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Recent outbreaks of Chikungunya virus (CHIKV) infection have resulted in millions of cases of disease with significant morbidity. No approved antiviral treatments exist for the prevention or treatment of this viral disease. Infection with CHIKV results in a high rate of symptomatic disease that primarily includes a debilitating arthralgia. To model this cardinal disease manifestation, adult DBA/1J mice were challenged with CHIKV by footpad injection. Viremia and hind limb virus titers increased ~100-fold while spleen virus increased >1,000-fold within 1 day post-virus infection (dpi). Footpad swelling was measured over a 10-day period, with peak swelling observed between 6 and 7 dpi. Histology of the hind leg at the site of virus challenge showed evidence of myositis and synovitis starting on 5 dpi. Cytokine profiling of the hind limb at the site of inoculation revealed a biphasic inflammatory response represented by an increase in IL-6, MCP-1, IFN-γ, MIP-1α, RANTES, and IL-17. To investigate the prophylactic capacity of IFN, mice were treated with mDEF201, an adenovirus-vectored IFN-α. Intranasal administration of a single 107 pfu/ml dose of mDEF201 administered 21 days to 24 h prior to infection, significantly reduced footpad swelling, virus titers in the hind leg and spleen, and several inflammatory cytokines. Efficacy was not observed when treatment was initiated 24 h after virus challenge. This arthralgia model of CHIKV recapitulates relevant disease features commonly observed in human disease making it applicable to preclinical testing of therapies that target both viral replication and the associated joint disease.

Rift Valley fever virus (RVFV) causes severe disease in humans and ungulates. The virus can be transmitted by mosquitoes, direct contact with infected tissues or fluids, or aerosol, making it a significant biological threat for which there is no approved vaccine or therapeutic. Herein we describe the evaluation of DEF201, an adenovirus-vectored interferon alpha which addresses the limitations of recombinant interferon alpha protein (cost, short half-life), as a pre- and post-exposure treatment in a lethal hamster RVFV challenge model. DEF201 was delivered intranasally to stimulate mucosal immunity and effectively bypass any pre-existing immunity to the vector. Complete protection against RVFV infection was observed from a single dose of DEF201 administered one or seven days prior to challenge while all control animals succumbed within three days of infection. Efficacy of treatment administered two weeks prior to challenge was limited. Post‑exposure, DEF201 was able to confer significant protection when dosed at 30 min or 6 h, but not at 24 h post-RVFV challenge. Protection was associated with reductions in serum and tissue viral loads. Our findings suggest that DEF201 may be a useful countermeasure against RVFV infection and further demonstrates its broad-spectrum capacity to stimulate single dose protective immunity.

Monoclonal antibodies (MAbs) are currently a promising treatment strategy against Ebola virus infection. This study combined MAbs with an adenovirus-vectored interferon (DEF201) to evaluate the efficacy in guinea pigs and extend the treatment window obtained with MAbs alone. Initiating the combination therapy at 3 days postinfection (d.p.i.) provided 100% survival, a significant improvement over survival with either treatment alone. The administration of DEF201 within 2 d.p.i. permits later MAb use, with protective efficacy observed up to 8 d.p.i.

Enterovirus 71 (EV71) causes hand-foot-and-mouth diseases as well as neurological complications in young children. Interferon (IFN) can inhibit the replication of many viruses with low cytotoxic effects. Previously, an adenovirus vectored mouse interferon-α (DEF201), subtype 5, was generated by Wu et al, 2007. In this study, the antiviral effects of DEF201 against EV71 were evaluated in a murine model. 6–day-old BALB/c mice were administered a single dose of DEF201 before or after infection with lethal dose of EV71. The survival rate, clinical symptoms, tissue viral loads and histology pathogenesis were evaluated. IFN gene expression following a single dose of DEF201 maintained high concentrations of 100–9000 pg/mL for more than 7 days in mice serum. Pre-infection administration of a single dose of 106 PFU of DEF201 offered full protection of the mice against EV71 infection compared with the empty Ad5 vector control. In addition, virus load in DEF201-treated mice muscle tissue was significantly decreased as compared with empty vector control. Histopathology analysis revealed that DEF201 significantly prevented the development of severe tissue damage with reduction of viral antigen in the murine muscle tissue. Post-infection treatment at 6 h offered full protection and partial protection at 12 h, indicating that DEF201 could be used as an anti-EV71 therapeutic agent in early stage of EV71 infection. In addition, our study showed that DEF201 enhanced the neutralization ability of serum in EV71-vaccinated mice, implying that DEF201 could promote the production of specific anti-EV71 antibodies. In conclusion, single dose of DEF201 is highly efficacious as a prophylactic agent against EV71 infection in vivo., Highlights • Single dose of DEF201 was highly efficacious as a prophylactic agent against EV71 infection in vivo. • DEF201 induced robust expression of mouse IFN in vivo. • DEF201 enhanced the neutralizing ability of anti-EV71 antibody in mouse serum.

Punta Toro virus (PTV; Bunyaviridae , Phlebovirus ) is related to Rift Valley fever virus (RVFV), a pathogenic agent which causes severe disease in humans and livestock primarily in the sub-Saharan region of Africa. The recent range expansion of RVFV and the potential for its intentional release into naïve populations pose a significant threat to public health and agriculture. Studies modeling disease in rodents and nonhuman primates have shown that PTV and RVFV are highly sensitive to the antiviral effects of alpha interferon (IFN-α), an important component of the innate antiviral host response. While recombinant IFN-α has high therapeutic value, its utility for the treatment of neglected tropical diseases is hindered by its short in vivo half-life and costly production of longer-lasting pegylated IFNs. Here, we demonstrate extended preexposure protection against lethal PTV challenge following a single intranasal administration of DEF201, which is a replication-deficient human adenovirus type 5 vector engineered to constitutively express consensus IFN-α (cIFN-α) from transduced host cells. DEF201 was also efficacious when administered within 24 h as a postexposure countermeasure. Serum concentrations of cIFN-α could be detected as early as 8 h following treatment and persisted for more than 1 week. The prolonged antiphlebovirus prophylactic effect, low production costs, and ease of administration make DEF201 a promising agent for intervention during natural disease outbreaks and for countering possible bioterrorist acts.

Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p

A spontaneously established porcine granulosa cell line (PGC-2) was cloned through the continuous culturing of primary granulosa cells collected from equine chorionic gonadotropin (eCG)-treated prepubertal gilts. This established cell line has undergone approximately 100 passages and shows contact-inhibition of growth. PGC-2 stained with a monoclonal antibody (mAb) directed against cytokeratin, indicating its epithelial nature, but not with a mAb directed against vimentin, suggesting that it is not fibroblast-derived. Immunoblotting revealed that PGC-2 expresses cadherin, an epithelial Ca+2-dependent cell adhesion molecule. The cells were dependent on serum for growth and had a doubling time of approximately 20 hr when cultured with 10% fetal bovine serum. The cell line was examined for the presence of FSH receptors, cAMP responses, and steroidogenic capabilities. The cell line lacks FSH receptors as assessed by radiolabelled-ligand binding, and no transcripts for FSH receptor were detected by Northern blotting of total cellular RNA. Neither FSH nor cholera toxin (0.5 ng/mL) stimulated increases in cAMP levels in these cells, whereas forskolin (10 microM) induced a fivefold increase in cAMP production. When a higher concentration of cholera toxin (300 ng/mL) was used, however, cAMP levels doubled by 2 hr. Despite a lack of responsiveness to purified of SH or oLH, the cells were capable of progesterone and estradiol production when provided with the appropriate substrates. We conclude that PGC-2 display properties that are similar to immature granulosa cells and may provide a suitable in vitro model for the study of granulosa cell function.

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal cowpox (Brighton strain) and vaccinia (WR strain) virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl) and pulmonary (50-µl), to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI), upper respiratory infections (URI), and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term) or 56d (long-term) prior to virus challenge. Single nasal sinus treatments of 10(6) and 10(7) PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201). Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7) mDEF201 treatments, but a 10(6) dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7) PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5) to 10(7) PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1-49 days pre-infection), with minimal weight loss occurring during infection. Surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mDEF201 did not compromise the immune response against the initial infection. Post-exposure therapy was given between 6-24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mDEF201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. Comparisons were made of the ability of mDEF201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. Lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). Lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mDEF201 treated mice were nearly at baseline. In contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. These results demonstrate the long-acting prophylactic and treatment capacity of mDEF201 to combat vaccinia virus infections.

Sea raven (Hemitriperus americanus) and ocean pout (Macrozoarces americanus) hearts were excised and perfused in an isolated system. The contractile response of hearts perfused with buffer equilibrated with 100% O2, 100% N2, or 99% O2: 1% CO2 was monitored and work was calculated from mean pressure development times cardiac output. Hearts subjected to either anoxia or hypercapnic acidosis could not sustain mechanical performance as well as hearts perfused with buffer equilibrated with 100% O2. The decrease in mechanical function was not due to an unavailability of ATP. Alternatively it is suggested that contractile failure is related to a decrease in intracellular pH.

Trout fitted with dorsal aortic cannulae were subjected to 6 min of intensive exercise and monitored over the following 12 h recovery period. Delayed mortality was 40%; the majority of deaths occurred 4–8 h post-exercise. Surviving fish exhibited a short-lived haemoconcentration reflected in increased haematocrit, haemoglobin, plasma protein, Na+ and Ch- levels; an extended rise in plasma [K+]; a quickly corrected respiratory acidosis; and a more prolonged metabolic acidosis in concert with a rise in blood lactate. Dying fish exhibited very similar trends except for a significantly greater metabolic acidosis, lower plasma [Cl-], and the apparent accumulation of an unknown anion in the blood prior to death. Cardiac failure did not occur. Blood metabolic acid levels, while elevated, were only ∼ 50% of peak lactate anion levels and well within the normal range of tolerance, as were all other changes observed in the blood of non-survivors. The hypothesis that post-exercise mortality is due to excessive ‘lactic acid’ accumulation in the blood is discounted. It is suggested that intracellular acidosis may be the proximate cause of death.

Chronically cannulated rainbow trout were subjected to 6 min of severe burst exercise and monitored over a 12 h recovery period. There were short-lived increases in haematocrit, haemoglobin, plasma protein, Na+ and Cl− levels. Plasma [Cl−] later declined below normal as organic anions accumulated. A much larger and more prolonged elevation in plasma [K+] probably resulted from intracellular acidosis. An intense extracellular acidosis was initially of equal respiratory (i.e. ) and metabolic (i.e. ) origin. However was rapidly corrected while the metabolic component persisted. Plasma ammonia increases had negligible influence on acid-base status. Elevations in blood lactate (ΔLa−) were equal to immediately post-exercise but later rose to twice the latter. Simultaneous white muscle biopsies and blood samples demonstrated that muscle to blood gradients of lactate and pyruvate were maximal immediately post-exercise. As blood levels rose and muscle levels declined, an approximate equilibrium was reached after 4 h of recovery. Intra-arterial infusions of lactic acid in resting trout produced a severe but rapidly corrected metabolic acidosis. The rates of disappearance of and ΔLa− from the blood were equal. Infusions of similar amounts of sodium lactate produced a small, prolonged metabolic alkalosis with a much slower ΔLa− disappearance rate. It is suggested that the excess of ΔLa− over in the blood after exercise is associated with differential release rates of the two species from white muscle rather than differential removal rates from the bloodstream, and that the majority of the lactic acid load in muscle is removed by metabolism in situ.

Glycolysis in muscle during the anaerobic component of severe exercise rapidly produces lactate (La−) and metabolic protons in equal amounts, some of which may enter the blood (Krebs, Wood & Alberti, 1975). In vivo experimentation using strenuously exercised fish has yielded two patterns: La− accumulating in blood either in greater or lesser amounts than . It was rare to find both species at the same concentration, as is the usual case in man (Keul, Keppler & Doll, 1967). In the dogfish (Piiper, Meyer & Drees, 1972) and rainbow trout (Turner, Wood & Clark, 1983a) after exercise, ΔLa− in blood greatly exceeded . The opposite discrepancy occurred in the starry flounder (Wood, McMahon & McDonald, 1977), muskellunge (Beggs, Holeton & Crossman, 1980), and flathead sole (Turner, Wood and Hōbe, 1983b), where ΔLa− was consistently lower than These discrepan cies could be caused either by differential removal rates of the two ionic species from the blood space or by differential release rates from the myotome. We have recently argued that the latter is the dominant process (Turner et al. 1983a,b). However, this is extremely difficult to prove with in vivo methodology, as measurements in a single body compartment with periodic sampling give little information about the dynamic equilibrium between production, metabolism and fluxes between compartments. In vitro muscle preparations can be used to overcome many of these difficulties (e.g., Mainwood & Worsley-Brown, 1975; Benadé & Heisler, 1978). Isolated-perfused rainbow trout trunks have been employed to examine the physiology of systemic vascular resistance control (Wood & Shelton, 1975; Wood, 1977) and substrate utilization by muscle (Moen & Klungsoyr, 1981). In the present study, by strenuously exercising intact trout, then rapidly preparing them as isolated-perfused trunks, it was possible to determine net and La− movement from muscle to perfusate during recovery while simultaneously monitoring muscle [La−] by repetitive biopsy.

A series of experiments was undertaken to determine the underlying mechanism of ocular freezing avoidance in a variety of antarctic fish. The three possible mechanisms that were examined include the depression of the freezing point through (1) colligative or (2) non-colligative means or (3) the supercooling of the ocular fluids. The ocular fluids of antarctic fish, like those of most vertebrates, are slightly hypotonic with respect to plasma and contain very low levels of serum proteins. The chief means of ocular freezing avoidance appears to involve supercooling of the ocular fluids. The presence of suitable barriers to prevent ice propagation into the eye has been demonstrated both in vitro and in vivo.

Chronically cannulated flathead sole were subjected to 10 min of either moderate or exhausting burst exercise and monitored over a 12 h recovery period. Acid-base disturbances were more severe after exhausting exercise, but ionic and haematological changes were the same in the two treatments. Most effects were qualitatively similar to those previously described in severely exercised rainbow trout (Turner, Wood & Clark, 1983). Specific differences are discussed and related to the different external environments (sea water vs fresh water), exercise capabilities and ecologies of the two species. The most striking divergence occurred in lactate (La−) and metabolic proton dynamics. Post-exercise La− levels in white muscle in sole were less than half those in trout but declined much more slowly. In contrast to the situation in trout, muscle [La−] remained significantly elevated and a large muscle to blood La− gradient persisted even after 12 h recovery. Blood [La−] underwent only minimal elevation (< 2 mequiv l−1), and blood metabolic proton load greatly exceeded ΔLa− throughout the recovery period, effects directly opposite those in trout. This observed excess of over ΔLa− in the blood of exercised sole is probably not due to a preferential removal mechanism, because and ΔLa− disappeared from the blood at similar rates after an intra-arterial infusion of lactic acid in resting animals. It is therefore argued that the phenomenon reflects a differential release of the two metabolites from the white muscle of the sole, La− being strictly retained in the muscle for gluconeogenesis in situ.

Cannulated adult rainbow trout were subjected to 6 min of exercise stress in four different water conditions: hard water ([Formula: see text] as CaCO3), control pH ([Formula: see text]); artificial soft water [Formula: see text], control pH; hard water, acid pH [Formula: see text]; and artificial soft water, acid pH. Physiological changes and postexercise mortalities were monitored over a 12-h recovery period. The physiological responses to exercise stress were qualitatively similar in all treatments and are discussed in detail. At control pH, water hardness had minimal influence on the magnitude of physiological response and postexercise mortality. When fish were exercised in hard water at acid pH, the symptoms of postexercise acidosis were actually ameliorated slightly and there was no increase in mortality. However, acid exposure in soft water greatly exacerbated most of the postexercise disturbances and caused a doubling of mortality.