Cell Culture Infectious Dose Research Articles

Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay

In this study, a rapid quantitative method using TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) has been developed for estimating the titers of measles, mumps and rubella (MMR) viruses in infected cell culture supernatants. The qPCR-RT assay was demonstrated to be a specific, sensitive, efficient and reproducible method. For MMR viral samples obtained during MMR viral propagations in Vero cells at a different multiplicity of infection, titers determined by the qPCR-RT assay have been compared with estimates of infectious virus obtained by a traditional commonly used method for MMR viruses - 50% cell culture infective dose (CCID(50)) assay, in paired samples. Pearson analysis evidenced a significant correlation between both methods for a certain period after viral inoculation. Furthermore, the established qPCR-RT assay was faster and less-laborious. The developed method could be used as an alternative method or a supplementary tool for the routine titer estimation during MMR vaccine production.

Heparan Sulfate Is an Attachment Factor for Foamy Virus Entry

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T(m) of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID(50)). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

Duration of Catarina Virus Infection in the Southern Plains Woodrat (Neotoma micropus)

Four adult male, 6 sub-adult, and 7 newborn southern plains woodrats (Neotoma micropus) each were inoculated subcutaneously with 3.1 log(10) median cell culture infectious doses (CCID(50)) of Catarina virus strain AV A0400135 (virus family Arenaviridae). The inoculated animals and the mothers of the newborn animals all became infected and remained asymptomatic. The infections in the adult male woodrats and in the mother woodrats were transient, the infections in 2 (33.3%) of the sub-adult woodrats persisted through month 4 post-inoculation, and 6 (85.7%) of the newborn woodrats were viruric through month 5 post-inoculation. Collectively these findings indicate that the duration of infection in the southern plains woodrat is dependent upon age at exposure to Catarina virus. The results of this study also indicate that chronically infected woodrats persistently shed Catarina virus into the environment.

Inactivation at various temperatures of bovine viral diarrhea virus in beef derived from persistently infected cattle1

Bovine viral diarrhea virus (BVDV) is a pestivirus that is enzootic in most cattle populations throughout the world. This virus is present throughout the body of persistently infected (PI) cattle. Previous research has not assessed the cooking temperature at which BVDV in meat from PI cattle can be inactivated. Therefore, muscle tissue from 6 PI cattle was harvested, refrigerated, frozen, and heated to various internal temperatures. The concentration of virus present was determined by virus isolation. Average cell culture infective doses (50% endpoint; CCID(50)) of BVDV per gram of frozen, uncooked meat from PI cattle were 10(5.85) CCID(50)/g of whole cuts and 10(6.02) CCID(50)/g of ground meat. The virus in whole and ground meat was consistently inactivated when cooked to temperatures greater than or equal to 75°C. A second objective of this research was to thoroughly reassess if Vero cells were permissive to BVDV infection in our laboratory to provide further indication of whether primates, including humans, might be susceptible to BVDV. Vero cells were not permissive to infection with any of 43 different strains of BVDV that readily replicated in Madin Darby bovine kidney cells. In conclusion, this bovine pathogen, which is not considered to be a human pathogen, can be inactivated by cooking ground or whole cuts of meat to 75°C or higher. Care should be taken to ensure that susceptible hosts such as pigs are not fed improperly cooked meat, meat by-products, or waste food originating from PI cattle.

Comparison of the immune effects of Coxsackievirus B3 VP1 protein, rAd/VP1 and pcDNA3/VP1 in mice

Objective To compare the immune effects of Coxsackievirus B3 （CVB3） capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte（CTL） killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose（ CCID50 ） assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups（ P 〈0.05 ）. While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group（ P 〈0. 05）. Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups （ P 〈 0.05 ）, while survival rate was significantly higher than these two groups （ P 〈 0.05 ）.Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines. Key words: Coxsackievirus B3 Adenovirus vector vaccine Protein vaccine DNA vaccine Immune response

Development of a New Tacaribe Arenavirus Infection Model and Its Use to Explore Antiviral Activity of a Novel Aristeromycin Analog

BackgroundA growing number of arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice.Methodology/Principal FindingsHere we describe a new mouse model based on TCRV challenge of AG129 IFN-α/β and -γ receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD50 of ∼100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4–8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 µM, administered i.p. once daily for 7 days, offered highly significant (P<0.001) protection against mortality in the AG129 mouse TCRV infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde arenavirus, MY-24 did not offer significant protection against mortality.Conclusions/SignificanceMY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our findings suggest that it may ameliorate disease by blunting the effects of the host response that play a role in disease pathogenesis. The new AG129 mouse TCRV infection model provides a safe and cost-effective means to conduct early-stage pre-clinical evaluations of candidate antiviral therapies that target clade B arenaviruses.

Development of a Particle Agglutination Method with Soluble Virus Receptor for Identification of Poliovirus

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples of patients with acute flaccid paralysis (AFP). In this study, we developed a particle agglutination (PA) method with a soluble human PV receptor (hPVR) in the form of an immunoadhesin (PVR-IgG2a) for the simple and rapid identification of PV. Sensitized gelatin particles with PVR-IgG2a showed specific agglutination with the culture fluid of PV-infected cells within 2 h of reaction in a one-step procedure. Detection limits for type 1, 2, and 3 PV(Sabin) strains were 1.5 x 10(6) 50% cell culture infectious doses (CCID(50)), 5.3 x 10(5) CCID(50), and 9.1 x 10(5) CCID(50), respectively. Wild-type PVs and PV isolates from acute flaccid paralysis cases examined were identified correctly with this PA method, except for some samples with a mixture of different serotypes of PVs, where a minor population of PV failed to be detected. These results suggest that this PA method is useful for the simple and rapid identification of PV, although the sensitivity was not high enough to detect a minor population of PV (<1/10 of the major population) among mixed PVs.

Vector Competence of Australian Mosquitoes for Chikungunya Virus

Chikungunya virus (CHIKV) is a globally emerging arbovirus responsible for unprecedented outbreaks in the western Indian Ocean, the Indian subcontinent and Italy. To assess the receptivity of Australia to CHIKV, we exposed 10 Australian mosquito species to a 2006 strain of CHIKV isolated from a viremic traveler from Mauritius. In susceptibility trials, the infectious dose required to infect 50% of the mosquitoes was 10(0.6) cell culture infectious dose (CCID)(50)/mosquito for Aedes procax, 10(1.7) CCID(50)/mosquito for Aedes albopictus, 10(2.1) CCID(50)/mosquito for Aedes vigilax, and 10(2.6) CCID(50)/mosquito for Aedes aegypti and Aedes notoscriptus. When exposed to blood meals containing between 10(3.5) and 10(4.1) CCID(50)/mosquito of CHIKV, infection rates in these five species, plus Coquillettidia linealis, were >or=81%. Subsequent transmission rates ranged between 20% for Ae. notoscriptus and 76% for Ae. vigilax. In contrast, Culex spp. were poor laboratory vectors, with infection and dissemination rates <or=20% and <or=12%, respectively. Although Australia has efficient laboratory vectors, the role a mosquito species plays in potential CHIKV transmission cycles will also depend on its geographical and temporal abundance, longevity, and association with humans.

Intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo–derived bovine embryos

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo–derived bovine embryos despite washing and trypsin treatment. Hence, the primary objective was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. In vivo–derived bovine embryos (n = 10) were nonsurgically collected from a single Bos tarus donor cow negative for BVDV. After collection and washing, embryos were placed into transfer media containing BVDV (SD-1; Type 1a). Each of the 10 embryos was individually loaded into an 0.25-mL straw, which was then nonsurgically transferred into the uterus of 1 of the 10 seronegative recipients on Day 0. The total quantity of virus transferred into the uterus of each of the 10 Bos tarus recipients was 878 cell culture infective doses to the 50% end point (CCID 50)/mL. Additionally, control heifers received 1.5 × 10 6 CCID 50 BVDV/.5 mL without an embryo (positive) or heat-inactivated BVDV (negative). The positive control heifer and all 10 recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15 after transfer. The negative control heifer did not exhibit a viremia or seroconvert. At 30 d after embryo transfer, 6 of 10 heifers in the treatment group were pregnant; however, 30 d later, only one was still pregnant. This fetus was nonviable and was positive for BVDV. In conclusion, the quantity of BVDV associated with bovine embryos after in vitro exposure can result in viremia and seroconversion of seronegative recipients after transfer into the uterus during diestrus.

Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay

The two most commonly used methods for the determination of a virus potency are plaque assay and 50% cell culture infective dose (CCID 50) assay, both based on cytopathic effect observation. We compared the potency estimates obtained by plaque and CCID 50 assays for nine mumps virus strains that produce different cytopathic effects in Vero cells. The ratios of CCID 50 and plaque assay quantification results differed for different strains and were in a range of 0.66–10, indicating that quantification results for some mumps virus strains are almost identical regardless of whether CCID 50 or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay.

Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 10(7.0) fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

Effect of T-705 treatment on western equine encephalitis in a mouse model

A mouse model of western equine encephalitis (WEE) was characterized for use in antiviral studies. Virus was detected in several tissues, most notably an average titer of 9.5 ± 1.1 log 10 50% cell culture infectious doses (CCID 50)/g tissue in the brains of infected animals. Signs of WEE included limb weakness, paralysis, involuntary spasms or extension of limbs, clenching of paws, hunching, ruffling of fur, and eye exudates, many of which are indicative of neurological disease. The pyrazinecarboxamide derivative, T-705, was found to be active in Vero cells against WEE virus (WEEV) with an 90% effective concentration (EC 90) of 49 μg/ml (selective index [SI] > 20). Treatment with T-705 in this WEE mouse model resulted in significant improvement in survival and mean day to death after oral treatment administered twice a day for 7 days at a dose of 400 mg/(kg d). Virus titer in the brain was not significantly reduced, despite a 1-log reduction in average brain titer in treated animals on 4 dpi. Signs of disease were relatively mild in treated animals, but were not eliminated. Treatment with T-705 improved morbidity and mortality of WEEV-infected mice, further illustrating the broad-spectrum activity of T-705 in the treatment of RNA viruses.

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed (for 2 h) to 2 × 10 5–7 cell culture infective dose (CCID 50)/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was ≤6.62 ± 1.57 copies/5 μL; 90% of the contaminated embryos had ≤4.64 ± 1.57 viral copies/5 μL of embryo-associated virus, using tolerance intervals ( P < 0.05). The SEM was 0.33 and the mean of averages was 1.12/5 μL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was ≤3.44 ± 0.89 copies/5 μL; 90% of the contaminated embryos had ≤2.40 ± 0.89 viral copies/5 μL of embryo-associated virus using tolerance intervals ( P < 0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 μL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.

140 INTRAUTERINE INOCULATION OF CATTLE WITH BOVINE VIRAL DIARRHEA VIRUS SIMULTANEOUS TO TRANSFER OF IN VIVO-DERIVED BOVINE EMBRYOS

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived and in vitro-produced bovine embryos despite washing. Hence, the primary objective of this study was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. A total of 10 in vivo-derived Day 7 bovine embryos were nonsurgically collected from a BVDV negative and seronegative donor cow. After collection, embryos were washed in accordance with the International Embryo Transfer Society (IETS) standards. Following washing, embryos were placed into transfer media containing BVDV (SD-1; type 1a). The embryos were immediately aspirated into 0.25-mL straws and transferred into seronegative recipients (Day 0). The total quantity of virus transferred into the uterus of each recipient was 900 to 1000 cell culture infective dose 50 (CCID50)/straw. This amount of virus was previously shown to be consistent with the average amount of BVDV associated with in vivo-derived and in vitro-produced embryos following standard IETS washing procedures after in vitro exposure to virus. The positive control heifer received 1.5 × 106 CCID50/straw of BVDV without an embryo. The negative control heifer received 1.5 × 106 CCID50/straw of heat-inactivated BVDV without an embryo. Serum and buffy coat samples were drawn from all heifers on Days 0, 3, 4, 6, 7, 8, 9, 10, 12, 15, and 30 after inoculation and analyzed for serum neutralizing antibodies and virus, respectively. The positive control heifer and all recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15. The negative control heifer did not exhibit viremia or seroconvert. All recipients receiving embryos were assessed for pregnancy using transrectal ultrasonography on Day 30 and 6 of 10 heifers were pregnant. On Day 60 the pregnant heifers were again assessed for pregnancy using transrectal ultrasonography. At this time only 1 of the 6 heifers was still pregnant. However, the fetus was determined to be nonviable and was removed via colpotomy. The fetus, fetal fluids and membranes were determined to be positive for BVDV via immunohistochemistry and PCR. Additionally, 213 base pairs of the 5′ nontranslated region of this PCR product were sequenced and found to be consistent with the inoculated strain. Results demonstrate that the average quantity of BVDV associated with bovine embryos after in vitro exposure and washing can result in viremia and seroconversion of seronegative recipients following transfer into the uterus during diestrus.

Clinical phase I intratumoral administration of two recombinant ALVAC canarypox viruses expressing human granulocyte-macrophage colony-stimulating factor or interleukin-2: the transgene determines the composition of the inflammatory infiltrate

Immunotherapy employs cytokines for modifying local inflammatory reactions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate dendritic cells, macrophages, and granulocytes leading to clinical trials using GM-CSF-based cancer vaccine approaches. Interleukin-2 (IL-2) is an important T cell stimulatory cytokine approved as exogenous antitumor agent. The ALVAC viral vector system uses a recombinant canarypox virus for local gene expression. We report a phase I clinical trial using intratumoral administration of ALVAC GM-CSF or ALVAC IL-2 in skin metastases of melanoma or leiomyosarcoma. ALVAC GM-CSF and ALVAC IL-2 were injected at 107.12 and 106.92, 50% cell culture infectious dose in eight metastases with acceptable tolerability. Local and systemic inflammatory reactions were observed. The transgene determined the local infiltrate: GM-CSF induced monocyte and macrophage enrichment of the peritumoral inflammatory infiltrate, whereas IL-2 increased local T lymphocytes. Stable disease of injected lesions was seen after ALVAC GM-CSF application, whereas ALVAC IL-2 treatment led to partial regression in three out of eight injected tumors, accompanied by decreased expression of melanocytic antigens. Local GM-CSF expression could be induced. In summary, ALVAC GM-CSF and ALVAC IL-2 injections are safe and can mediate local biologic and immunologic effects.

Specific small interfering RNAs-mediated inhibition of replication of porcine encephalomyocarditis virus in BHK-21 cells

Encephalomyocarditis virus (EMCV) is recognized as a pathogen inducing acute myocarditis and sudden death in preweaned piglets and severe reproductive failure in sows. In this study, eight specific small interfering RNA (siRNA) duplexes targeting different genomic regions of EMCV BJC3 were designed and their ability to inhibit virus replication in BHK-21 cells was investigated. The results showed that BHK-21 cells transfected with siRNA duplexes to 2C gene (JH-4666, BJC-1739), 2B gene (BJC-807), 3C gene (BJC-2363) and 3D gene (BJC-3269) were specifically resistant to EMCV infection when exposed to 500 times the 50% cell culture infective dose (CCID 50) of EMCV. The levels of the 3D gene in the transfected cells were obviously decreased. IFA and Western blotting analysis confirmed that the expression of VP1 protein in cell culture transfected with the siRNAs was apparently reduced. Of the five siRNAs, JH-4666, BJC-2363 and BJC-3269 were the most effective. Combination of the siRNA duplexes enhanced the inhibition of EMCV replication. Our data indicated that specific siRNAs are able to inhibit the replication of porcine encephalomyocarditis virus in BHK-21 cells, suggesting that RNAi might provide a new approach to prevent EMCV infection.

153 EFFECTS OF OVINE INTERFERON-β ON REPLICATION OF BOVINE VIRAL DIARRHEA VIRUS AND BOVINE HERPESVIRUS-1

Bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) are the most commonly isolated viruses from abattoir-origin materials utilized in embryo production and known to associate with zona pellucida-intact (ZP-I) embryos after exposure and washing. Some evidence indicates that developing embryos may produce substances that are able to inhibit viral replication in adjacent cells. Interferons such as recombinant human interferon-α are known to have anti-BVDV activity but no effect against BHV-1. In some preliminary studies, bovine interferon-τ has shown antiviral activities against BVDV but not against BHV-1. However, interferon-τ in other species has not been evaluated for anti-BVDV and anti-BHV-1 effects. Thus, the objective of this study was to evaluate the cytotoxicity and anti-viral effect of ovine interferon-τ against a non-cytopathic high affinity strain of BVDV (SD-1) and BHV-1 (Colorado) in cell culture. Serial dilutions (1:10) beginning with an initial concentration of 1 mg mL–1 of interferon-τ were made in 96-well plates and then Madin Darby bovine kidney (MDBK) cells were seeded in the wells. Cells and interferon-τ were incubated at 37.5�C in 5% CO2 and air for 24 h prior to addition of virus. The following concentrations of BVDV were added to the wells: 6000, 3500, 1000, 625, and 350 cell culture infective doses (CCID50) (50% endpoint) per well. In addition, four viral concentrations of BHV-1, 1000, 500, 250, and 100 CCID50/mL were evaluated in separate cell cultures. Virus isolation was utilized to determine if the addition of interferon-τ decreased the amount of infective virus. Ovine interferon-τ produced no observable cytotoxicity in MDBK cells in any of the assays. Also, the three highest concentrations of interferon-τ significantly decreased the amount of BVDV in all of the concentrations of BVDV tested but had no apparent effect on the concentration of BHV-1 in cell cultures. Therefore ovine interferon-τ has anti-BVDV effects similar to those seen with bovine interferont-τ and neither has any apparent antiviral activity on BHV-1 in cell culture. Additionally, ovine and bovine interferon-τ might serve to limit or prevent the transmission of BVDV and curtail the negative effects of BVDV on oocyte and embryo development. However, a similar effect is not expected for BHV-1.

Role ofVerrallina funerea(Diptera: Culicidae) in Transmission of Barmah Forest Virus and Ross River Virus in Coastal Areas of Eastern Australia

Verrallina funerea (Theobald) (Diptera: Culicidae) is a brackish water mosquito species found most commonly in Indonesia, Papua New Guinea, and the northeastern coastal regions of Australia. Aspects of the vector competence of this species for Barmah Forest virus (family Togaviridae, genus Alphavirus, BFV) and Ross River virus (family Togaviridae, genus Alphavirus, RRV), two medically important arboviruses in Australia, were investigated. Laboratory-reared Ve. funerea were moderately susceptible to experimental infection with BFV (median cell culture infectious dose of 103.6 per mosquito) and were capable of transmission to suckling mice (52% after a 9–12-d extrinsic incubation period). Maximum salivary gland infections for BFV (65%) and RRV (50%) were observed 8 and 10 d postinfection, respectively. To examine any regional differences in vector competence, field populations (separated by up to 200 km) of Ve. funerea and Aedes vigilax (Skuse) from southeastern Queensland and northern New South Wales were fed BFV and RRV, and subsequent infection rates were compared. For both viruses, no statistically significant variations in body, disseminated, or salivary gland infection rates were found in either Ve. funerea or Ae. vigilax. The results from this study indicate that Ve. funerea may have an important role as an amplification vector during outbreaks of both viruses and that local government authorities should rapidly treat brackish water habitats to control this species during periods of increased disease activity.

Role of &lt;I&gt;Verrallina funerea&lt;/I&gt; (Diptera: Culicidae) in Transmission of Barmah Forest Virus and Ross River Virus in Coastal Areas of Eastern Australia

Verrallina funerea (Theobald) (Diptera: Culicidae) is a brackish water mosquito species found most commonly in Indonesia, Papua New Guinea, and the northeastern coastal regions of Australia. Aspects of the vector competence of this species for Barmah Forest virus (family Togaviridae, genus Alphavirus, BFV) and Ross River virus (family Togaviridae, genus Alphavirus, RRV), two medically important arboviruses in Australia, were investigated. Laboratory-reared Ve. funerea were moderately susceptible to experimental infection with BFV (median cell culture infectious dose of 10(3.6) per mosquito) and were capable of transmission to suckling mice (52% after a 9-12-d extrinsic incubation period). Maximum salivary gland infections for BFV (65%) and RRV (50%) were observed 8 and 10 d postinfection, respectively. To examine any regional differences in vector competence, field populations (separated by up to 200 km) of Ve. funerea and Aedes vigilax (Skuse) from southeastern Queensland and northern New South Wales were fed BFV and RRV, and subsequent infection rates were compared. For both viruses, no statistically significant variations in body, disseminated, or salivary gland infection rates were found in either Ve. funerea or Ae. vigilax. The results from this study indicate that Ve. funerea may have an important role as an amplification vector during outbreaks of both viruses and that local government authorities should rapidly treat brackish water habitats to control this species during periods of increased disease activity.