Such animals are of prime importance in the epidemiology of BVD because they shed large amounts of virus, and thus serve as a constant source of infection for non-immune animals. Infection of pregnant animals during the first two months of gestation may result in an increased rate of return to estrus. Animals infected in the period of five months to birth may abort or give birth to calves with malformations. The effects of BVD virus on fertility and gestation are well documented from experimental infection. However, much less is known of the extent of losses under field conditions. The main reason for this may be the multitude of other causes of increased return rates and gestation failures. In addition, the incidence of infection with BVD virus may vary over time and depends on management practices. In this study, we investigated the impact of BVD virus on gestation failures under field conditions in a large cattle-breeding area of Switzerland, where no specific measures to control BVD are in effect. Our approach consisted of relating seroconversions to BVD virus with the rate of return to estrus, abortion, and birth of calves with apparent malformations. These parameters of fertility were compared to those of animals immune to BVDV infection due to previous exposure to the virus and animals without seroconversion. Our data show that infection with BVD virus during the first 45 days of gestation did not influence the rate of return to estrus. By contrast, we observed a statistically significant increase in the abortion rate in mid-term gestation (Days 46 to 210) while no such effect was observed in the later stages of gestation. No clinically manifest malformations were observed in the offspring of animals that had seroconverted to BVD virus. In our study population the prevalence of BVDV antibody positive cattle varied only slightly between 78% and 80% over the period of observation. Our data showed that 7% (CI: 2.4-14%) of fetal deaths may be attributable to infection with BVD virus.     

Fusion to C3d enhances the immunogenicity of the E2 glycoprotein of type 2 bovine viral diarrhea virus

The use of DNA and protein subunit vaccines in animals provides an opportunity to introduce vaccines that are arguably the safest that can be developed. For that reason, considerable effort is under way to devise methods of enhancing the immunogenicity of such vaccines. Seven years ago it was shown that fusing complement fragment C3d to hen egg lysozyme (HEL) enhanced the immunogenicity of HEL 10,000-fold. Based on this observation, we decided to evaluate the effect of C3d on the immunogenicity of the E2 protein of bovine viral diarrhea virus (BVDV). E2 is the major target of neutralizing antibody during BVDV infection. To test the effect of C3d on E2 immunogenicity, expression cassettes encoding a secreted form of E2 alone (E2s) or E2 fused to three copies of murine C3d (E2s-C3d) were constructed. The proteins were purified from the supernatants of transfected cells and used to immunize mice. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for E2s-specific antibody and by a virus neutralization test. The ELISA results indicated that the E2s-C3d protein is 10,000-fold more immunogenic than the E2s protein alone. The maximum primary immune response was elicited with <0.1 microg of E2s-C3d protein without an adjuvant. In addition, we have shown for the first time that high levels of anti-E2s and neutralizing antibodies can be elicited when this same low concentration of E2s-C3d is used to both prime and boost the immune response. We conclude that the E2s-C3d fusion protein has significant potential as a subunit vaccine against BVDV infection.     

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.     

Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.     

Expression of unmodified hepatitis C virus envelope glycoprotein-coding sequences leads to cryptic intron excision and cell surface expression of E1/E2 heterodimers comprising full-length and partially deleted E1

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates exclusively in the cytoplasm of infected cells. The viral envelope glycoproteins, E1 and E2, appear to be retained in the endoplasmic reticulum, where viral budding is thought to occur. Surprisingly, we found that the expression system used to generate HCV envelope glycoproteins influences their subcellular localization and processing. These findings have important implications for optimizing novel HCV fusion and entry assays as well as for budding and virus particle formation.     

Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1

The objective was to determine the effect of cryopreservation by conventional slow controlled cooling (0.5 °C/min) and by vitrification on the presence of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) infectivity associated with frozen-thawed Day 7 bovine embryos. In this study, Day 7 embryos generated by in vitro fertilization (IVF) were exposed in vitro for 1.5 h to BVDV (N = 393) and BHV-1 (N = 242) and subsequently tested before and after cryopreservation for the presence of infectivity. Exposure of embryos to viral agents resulted in 72% of them infected prior to cryopreservation. Stepwise exposure of embryos to cryoprotectants, as well as their removal, substantially reduced the proportion of contaminated embryos (46% vs. 72%, P < 0.05). Overall, both freezing methods reduced the percentage of infectious embryos compared with that of embryos similarly exposed to viruses but not cryopreserved (31% vs. 72%, respectively; P < 0.001). The percentage of embryos with infectious viruses was not significantly higher after vitrification than after slow cooling (38% vs. 22%). In addition, after cryopreservation, a higher percentage (P < 0.002) of embryos exposed to BHV-1 (42%) remained infectious than did embryos exposed to BVDV (24%). In conclusion, cryopreservation reduced the proportion of infected embryos but did not render all of them free from infectious pathogens.     

Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.     

An outbreak of fetal and neonatal losses associated with the diagnosis of bovine viral diarrhea virus

A severe outbreak of dairy herd pregnancy wastage was investigated. At the beginning of the outbreak, a total of 121 lactating cattle were pregnant and considered to be at risk. Overall, 33.1% of the population at risk aborted, while 25.6% gave birth to calves that either died during the early neonatal period or demonstrated signs compatible with congenital defects (abnormal births). A laboratory diagnosis of bovine viral diarrhea virus (BVDV) infection was made in two surviving neonatal calves with symptoms of cerebellar hypoplasia and blindness. An on-farm investigation was conducted to determine if the abortions and abnormal births were associated with BVDV infection. The rate of abortions versus abnormal births was biphasic when graphed by the date of occurrence. The cases of abortion occurred early in the outbreak and were followed by the neonatal losses. Within the population at risk, the mean values for gestational age at the beginning of the outbreak were different between the subpopulations described by gestational outcome. The outcome of each pregnancy that existed at the beginning of the outbreak was determined. Classifications included normal birth (birth of a normal calf), abnormal birth (a neonatal loss of the type described above), abortion, and continued gestation (normal, uncompleted pregnancy). The average gestational age at the time of the index case (the first cases of pregnancy wastage) for these four pregnancy outcome classfications was 142.0, 106.2, 86.7 and 31.3 days, respectively. Reasons for assuming that this outbreak was related to BVDV are discussed.     

Structure and interactions at the viral surface of the envelope protein E1 of Semliki Forest virus

Semliki Forest virus (SFV) is enveloped by a lipid bilayer enclosed within a glycoprotein cage made by glycoproteins E1 and E2. E1 is responsible for inducing membrane fusion, triggered by exposure to the acidic environment of the endosomes. Acidic pH induces E1/E2 dissociation, allowing E1 to interact with the target membrane, and, at the same time, to rearrange into E1 homotrimers that drive the membrane fusion reaction. We previously reported a preliminary Calpha trace of the monomeric E1 glycoprotein ectodomain and its organization on the virus particle. We also reported the 3.3 A structure of the trimeric, fusogenic conformation of E1. Here, we report the crystal structure of monomeric E1 refined to 3 A resolution and describe the amino acids involved in contacts in the virion. These results identify the major determinants for the E1/E2 icosahedral shell formation and open the way to rational mutagenesis approaches to shed light on SFV assembly.     

Reproductive and economic impact following controlled introduction of cattle persistently infected with bovine viral diarrhea virus into a naive group of heifers

The reproductive impact following controlled introduction of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) was evaluated in BVDV-naive heifers. Heifers were randomly allocated into two groups: an unexposed control herd (n = 34) and a herd exposed to five persistently infected (PI) animals for 7 mo, beginning 50 days before the breeding season (n = 34). Initiation of the BVDV-challenge was timed to mimic either direct contact with PI calves born in the previous calving season or accidental introduction of PI herd additions prior to the breeding season. The PI animals represented BVDV Types 1a (n = 3), 1b (n = 1) and 2 (n = 1). Two BVDV-free, seropositive bulls were used in each group for 78 days breeding seasons. In both groups, 33 of 34 heifers became pregnant, with similar distribution of fetal ages. Two heifers in each group aborted (etiology undetermined). In addition, one calf was born dead and one calf died 3 days post-partum in the BVDV-exposed group. One calf in the unexposed group died 4 mo post-partum. No calves, including the stillborn calf and the two calves that died prior to weaning, were persistently infected with BVDV. In summary, introduction of PI cattle to a group of BVDV-naive heifers 50 days prior to the breeding season did not negatively impact reproductive performance. To the contrary, the active immunity that developed following field exposure to BVDV provided effective reproductive and fetal protection during the breeding season and subsequent gestations, despite continuous exposure to PI animals until approximately midgestation. Although BVDV can have potentially devastating reproductive effects, timing of infection is a critical determinant in the outcome of a BVDV infection. A controlled breeding season with introduction of herd additions at less critical reproductive time points can mitigate the negative reproductive health consequences of BVDV.     

Cleavage of the NS2-3 protein in the cells of cattle persistently infected with non-cytopathogenic bovine viral diarrhea virus

The NS2-3 of BVDV is cleaved in cultured cells infected with cp BVDV but not in those infected with ncp BVDV when tested more than 10 hours post infection. However, it is not known whether cleavage of NS2-3 occurs in vivo. In the present study, cleavage of NS2-3 in cattle persistently infected with BVDV was investigated. All BVDV isolated from PI animals were of the ncp biotype, and NS2-3 proteins were detected in bovine fetal muscular cells infected with these viruses. On the other hand, in the leukocytes of those PI animals, NS3 proteins, products of the cleavage of NS2-3 proteins, were detected. In addition, the NS3 proteins were also detected in leukocytes artificially infected with ncp BVDV. These results reveal that the NS2-3 protein of BVDV is cleaved in leukocytes. Furthermore, NS3 proteins were detected in many tissues of PI cattle, such as lymphoid tissue, brain, thyroid, lung, and kidney. These results suggest that the NS2-3 protein of ncp BVDV cleaves in vivo.     

Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity

The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).