Bovine maternal, fetal and neonatal responses to bovine viral diarrhea virus infections.

Due to the affinity of BVDV for the fetus and for cells of lymphatic organs of infected cattle, reproductive failure or immunosuppression, respectively, are likely consequences of BVDV infections of susceptible cattle. Infection of susceptible pregnant cattle with noncytopathic (ncp) BVDV results in transplacental infection with induction of maternal and fetal innate and adaptive immune responses. Differences in maternal innate and adaptive immune responses are evident in late gestation between cows carrying fetuses persistently-infected (PI) with BVDV and cows with fetuses transiently-infected with BVDV. Fetal innate and adaptive immune responses to ncp BVDV infection are defined by fetal age and developmental stage of the fetal immune system. Since a functional fetal adaptive immune response does not occur in the early fetus, immunotolerance to ncp BVDV is established, virus replicates unrestricted in fetal tissues and calves are born immunotolerant and PI with the virus. In the last trimester of gestation, the fetal immune system is adequately developed to respond in an efficacious manner, most commonly resulting in the birth of a clinically normal calf with pre-colostral antibodies. Immunosuppression due to postnatal acute ncp BVDV infections of susceptible calves may contribute to the occurrence and severity of multi-factorial respiratory tract and enteric diseases.

Gastrointestinal parasites of a reintroduced semi-wild plains bison (Bison bison bison) herd: Examining effects of demographic variation, deworming treatments, and management strategy.

Bison (Bison spp) are being reintroduced into semi-wild, spatially constrained herds across North America and Europe. Herd managers are concerned about gastrointestinal (GI) nematode parasites as they care for the health of their bison. We examine how demographics, grazing location, herd management, and anthelmintic treatments affect the fecal egg counts (FECs) of GI nematodes within a reintroduced Plains bison (Bison bison bison) herd in the Great Plains. Our results suggest that younger bison (<2 years of age) experience higher GI parasite eggs/oocysts per gram (epg/opg) and that some taxa are more prevalent throughout different periods of a bison's early years. Demographic findings suggest that calf and yearling (0-2 yrs age) bison have the highest FECs and that these decline until reaching a low in peak adulthood and thereafter (x > 6 yrs of age). FECs of both Trichuris spp. and particularly Nematodirus spp. were much more abundant, relatively, during the first year of a bison's life. This pattern was also true of Moniezia spp. and Eimeria spp., however, strongyle-type spp. FECs appeared to peak in relative abundance during the second year of life. Our data also indicate that FECs are influenced by differences in land-use histories of pastures previously grazed by cattle or by the proportion of frequent flooding in different pastures. Treatment results suggest that fenbendazole may more effective than moxidectin at lowering FECs of bison over the long-term, and lasting effects of at least one administered anthelmintic treatment. Multiplex PCR assays revealed that American bison share GI nematodes with cattle including: Ostertagia spp., Haemonchus placei, Cooperia onchophora, and Oesophagostomum spp, but did not detect the presence Trichostrongylus columbriformis. Our results may have wider conservation implications for reintroduction efforts of American bison, as well as the endangered European bison (Bison bonasus).

Effect of the viral protein N(pro) on virulence of bovine viral diarrhea virus and induction of interferon type I in calves.

OBJECTIVE: To characterize the influence of the viral protein N(pro) on virulence of bovine viral diarrhea virus (BVDV) and on type I interferon responses in calves. ANIMALS: 10 calves, 4 to 6 months of age. PROCEDURES: BVDV virulence and type I interferon responses of calves (n = 5) infected with a noncytopathic BVDV with a deleted N(pro) were compared with those of calves (5) infected with a noncytopathic BVDV with a functional N(pro). Rectal temperatures, clinical signs, platelet counts, and total and differential WBC counts were evaluted daily. Histologic examinations and immunohistochemical analyses of tissues were conducted to assess lesions and distribution of viral antigens, respectively. Serum type I interferon concentrations were determined. RESULTS: Calves infected with N(pro)-deleted BVDV developed leukopenia and lymphopenia, without developing increased rectal temperatures or lymphoid depletion of target lymphoid organs. There was minimal antigen deposition in lymphoid organs. Calves infected with N(pro) BVDV developed increased rectal temperatures, leukopenia, lymphopenia, and lymphoid depletion with marked BVDV antigen deposition in lymphatic tissues. Interferon type I responses were detected in both groups of calves. CONCLUSIONS AND CLINICAL RELEVANCE: Deletion of N(pro) resulted in attenuation of BVDV as evidenced by reduced virulence in calves, compared with BVDV with a functional N(pro). Deletion of N(pro) did not affect induction of type I interferon. The N(pro)-deleted BVDV mutant may represent a safe noncytopathic virus candidate for vaccine development.

Prevalence of bovine viral diarrhea virus infections in alpacas in the United States.

OBJECTIVE: To determine the prevalence of bovine viral diarrhea virus (BVDV)-infected alpaca herds in the United States and investigate factors associated with seropositive herd status and, subsequently, determine the proportion of animals within seropositive alpaca herds that are persistently infected (PI) carriers for BVDV, obtain information regarding previous herd exposure to BVDV, determine titers of anti-BVDV antibodies of dams, and ascertain whether individual seropositive crias had received supplemental colostrum at birth. DESIGN: Prevalence study. ANIMALS: 63 alpaca herds with >or= 12 registered female alpacas. PROCEDURES: 250 alpaca breeders were randomly selected from 562 eligible herds listed in the Alpaca Owner and Breeders Association membership directory and mailed a voluntary participation request. Sixty-three alpaca breeders participated in the study. From each herd, blood samples from >or= 4 crias were tested for BVDV, BVDV RNA, and serum neutralizing antibodies against BVDV. A region of the genome of BVDV recovered from PI crias was sequenced to determine genetic homology. RESULTS: Among the 63 herds, 16 (25.4%) had seropositive crias and 4 (6.3%) had PI crias. Infections in 3 of the 4 herds with PI crias were linked as evidence by the genetic homologies of viruses. In addition to PI crias, feeding supplemental colostrum was associated with herd seropositivity. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the importance of BVDV infections in alpacas in the United States and highlighted the importance of determining the BVDV infection status of animals before they are commingled to limit exposure of herds to BVDV infection.

Immune evasion by pathogens of bovine respiratory disease complex.

Bovine respiratory tract disease is a multi-factorial disease complex involving several viruses and bacteria. Viruses that play prominent roles in causing the bovine respiratory disease complex include bovine herpesvirus-1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parinfluenza-3 virus. Bacteria that play prominent roles in this disease complex are Mannheimia haemolytica and Mycoplasma bovis. Other bacteria that infect the bovine respiratory tract of cattle are Histophilus (Haemophilus) somni and Pasteurella multocida. Frequently, severe respiratory tract disease in cattle is associated with concurrent infections of these pathogens. Like other pathogens, the viral and bacterial pathogens of this disease complex have co-evolved with their hosts over millions of years. As much as the hosts have diversified and fine-tuned the components of their immune system, the pathogens have also evolved diverse and sophisticated strategies to evade the host immune responses. These pathogens have developed intricate mechanisms to thwart both the innate and adaptive arms of the immune responses of their hosts. This review presents an overview of the strategies by which the pathogens suppress host immune responses, as well as the strategies by which the pathogens modify themselves or their locations in the host to evade host immune responses. These immune evasion strategies likely contribute to the failure of currently-available vaccines to provide complete protection to cattle against these pathogens.

Characterization of protection against systemic infection and disease from experimental bovine viral diarrhea virus type 2 infection by use of a modified-live noncytopathic type 1 vaccine in calves.

OBJECTIVE: To evaluate protection resulting from use of a modified-live noncytopathic bovine viral diarrhea virus (BVDV) type 1 vaccine against systemic infection and clinical disease in calves challenged with type 2 BVDV. ANIMALS: 10 calves, 5 to 7 months of age. PROCEDURES: Calves were allocated (n = 5/group) to be nonvaccinated or vaccinated SC on day 0 with BVDV 1 (WRL strain). Calves in both groups were challenged intranasally with BVDV type 2 isolate 890 on day 21. Rectal temperatures and clinical signs of disease were recorded daily, and total and differential WBC and platelet counts were performed. Histologic examinations and immunohistochemical analyses to detect lesions and distribution of viral antigens, respectively, were performed. RESULTS: After challenge exposure to BVDV type 2, nonvaccinated calves developed high rectal temperatures, increased respiratory rates, viremia, leukopenia, lymphopenia, and infection of the thymus. Vaccinated calves did not develop high rectal temperatures or clinical signs of respiratory tract disease. Vaccinated calves appeared to be protected against systemic replication of virus in that they did not develop leukopenia, lymphopenia, viremia, or infection of target organs, and infectious virus was not detected in peripheral blood mononuclear cells or the thymus. CONCLUSIONS AND CLINICAL RELEVANCE: The modified-live BVDV type 1 vaccine protected against systemic infection and disease after experimental challenge exposure with BVDV type 2. The vaccine protected calves against infection and viremia and prevented infection of target lymphoid cells.

Type 2 BVDV Npro suppresses IFN-1 pathway signaling in bovine cells and augments BRSV replication.

Bovine viral diarrhea virus (BVDV) infection induces immunosuppression and in conjunction with bovine respiratory syncytial virus (BRSV) contributes to the bovine respiratory disease complex. Bovine turbinate cells were single or co-infected with type 2 BVDV wild-type (BVDV2-wt), its dysfunctional Npro mutant (BVDV2-E), and/or BRSV. BVDV2-E significantly up-regulated PKR, IRF-7, TBK-1, IRF-3, and IFN-ß mRNAs based on real-time Q-RT-PCR. BRSV-infected cells expressed significantly up-regulated PKR, IRF-3, IRF-7, and IFN-ß mRNAs, whereas BVDV2-wt, but not BVDV2-E, abolished this up-regulation in co-infection. No significant differences were observed in MAVS, NF-κB, and PIN-1 mRNAs. A dual-luciferase reporter assay showed that BVDV2-wt significantly increased NF-κB activity compared to BVDV2-E, while BVDV2-E significantly increased IFN-ß activity compared to BVDV2-wt. The BRSV titer and RNA levels significantly increased in cells co-infected with BRSV/BVDV2-wt compared to cells co-infected with BRSV/BVDV2-E or infected with BRSV alone. This data supports the synergistic action of BVDV2-wt and BRSV inhibition of IFN-1.

Experimental acute infection of alpacas with Bovine viral diarrhea virus 1 subgenotype b alters peripheral blood and GALT leukocyte subsets.

Bovine viral diarrhea virus (BVDV) is a pathogen in cattle and alpacas ( Vicugna pacos), causing acute and persistent BVDV infections. We characterized the effect of acute BVDV infection on the immune system of alpacas by determining lymphocyte subpopulations in peripheral blood and gut-associated lymphoid tissues (GALT) as well as serum interferon levels. Alpacas were experimentally infected with BVDV-1b (strain CO-06). Peripheral blood leukocytes were isolated at 0, 3, 6, and 9 d postinfection (dpi), and leukocytes of GALT at 9 dpi, and evaluated using flow cytometry. Serum interferon levels were determined daily. Flow cytometric analyses of peripheral blood leukocytes showed a significant decrease in CD4+, CD8+, and αß T-lymphocytes at 3 dpi. CD8+ lymphocytes were significantly increased, and activated lymphocytes were significantly decreased in the C3-stomach region in BVDV-infected alpacas. Serum interferon concentrations significantly increased in BVDV-infected alpacas at 3-6 dpi, peaking at 3 dpi. Our study confirms that BVDV can be a primary acute pathogen in alpacas and that it induces an interferon response and alters leukocyte subset populations. The changes in the proportion of T-lymphocytes during the early stages of BVDV infection may result in transient immunosuppression that may contribute to secondary bacterial and viral infections, similar to cattle.

Characterization of protection from systemic infection and disease by use of a modified-live noncytopathic bovine viral diarrhea virus type 1 vaccine in experimentally infected calves.

OBJECTIVE: To evaluate protection against systemic infection and clinical disease provided by use of a modified-live noncytopathic bovine viral diarrhea virus (BVDV) type 1 vaccine in calves challenged with NY-1 BVDV. ANIMALS: 10 calves, 5 to 7 months of age. PROCEDURES: Calves were allocated (n = 5/group) to be nonvaccinated or vaccinated SC on day 0 with BVDV type 1 (WRL strain). Calves in both groups were challenged intranasally with NY-1 BVDV on day 21. Calves' rectal temperatures and clinical signs of disease were recorded daily, total and differential WBC and platelet counts were performed, and serum neutralizing antibody titers against NY-1 BVDV were determined. Histologic examinations and immunohistochemical analyses to detect gross lesions and distribution of viral antigens, respectively, were performed. RESULTS: After challenge exposure to NY-1 BVDV, nonvaccinated calves developed high rectal temperatures, increased respiratory rates, viremia, leukopenia, lymphopenia, and infection of the thymus. Vaccinated calves did not develop high rectal temperatures or clinical signs of respiratory tract disease. Vaccinated calves appeared to be protected against systemic replication of virus in that they did not develop leukopenia, lymphopenia, viremia, or infection of target organs, and infectious virus was not detected in peripheral blood mononuclear cells or the thymus. CONCLUSIONS AND CLINICAL RELEVANCE: The modified-live BVDV vaccine protected calves against systemic infection and disease after experimental challenge exposure with NY-1 BVDV. The vaccine protected calves against infection and viremia and prevented infection of target lymphoid cells.

Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays.

A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad's iCycler iQ. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovine turbinate cell lysate harvested at eight time points (1.5, 6, 12, 24, 36, 48, 60, 72 h) post-infection. A homologous BRSV cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. Detection as low as 171 copies/microl of standard BRSV cRNA was possible. For QC-RT-PCR, a competitor RNA molecule having a deletion was designed and used for quantitation of the BRSV viral mRNA. The results of real-time Q-RT-PCR and QC-RT-PCR assays showed a positive correlation. Real-time Q-RT-PCR was a sensitive, specific, rapid, and efficient method that eliminates the post-PCR processing steps when compared to QC-RT-PCR. Quantitation of BRSV using real-time Q-RT-PCR will have application in studies aimed at understanding the pathogenesis of BRSV.

Effect of infection with bovine viral diarrhea virus alone, bovine rotavirus alone, or concurrent infection with both on enteric disease in gnotobiotic neonatal calves.

OBJECTIVE: To compare experimentally induced concurrent infection with bovine viral diarrhea virus (BVDV) and bovine rotavirus (BRV) with infection of either virus alone in calves. ANIMALS: Seventeen 1-day-old gnotobiotic calves. PROCEDURE: Calves were allotted to 8 treatments as follows: group 1, mock-infected control calves (n = 2); group 2, inoculated with BVDV on day 1 (2); groups 3, 5, and 7, inoculated with BRV on days 1 (2), 4 (1), or 7 (2), respectively; and groups 4, 6, and 8, inoculated with BVDV on day 1 and with BRV on days 1 (2), 4 (2), or 7 (4), respectively. Concentrations of BVDV in serum and ileal tissues were measured, and BRV shedding in feces was determined. Histologic examination and immunohistochemical analysis were conducted to detect lesions and viral antigens. RESULTS: Neonatal calves inoculated with BVDV alone or with BVDV on day 1 and BRV on day 7 developed villus atrophy and submucosal inflammation of the intestines. Concurrent BVDV and BRV infections acted synergistically in the intestinal tract, causing more severe enteric disease than infection with either virus alone. Severe lymphoid depletion was associated with BVDV infection in calves regardlesss of concurrent BRV infection. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with BVDV played direct and indirect roles in enteritis in neonatal calves, causing villus atrophy in the duodenum and submucosal inflammation of the intestines. Also, BVDV potentiated effects of BRV. Concurrent infection with BVDV and BRV resulted in more severe enteric disease in neonatal calves than infection with BRV or BVDV alone.

Comparative virulence of isolates of bovine viral diarrhea virus type II in experimentally inoculated six- to nine-month-old calves.

OBJECTIVE: To determine the comparative virulence of 5 isolates of bovine viral diarrhea virus (BVDV) type II by inoculating 6- to 9-month-old beef calves with isolates originating from the tissues of cattle affected with naturally occurring, transient, acute, nonfatal infections or naturally occurring, peracute, fatal infections. ANIMALS: 22 calves that were 6 to 9 months old. PROCEDURE: The study used BVDV isolates 17011, 713, and 5521 that originated from fetuses aborted from cows with transient, nonfatal, acute BVDV infections and isolates 23025 and 17583 that originated from the tissues of cattle with peracute, fatal BVDV infections. Calves were allotted to 6 groups (1, mock-infected control calves [n = 2]; 2, inoculated with BVDV 17011 [4]; 3, inoculated with BVDV 713 [4]; 4, inoculated with BVDV 5521 [4]; 5, inoculated with BVDV 23025 [4]; and 6, inoculated with BVDV 17583 [41]. Rectal temperatures and clinical signs of disease were recorded daily. Total and differential WBC and platelet counts were performed. Histologic examination and immunohistochemical analysis were conducted to detect lesions and distribution of viral antigens, respectively. RESULTS: Calves inoculated with BVDV 23025 or 17583 developed more severe clinical signs of disease (fever and diarrhea), more severe lymphopenia, and more severe lesions (alimentary epithelial necrosis, lymphoid depletion, and BVDV antigen deposition in lymphatic tissues), compared with calves inoculated with BVDV 713, 5521, or 17011. CONCLUSIONS AND CLINICAL RELEVANCE: Relative severity of experimentally induced infections corresponded to severity of clinical signs of naturally occurring infections with respective BVDV isolates.

Evolution of bovine viral diarrhea virus vaccines.

Control of bovine viral diarrhea virus (BVDV) infection is economically important to the cattle industry because the virus causes a variety of clinical diseases that adversely affect essentially all stages of the production cycle. Production losses primarily stem from reproductive failure and from immunosuppression during acute BVDV infection, which predisposes calves to respiratory or enteric diseases. Control is achieved by implementing herd health pro-grams focused on limiting exposure by avoiding persistently infected (PI) carrier cattle and by optimizing protective immunity through immunization. Vaccination cannot be relied upon solely to protect against fetal infection and losses due to BVD. This is because no single BVDV vaccine has been shown to give complete fetal protection. In addition to strategic use of vaccines, herd management practices should also be implemented to identify and eliminate PI carrier cattle and to avoid exposure to BVDV infection.

Distribution of lymphoid depletion and viral antigen in alpacas experimentally infected with Bovine viral diarrhea virus 1.

It was hypothesized that acute postnatal Bovine viral diarrhea virus 1 (BVDV-1) infection leads to leukopenia and lymphoid depletion of gut-associated lymphoid tissues similar to acute disease in calves. The objectives of the current study were to characterize the pathologic effects, viremia, viral shedding, and viral antigen deposition in 6-24-month-old, acutely infected alpacas following experimental infection with noncytopathic BVDV-1 subgenotype 1b (BVDV C0-6). The BVDV-1 isolate was obtained from a cria with naturally occurring persistent infection. Lymphocytopenia occurred 3-7 days postinfection, with a 50% reduction in peripheral lymphocytes in infected alpacas. Depletion of B-cell populations in gut-associated lymphoid tissues was evident microscopically. Populations of T cells in parafollicular zones and in nodular aggregates along the superficial submucosa remained intact. The BVDV antigen was deposited most consistently in submucosal gastrointestinal aggregated lymphoid tissues of ileum, proximal colon, and stomach compartment three. Viral antigen was more variably evident in other lymphoid tissues. Antigen distribution correlated well with histologic lesions in gastrointestinal aggregated lymphoid tissues, confirming the role of virus in lymphoid depletion. Nasal shedding was detected in all challenged alpacas on day 6 and in 4 out of 12 challenged alpacas on day 9. Viremia was present as early as day 3, and present in all challenged alpacas on days 5, 6, 7, and 9 postchallenge. Lymphocytopenia and depletion of gastrointestinal aggregated lymphoid tissues associated with acute BVDV-1 infection likely results in immune compromise and is expected to exacerbate concurrent infections even though uncomplicated BVDV-1 infection was clinically unapparent.

Comparison of viral replication and IFN response in alpaca and bovine cells following bovine viral diarrhea virus infection.

Alpacas develop diminished disease following bovine viral diarrhea virus (BVDV) infection compared to cattle. We hypothesized that alpaca and bovine cells have differential permissiveness and responses to BVDV infection. To characterize alpaca testicular (AT) and bovine turbinate (BT) cells BVDV infection permissiveness, viral replication and interferon (IFN) synthesis was evaluated. BVDV replicated 3-4 logs lower in AT cells with diminished antigen deposition compared to BT cells. BVDV infection inhibited IFN response in both AT and BT cells. Compared to BT cells, BVDV-infected AT cells had a 2-5 fold increase in IFN synthesis following dsRNA stimulation. The greater IFN response of AT cells compared to BT cells following poly I:C stimulation with or without ncp BVDV infection, may be the basis for the decreased BVDV permissiveness of AT cells and may contribute to the clinical differences following BVDV infection of alpacas and cattle.

Bighorn sheep fetal lung cell line for detection of respiratory viruses.

Pneumonia is an important disease of bighorn sheep (BHS) that is primarily responsible for the drastic decline in numbers of these animals in North America. Members of the genus Mannheimia and Pasteurella have frequently been isolated from the pneumonic lungs of BHS. Antibodies to several respiratory viruses, including bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BoHV-1), have been detected in herds of BHS. The availability of BHS fetal lung cell lines is likely to enhance the chances of isolation of these viruses. Here we report the development of such a cell line. This line is permissive for BPIV-3, BRSV, BVDV, and BoHV-1 infection, as revealed by an enzyme immunoassay of virus-infected cells with antibodies specific for each of these viruses. This cell line should be valuable for detecting these 4, and possibly other, respiratory viruses in BHS.

Ethidium monoazide does not inhibit RT-PCR amplification of nonviable avian influenza RNA.

A critical obstacle to using PCR to quantify viral titers is the distinguishment of viable and nonviable genomic material. Pretreatments of ethidium monoazide (EMA) have been effective in preventing PCR amplification of DNA from nonviable bacteria. To test whether an EMA pretreatment could be used with RT-PCR to quantify viable RNA virions, avian influenza virus (AIV) survival was measured in water through 28d using cell culture titration and real-time RT-PCR with or without EMA pretreatment. Cell culture titration yielded significantly lower titers than both RT-PCR procedures, and there was no significant difference between RT-PCR results with or without EMA. Ineffective binding of EMA to AIV RNA may have allowed nonviable AIV RNA to amplify. Furthermore, since AIV inactivation may take place by means other than membrane disruption, any pretreatment distinguishing viable and nonviable AIV virions by membrane integrity may not be practical.

Survival of the avian influenza virus (H6N2) after land disposal.

An integral component in preventing an avian influenza pandemic is containment and disposal of infected bird (poultry) carcasses. Disposal of carcasses in Subtitle D municipal solid waste (MSW) landfills may be an advantageous option due to their large capacities and facility distribution in the U.S. In this study, the survival of H6N2 avian influenza virus (AIV) was measured in a methanogenic landfill leachate and water as a function of temperature, conductivity, and pH. Elevated temperature and nonneutral pH resulted in the quickest inactivation times for AIV in both media, whereas conductivity did not have a significant influence on AIV survival. Media effects were significant and AIV inactivation in leachate was consistently the same or faster than AIV inactivation in water. Based on an initial titer of 10(5) TCID50/mL, calculated inactivation times ranged from 30 days to greater than 600 days, indicating that AIV will remain infectious during and after waste disposal. Disposal of infected carcasses in a MSW landfill may be an appropriate option as inactivation times are within the design life of required barrier systems at Subtitle D landfills.

A replicon trans-packaging system reveals the requirement of nonstructural proteins for the assembly of bovine viral diarrhea virus (BVDV) virion.

A selective trans-packaging system was developed to produce and isolate bovine viral diarrhea virus (BVDV) pseudo-particles with complementing reporter replicons and their packaging proteins expressed in trans with recombinant vaccinia virus. The encapsidation of replicon rNS3-5B was dependent not only on the in trans expression of structural proteins C, E(rns), E1 and E2, but also the nonstructural proteins, p7 and contiguous precursor NS2-3-4A. Nonstructural p7, NS4B, NS5A or NS5B could be expressed in cis and in trans with precursor NS2-3-4A without significantly affecting virion assembly efficiency. NS2-3-4A was identified as an in trans functional precursor in virion assembly. BVDV genomes with mutant NS5B, which did not undergo active replication, were packaged 5-fold less efficiently than the intact genomes demonstrating the importance of replication in virion packaging. These results suggest that genome replication and assembly are closely associated, consistent with a model in which these two steps are coupled for maximum efficiency.

Influence of bovine respiratory syncytial virus F glycoprotein N-linked glycans on in vitro expression and on antibody responses in BALB/c mice.

Bovine respiratory syncytial virus (BRSV) is an etiological component of the bovine respiratory tract disease complex. Infection with BRSV following vaccination, or re-infection following natural infection is common since protection is incomplete. The objectives of this study were to create plasmid DNA constructs encoding single or multiple N-glycosylation-site deletion BRSV fusion (F) proteins, and evaluate their expression in cell culture, and potential to induce anti-BRSV F antibody responses in BALB/c mice. Four plasmid DNAs were constructed, each encoding 1-4 N-glycosylation-site deletions: Gly4, Gly2/4, Gly1/2/4 and Gly1/2/3/4. Each of the N-glycosylation-site deletion BRSV F proteins were expressed in COS-7 cells following transfection with plasmid DNA. Inoculation of BALB/c mice with plasmid DNA, resulted in a significant anti-BRSV F IgG response to the wild-type (WT) F and glycosylation-site deletion protein Gly2/4. Gly2/4 elicited a higher antibody titer than the fully glycosylated WT F protein. Significant neutralizing antibody titers were detected following immunization with the Gly2/4 plasmid DNA. These glycosylation-site deletion BRSV F proteins will be useful to characterize the effects of glycosylation on immunogenicity in the natural host, and may lead to a new approach for the generation of BRSV vaccines.