Effects of dietary forage level and monensin on lactation performance, digestibility and fecal excretion of nutrients, and efficiency of feed nitrogen utilization of Holstein dairy cows.

Two experiments (Exp. 1 and 2) were conducted using a 4 x 4 Latin square design with 2 replications (n = 8) to evaluate effects of feeding Holstein dairy cows a total mixed ration containing 50 or 60% of ration dry matter (DM) from forages with or without supplementation of monensin. In Exp. 1, alfalfa silage (AS) was used as the major forage (55% forage DM), and corn silage (CS; 45% forage DM) was used to make up the rest of the forage portion of diets (55AS:45CS). In Exp. 2, CS was used as the major forage (70% forage DM) and alfalfa hay (AH; 30% forage DM) was used to make up the rest of the forage portion of diets (70CS:30AH). Experimental diets were arranged in a 2 x 2 factorial with 50 or 60% ration DM from forages and monensin supplemented at 0 or 300 mg/cow daily. In Exp. 1 (55AS:45CS), feeding 60% forage diets decreased DM intake (DMI; 27.3 vs. 29.6 kg/d) but maintained the same levels of milk (45.8 vs. 47.0 kg/d) compared with 50% forage diets. The efficiency of converting feed to milk or 3.5% fat-corrected milk was greater for cows fed 60% compared with 50% forage diets (1.7 vs. 1.6 kg milk or 3.5% fat-corrected milk/kg of DMI, respectively). Increasing dietary forage level from 50 to 60% of ration DM increased milk fat percentage (3.4 to 3.5%); however, adding monensin to the 60% forage diet inhibited the increase in milk fat percentage. Feeding 60% forage diets decreased feed cost, but this decrease ($0.5/head per day) in feed cost did not affect income over feed cost. Feeding 60% forage diets decreased fecal excretion of DM (10.6 to 9.6 kg/d) and nitrogen (N; 354 to 324 g/d) and improved apparent digestibility of neutral detergent fiber from 43 to 49% and apparent efficiency of feed N utilization from 32.3 to 35.9% compared with 50% forage diets. In Exp. 2 (70CS:30AH), feeding 60% forage diets decreased DMI from 29.6 to 28.2 kg but maintained the same level of milk (41.1 vs. 40.8 kg/d) and therefore increased the efficiency of converting feed to milk (1.46 vs. 1.38 kg milk/kg DMI) compared with 50% forage diets. Daily feed cost for feeding 60% forage diets was $0.3/head lower than for the 50% forage diets. Fecal excretion of DM (10.3 vs. 11.5 kg/d) was lower and fecal excretion of N (299 vs. 328 g/d) tended to be lower for 60% compared with 50% forage diets. Results from these 2 experiments suggest that a 60% forage diet consisting of either AS or CS as the major forage can be fed to high producing Holstein dairy cows without affecting milk production while improving or maintaining the efficiency of converting feed to milk and the apparent efficiency of utilization of feed N. Cows receiving a 60% forage diet had a similar or improved digestibility of nutrients with a similar or reduced fecal excretion of nutrients. Effects of monensin under the conditions of the current experiments were minimal.

In vitro and in vivo influenza virus-inhibitory effects of viramidine.

Viramidine, the 3-carboxamidine derivative of ribavirin, was effective against a spectrum of influenza A (H1N1, H3N2 and H5N1) and B viruses in vitro, with the 50% effective concentration (EC50) ranging from 2 to 32 microg/ml. The mean 50% cytotoxic concentration (CC50) in the MDCK cells used in these experiments was 760 microg/ml. Ribavirin, run in parallel, had a similar antiviral spectrum, with EC50 values ranging from 0.6 to 5.5 microg/ml; the mean CC50 for ribavirin was 560 microg/ml. Oral gavage administrations of viramidine or ribavirin to mice infected with influenza A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), B/Hong Kong/5/72 or B/Sichuan/379/99 viruses were highly effective in preventing death, lessening decline in arterial oxygen saturation, inhibition of lung consolidation and reducing lung virus titers. The minimum effective dose of viramidine in these studies ranged from 15 to 31 mg/kg/day, depending upon the virus infection, when administered twice daily for 5 days beginning 4 h pre-virus exposure. The LD50 of the compound was 610 mg/kg/day. Ribavirin's minimum effective dose varied between 18 and 37.5 mg/kg/day with the LD50 determined to be 220 mg/kg/day. Viramidine's efficacy was also seen against an influenza A/NWS/33 (H1N1) virus infection in mice, when the compound was administered in the drinking water, the minimum effective dose being 100 mg/kg/day. Delay of the initiation of either viramidine or ribavirin therapy, using the approximate 1/3 LD50 dose of each, was protective as late as 48 h after exposure to the A/NWS/33 virus. While both compounds appear to have similar efficacy against influenza virus infections, when one considers the lesser toxicity, viramidine may warrant further evaluation as a possible therapy for influenza.

Economic returns to Holstein and Jersey herds under multiple component pricing.

This study analyzed component data from herds participating in the Mideast Federal Milk Marketing Order from 2000 through 2002, and its implications for herd profitability. A monthly simulation model was developed to evaluate the economic returns for a representative Holstein and Jersey herd in Pennsylvania under multiple component pricing. Component levels were highly seasonal and variable from farm to farm. A third of the herds during the course of a year realized a 1- to 3-mo temporary reduction in milk fat or protein greater than one standard deviation. Consistently producing milk fat and protein one standard deviation below the mean reduced the Class III value by $0.82/cwt (100 pounds), or 7.09%. The simulation model indicated that a herd of 100 Holstein cows generated $31,221 more income over feed costs (IOFC) a year than a herd of 100 Jersey cows. Although Jersey milk had greater gross value than Holstein milk due to higher component levels, total volume of milk and components produced by Holsteins offset this difference. Simulation results confirm that increasing milk fat and protein percentages by one standard deviation increased IOFC 7.7% for Holsteins and 9.2% for Jerseys relative to the baseline IOFC, with similar losses for component reductions. Increasing milk yield by one standard deviation increased IOFC by 19.6% for Holsteins and 23.9% for Jerseys relative to the baseline IOFC, again with similar losses for reductions in milk production. In all of the scenarios analyzed, the most important factor affecting IOFC was total amount of milk fat and protein produced, not the component percentage levels.

A multiple-component analysis of US dairy trade.

This paper develops a multiple component-based methodology to account for imports and exports of dairy products and products with a significant dairy content. More specifically, it accounts for imports, exports, and net trade for all dairy products on the basis of milk fat, protein, other solids, and moisture. This approach provides a less biased method of assessing the amount of dairy imports entering the US than the USDA's milk equivalent conversions.

Inhibition of influenza virus infections in immunosuppressed mice with orally administered peramivir (BCX-1812).

Experiments were run to determine the effect of oral gavage treatment with the cyclopentane influenza virus neuraminidase inhibitor peramivir (BCX-1812, RWJ-270201) in influenza A (H1N1) virus-infected mice that had their immune system suppressed by cyclophosphamide (CP) therapy or in severe combined immune deficient (SCID) mice. Treatment of CP-immunosuppressed mice with peramivir using doses of 100, 10, or 1mg/kg/day was begun 2.5 or 8 days post-virus exposure and continued twice daily for 3 or 5 days. The 5-day therapy was more effective than the 3-day treatment, as seen by significantly increased survivor numbers, lessened decline in arterial oxygen saturation, reduced lung consolidation, and inhibition of lung virus titers. Infected SCID mice were also responsive to peramivir therapy begun 8 days after virus exposure and continued for 5 days, although antiviral effects did not include prevention of death and were dependent upon the viral challenge dose received. These data indicate that peramivir may have potential for treatment of influenza virus-infected immunosuppressed patients.

Estimation of the protein content of US imports of milk protein concentrates.

Recent declines in milk prices in the United States have sparked renewed concern that imports of milk protein concentrates (MPC) are increasingly entering the United States with very low tariff rates and is having an adverse impact on the US dairy industry. Milk protein concentrates are used in the United States in many different products, including the starter culture of cheese, or in nonstandard cheeses such as baker's cheese, ricotta, Feta and Hispanic cheese, processed cheese foods, and nutritional products. One of the difficult aspects of trying to assess the impact of MPC imports on the US dairy industry is to quantify the protein content of these imports. The protein content of MPC imports typically ranges from 40 to 88%. The purpose of this study is to develop a methodology that can be used to estimate the protein content of MPC on a country by country basis. Such an estimate would not only provide information regarding the quantity of protein entering the United States, but would also provide a profile of low- and high-value MPC importers. This is critical for market analysis, since it is the lower valued MPC imports that more directly displaces US-produced skim milk powder.

Characterization of an influenza A (H3N2) virus resistant to the cyclopentane neuraminidase inhibitor RWJ-270201.

The novel influenza virus neuraminidase (NA) inhibitor, (1S,2S,3R,4R)-3-[(1S)-(acetylamino)-2-ethylbutyl]-4-[(aminoiminomethyl)amino]-2-hydroxy-cyclopentanecarboxylic acid (RWJ-270201, BCX-1812), is a potent inhibitor of influenza A and B viruses in cell culture and in infected mice. A mouse-adapted strain of influenza A/Shangdong/09/93 (H3N2) virus was serially passaged in the presence of 1 microM compound. After the fourth passage, breakthrough of resistant virus occurred. By the tenth passage, a twice plaque purified isolate was obtained which could replicate in 10 microM inhibitor. The 50% effective concentration (EC(50)) values for RWJ-270201 against wild-type and resistant viruses, determined by using a cytopathic effect inhibition assay, were 0.007 and 23 microM, respectively. Cross-resistance to zanamivir and oseltamivir carboxylate was observed. The hemagglutinin (HA) and NA genes of the virus were sequenced to determine the mutation(s) which conferred drug resistance. No differences were found between the resistant and wild-type viruses in the NA gene. However, a point mutation resulting in a single amino acid change (Lys189Glu) was found in the resistant viral HA. The wild-type and resistant viruses were compared for virulence in BALB/c mice. The resistant virus was approximately tenfold less virulent than the wild-type virus based upon virus challenge dose. Mice infected with a lethal dose of the resistant virus could still be effectively treated with RWJ-270201. Thus, the HA mutation may allow for the spread of the virus in cell culture in the presence of the NA inhibitor, but not in mice.

Effects of cidofovir on the pathogenesis of a lethal vaccinia virus respiratory infection in mice.

Intranasal infection of BALB/c mice with the WR strain of vaccinia virus leads to pneumonia, profound weight loss, and death. Although the major sites of virus replication are in the lungs and nasal tissue, dissemination of the virus to other visceral organs and brain occurs via the blood. In this report the effects of cidofovir on the pathogenesis of the infection was studied. Mice were infected intranasally with virus followed 1 day later by a single intraperitoneal treatment with cidofovir (100 mg/kg) or placebo. Placebo-treated mice were dead by day 8, whereas all cidofovir-treated animals survived through 21 days. Cidofovir treatment did not prevent profound weight loss from occurring during the acute phase of the infection, but the mice gained weight quickly after the 8th day. Significantly higher arterial oxygen saturation levels, as determined by pulse oximetry, were seen in cidofovir-treated animals compared to placebos on days 4-7. Cidofovir treatment markedly improved lung consolidation scores and prevented lung weights from increasing during the infection. Virus titers in lungs and nasal tissue were high starting from the first day of the infection, whereas the titers in liver, spleen, brain, and blood was low for 3 days then markedly rose between days 4 and 6. Lung and nasal virus titers were reduced 10-30-fold by cidofovir treatment on days 2, 4 and 6. Virus titers in the other tissues and blood at their peak (day 6) were 30- to >1000-fold less than in tissues of placebos. These results illustrate the ability of a single cidofovir treatment to control the pathogenesis of an acute lethal infection in various tissues during the vaccinia virus infection in mice.

Treatment of lethal vaccinia virus respiratory infections in mice with cidofovir.

Intranasal infection of BALB/c mice with the WR strain of vaccinia virus leads to pneumonia, profound weight loss and death. Five days after intranasal inoculation, virus from untreated mice was recovered from 11 organs, tissues and whole blood. The highest titres [>10(8) plaque forming units (pfu)/g] were in lungs and nose/sinus tissue, with about 10(7) pfu/g in spleen and blood. Seven other organs contained 30- to > or = 50-fold lower amounts of virus. Mice infected with the related cowpox virus (for comparative purposes) had the majority of virus located in the respiratory tract. The vaccinia mouse model was used to study the efficacy of cidofovir treatments on the infection. Subcutaneous injections of 30 or 100 mg/kg/day, given on days 1 and 4 after virus challenge, reduced mortality by 60-100%. However, lung virus titres on days 2-5 were reduced no more than 10-fold by these treatments. A moderate improvement in drug efficacy occurred with daily treatments for 5 days. The efficacy of cidofovir also increased as the virus challenge dose decreased, where subcutaneous or intraperitoneal treatment routes showed similar degrees of protection. Although it has been known for many years that the WR strain of vaccinia virus can cause lethal infections by intranasal route, its application to antiviral therapy represents a new model for studying anti-orthopoxvirus agents.

Influence of virus strain, challenge dose, and time of therapy initiation on the in vivo influenza inhibitory effects of RWJ-270201.

The influenza virus neuraminidase inhibitor RWJ-270201 (cyclopentane carboxylic acid, 3-[cis-1-(acetylamino)-2-ethylbutyl]-4[(aminoiminomethyl)amino]-2-hydroxy-[cis, 2S, 3R, 4R]) was significantly inhibitory to an infection in mice induced by influenza A/NWS/33 (H1N1) virus when oral gavage (p.o.) treatment with 10 mg/kg per day was delayed at least 60 h after virus exposure. Treatment was 5 mg/kg twice daily for 5 days. Viral challenge doses of influenza A/Shangdong/09/93 (H3N2) virus ranging from the LD(70) to the LD(100) did not affect the marked antiviral efficacy of 12.5 mg/kg of RWJ-270201 administered p.o. twice daily for 5 days beginning 4 h pre-virus exposure; infection by an approximate 2 LD(100) dose (10(8) cell culture infectious doses/ml) was only weakly inhibited by the same treatment as seen by significant increase in mean day to death. Murine infections induced by influenza A/Bayern/57/93 (H1N1) and B/Lee/40 viruses were significantly inhibited by 100, 10, and 1 mg/kg per day of RWJ-270201 using the above treatment regimen; influenza A/PR/8/34 (H1N1) virus infections in mice were only moderately inhibited, the antiviral effects using this virus being lessening of arterial oxygen decline, reduced lung consolidation, and inhibition of lung virus titers primarily at the higher dosages.

Primary immune system effects of the orally administered cyclopentane neuraminidase inhibitor RWJ-270201 in influenza virus-infected mice.

The cyclopentane derivative [1S,2S,3R,4R]-3-[(1S)-1-(acetylamino)-2- ethylbutyl]-4-[(aminoiminomethyl)amino]-2-hydroxy-cyclopentanecarboxylic acid (RWJ-270201) has been previously reported to be a potent and selective inhibitor of influenza virus neuraminidase, and to inhibit infections with this virus in vitro, in mice, and in clinical challenge studies. The effect of oral gavage therapy of 100 mg/kg/day of RWJ-270201 administered twice daily for 5 days beginning 16 h prior to virus exposure, on various immune factors of importance in response to primary influenza infection was determined in mice infected with influenza A/Shangdong/09/93 (H3N2) virus. Spleens taken from the mice 2 h after termination of treatment were processed for cytotoxic T lymphocytes (CTL) and natural killer (NK) cell activity and for enumeration of macrophages, T, T-helper, T-suppressor/cytotoxic, and B cells. Saline-treated mice and normal mice were run in parallel. Treatment had no significant effect on any immune parameter. In a second experiment, mice infected with influenza A/NWS/33 (H1N1) were treated similarly with RWJ-270201 beginning 4 h pre-virus exposure. Treatment prevented any deaths from occurring, and markedly lessened arterial oxygen decline, lung consolidation, and lung virus titers. The mice developed mean neutralizing antibody (NA) titers of 1:592, and six of seven rechallenged mice resisted rechallenge with the same virus, indicating the initial virus-inhibitory effect also did not prevent the animals from developing an adequate humoral immune response to the virus.

In vivo influenza virus-inhibitory effects of the cyclopentane neuraminidase inhibitor RJW-270201.

The cyclopentane influenza virus neuraminidase inhibitor RWJ-270201 was evaluated against influenza A/NWS/33 (H1N1), A/Shangdong/09/93 (H3N2), A/Victoria/3/75 (H3N2), and B/Hong Kong/05/72 virus infections in mice. Treatment was by oral gavage twice daily for 5 days beginning 4 h pre-virus exposure. The influenza virus inhibitor oseltamivir was run in parallel, and ribavirin was included in studies with the A/Shangdong and B/Hong Kong viruses. RWJ-270201 was inhibitory to all infections using doses as low as 1 mg/kg/day. Oseltamivir was generally up to 10-fold less effective than RWJ-270201. Ribavirin was also inhibitory but was less tolerated by the mice at the 75-mg/kg/day dose used. Disease-inhibitory effects included prevention of death, lessening of decline of arterial oxygen saturation, inhibition of lung consolidation, and reduction in lung virus titers. RWJ-270201 and oseltamivir, at doses of 10 and 1 mg/kg/day each, were compared with regard to their effects on daily lung parameters in influenza A/Shangdong/09/93 virus-infected mice. Maximum virus titer inhibition was seen on day 1, with RWJ-270201 exhibiting the greater inhibitory effect, a titer reduction of >10(4) cell culture 50% infective doses (CCID(50))/g. By day 8, the lung virus titers in mice treated with RWJ-270201 had declined to 10(1.2) CCID(50)/g, whereas titers from oseltamivir-treated animals were >10(3) CCID(50)/g. Mean lung consolidation was also higher in the oseltamivir-treated animals on day 8. Both neuraminidase inhibitors were well tolerated by the mice. RWJ-270201 was nontoxic at doses as high as 1,000 mg/kg/day. These data indicate potential for the oral use of RWJ-270201 in the treatment of influenza virus infections in humans.

Utilization of alpha-1-acid glycoprotein levels in the serum as a parameter for in vivo assay of influenza virus inhibitors.

Alpha-1-acid glycoprotein (AGP), an acute phase protein in serum assayed by single radial immunodiffusion using a commercially available kit, was found to significantly increase in mice infected with influenza A and B viruses. Experiments were run to determine the rate of increase of serum AGP and its relation to other influenza disease parameters, including lung consolidation, development of lung virus titres, decline in arterial oxygen saturation (SaO2), histopathological changes in the lung, and death of the animal. Maximal AGP levels occurred by day 3 in the animals, at about the same time lung virus titres reached their peak and inflammatory effects were evident in the lung. Serum levels of AGP were then compared with other disease parameters in the evaluation of the anti-influenza A and B virus efficacy of oseltamivir and ribavirin in mice. Treatment was by oral gavage twice daily for 5 days, beginning 4 h before virus exposure using doses of 100, 10, and 1 mg/kg per day of oseltamivir and 75 mg/kg per day of ribavirin. Against the influenza A infection, significant inhibition of death, SaO2 decline, and lung consolidation was seen at all doses of each compound; day-6 AGP levels were reduced in a dose-responsive manner. Lung virus titres were lessened at this time, but to a significant degree only at the high dose of oseltamivir and by ribavirin. The influenza B virus infection, which appeared more severe than the influenza A infection, was also significantly inhibited by both compounds, but to a lesser extent. The serum AGP levels were again lessened by therapy with both compounds. The influence of challenge dose of influenza A virus on AGP level and on the antiviral activity of 20 mg/kg per day of oseltamivir, administered by oral gavage, was determined in mice. The AGP level was in proportion to the viral challenge dose; oseltamivir significantly inhibited AGP levels and all other disease parameters regardless of size of viral inoculum. These data indicate murine AGP levels to be markedly stimulated by infection with influenza A and B viruses, and the level of the protein to be an additional measure of antiviral efficacy.

Intranasal treatment of cowpox virus respiratory infections in mice with cidofovir.

Orthopoxvirus infections in mice have been effectively treated with cidofovir, a clinically approved drug given by intravenous infusion to treat cytomegalovirus infections. In a bioterrorist scenario it would be technically difficult to give this drug to a large number of exposed individuals. New treatment approaches are being sought, which include giving cidofovir by alternative routes or designing oral prodrugs of cidofovir. In this report, intranasal cidofovir was investigated as a treatment of pulmonary cowpox virus infections in BALB/c mice. Ninety to 100% of animals given a single intranasal drug treatment (10, 20 or 40 mg/kg) 24 h after virus challenge survived the infection, whereas all placebo-treated mice died. Doses of 2.5 and 5 mg/kg resulted in 60 and 80% survival, respectively. Single treatments of 20 and 40 mg/kg could be given up to 3 days after virus inoculation and still be 80-90% protective. A single 40 mg/kg treatment of infected mice given 1 or 2 days after infection also resulted in statistically significant decreases in virus titer in lungs and nose/sinus compared to the placebo group. Drug efficacy was found to be contingent upon treatment volume. A 10 mg/kg intranasal dose given 24 h after virus challenge was 100 and 50% effective in volumes of 40 and 20 microl, respectively. The same dose in 5 and 10 microl volumes caused no decrease in mortality. The results of these studies establish the utility of cidofovir treatment of poxvirus infections in mice by intranasal route. The data suggest the possibility that aerosol delivery of cidofovir to human lungs may be a viable alternative to intravenous dosing.

Treatment of cowpox virus respiratory infections in mice with ribavirin as a single agent or followed sequentially by cidofovir.

To better understand the potential of ribavirin in the treatment of orthopoxvirus infections (such as those acquired through bioterrorist activities), the efficacy of the drug was studied in a cowpox respiratory infection model in mice under varying disease severity. Mice did not survive a high intranasal cowpox virus challenge [3 x 10(6) plaque forming units (pfu)/animal] treated with subcutaneous ribavirin (100 mg/kg/day for 5 days), but lived 3.9 days longer than placebos. In contrast, 100% of animals receiving the same dose of drug survived a 3 x 10(5) pfu challenge compared with 0% survival of those that received placebo. Survival rates of 50 and 30% occurred with ribavirin doses of 50 and 25 mg/kg/day, respectively. At the 100 mg/kg/day dose, ribavirin reduced lung virus titres 40-fold on day 6 of the infection relative to titres in the placebo group. Weight loss resulting from illness and mean lung weights of mice treated with ribavirin were also significantly reduced. Mice were infected intranasally with the high 3 x 10(6) pfu virus challenge dose and treated with 100 mg/kg/day ribavirin for 5 days, followed by single injections of 75 mg/kg cidofovir on day 6, 7, 8 or 9. Cidofovir alone (without ribavirin) administered on day 6 had no beneficial effect on disease outcome. Ribavirin alone increased the mean time to death by 3.7 days. Ribavirin treatment for 5 days followed by cidofovir treatment on days 6 and 7 significantly increased the mean time to death beyond that achieved with ribavirin alone by 8.2 and 4.4 days, respectively, with 30 and 40% of mice surviving the infection. These results suggest that many individuals infected with an orthopoxvirus by aerosol route would benefit by a course of ribavirin therapy. Later, the fewer number of very sick individuals could be treated with intravenous cidofovir.

Immunological effects of the orally administered neuraminidase inhibitor oseltamivir in influenza virus-infected and uninfected mice.

Oseltamivir (GS4104), the ethyl ester prodrug of the carbocyclic transition state sialic acid analog GS4071, has been reported to be a striking inhibitor of influenza A and B virus infections in mice and ferrets. Multiple studies indicate this material to also be active against the disease in humans, and it has recently been approved for human use. The effect of oral gavage (p.o.) therapy of oseltamivir on various immune factors considered to be of importance in primary influenza virus infection was studied in mice. Both uninfected animals and those infected with influenza A/NWS/33 (H1N1) virus were used. Doses of 100 mg kg(-1) day(-1) were administered twice daily for 5 days beginning 16 h pre-virus exposure. Two hours after end of treatment, the mice were killed and their spleens assayed for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell activity. Subpopulations of splenic T, T-helper, T-cytotoxic and B lymphocytes as well as macrophages were determined using flow cytometry. Similar significant (P<0.01) increases in CTL activity were seen at effector:target cell ratios of 60:1 and 30:1 in the infected mice treated with oseltamivir or with placebo. NK cell activity was greater in the infected mice than in uninfected mice; the levels in all animals were not significantly affected by treatment with oseltamivir. Macrophage, T, T-helper, T-cytotoxic and B lymphocyte populations were similar in both treated and untreated animals. These data indicate treatment with oseltamivir does not adversely affect the primary in vivo cellular immune responses to influenza virus infection assayed in this study. The experiment was repeated to show that treatment with this compound significantly prevented the development of the infection and inhibited virus titers in the lung. Surviving treated mice on day 21 had mean neutralizing antibody titers of 1:208, and withstood rechallenge with the virus at this time, indicating the initial virus-inhibitory effect also did not prevent the animals from developing an adequate humoral immunity to the virus.

Influence of treatment schedule and viral challenge dose on the in vivo influenza virus-inhibitory effects of the orally administered neuraminidase inhibitor GS 4104.

Experiments were done to determine how an alteration of the treatment schedule of 5 or 32 mg/kg/day per os (p.o.) doses of GS 4104 [the ethyl ester prodrug of the neuraminidase inhibitor (3R, 4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cylohexene-1 -carboxylic acid (GS 4071)] would affect influenza A (H1N1) virus infection in mice. Treatments with a low dose, one, two, three or four times daily, were highly inhibitory, unless therapy was terminated relatively early in the infection (days 2-3), in which case efficacy was curtailed. Single administrations at various times relative to virus exposure had essentially no effect. The 32 mg/kg/day dose was significantly inhibitory using all treatment schedules. These data indicated a requirement for the compound to be in the host when lung virus titres were reaching maximal levels and, for minimally effective doses, that at least continued daily therapy was needed to maintain adequate serum levels to achieve an appropriate antiviral effect. Twice daily p.o. treatment for 5 days with 20 mg/kg/day of GS 4104 totally prevented deaths in mice receiving high viral challenge doses that were sufficient to kill placebo-treated controls in less than 5 days. Other parameters of antiviral efficacy (lung consolidation, arterial oxygen saturation, lung virus titres) were also markedly inhibited regardless of viral challenge doses. These data provide further insights into how the maximum therapeutic benefit can be derived from use of this orally effective influenza virus neuraminidase inhibitor.

Utilization of transgenic mice replicating high levels of hepatitis B virus for antiviral evaluation of lamivudine.

A recently developed transgenic mouse strain which expresses high levels of hepatitis B virus (HBV) was studied as a model for evaluation of potential chemotherapeutic agents. Lamivudine ([-]2'-deoxy-3'-thiacytidine), known to reduce hepatitis B viremia in human patients, and zidovudine (3'-azido-3'-deoxythymidine), previously shown to be ineffective for HBV infections in man, were used in parallel in this transgenic animal model. Orally administered lamivudine at dosages of 100, 50, and 25 mg/kg per day given once a day for 21 days significantly decreased serum and liver HBV DNA titers in a dose-responsive manner. Zidovudine (approximately 22 mg/kg per day) administered in the drinking water for 21 days was not effective in reducing these HBV parameters as compared to transgenic placebo-treated controls. The serum HBV DNA titers rebounded to high levels 1 week after cessation of lamivudine treatment. Male and female mice responded in a similar manner to these therapies. The results using this transgenic mouse model were similar to what would be predicted from treatment of HBV-infected human patients with lamivudine and zidovudine, and indicate these mice may be useful as a small animal chemotherapeutic model for study of potential HBV inhibitors.

Inhibition of influenza virus infections in mice by GS4104, an orally effective influenza virus neuraminidase inhibitor.

The carbocyclic transition state sialic acid analog GS4071 ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexane-1 -carboxylic acid), a potent influenza virus neuraminidase inhibitor, was highly inhibitory to influenza A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), A/Shangdong/09/93 (H3N2) and B/Hong Kong/5/72 viruses in Madin Darby canine kidney (MDCK) cells. The 50% effective concentrations in these experiments ranged from 1.8 to 59.5 microM, with no cytotoxicity evident at 1000 microM, using inhibition of viral cytopathic effect determined visually and by neutral red dye uptake. The ethyl ester prodrug of GS4071, GS4104, administered by oral gavage (p.o.), had significant inhibitory effects on infections in mice induced by these viruses. Antiviral effects were seen as prevention of death, increase in mean day to death, inhibition of decline of arterial oxygen saturation, lessened lung consolidation and inhibition of infectious virus recovered from the lungs. No toxicity was seen in dosages up to 100 mg/kg/day (highest evaluated). Comparison experiments run versus the influenza A (H1N1) virus-induced infection using GS4104, GS4071 and the neuraminidase inhibitor zanamivir (GG167, 4-guanidino-Neu5Ac2en), all administered p.o., indicated a 10-fold or greater potency for inhibiting the infection by GS4104. The minimum effective dosage for GS4104 was 0.1 mg/kg/day, with the compound administered twice daily for 5 days beginning 4 h pre-virus exposure. Oral therapy with GS4104 could be delayed from 48 to at least 60 h after exposure of mice to influenza A (H1N1) virus and still render a significant antiviral effect, the time of delay being dependent on the viral challenge dose. Intranasal instillation of GS4071 and GG167 to mice infected with influenza virus was highly inhibitory to the infection, the minimum effective dosages to significantly prevent death being 0.01 mg/kg/day for GS4071 and 0.1 mg/kg/day for GG167. Caging of infected mice treated with 10 mg/kg/day of GS4104 with infected saline-treated animals did not transfer any influenza-inhibitory effect to the latter animals. These data provide strong evidence of the potential of orally administered GS4104 for treatment of influenza A and B virus infections in humans.

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice.

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.