Mortality in mice infected with an amyocarditic coxsackievirus and given a subacute dose of mercuric chloride

An amyocarditic strain of coxsackievirus B3 (CVB3/0) induces heart damage when inoculated into selenium (Se)-deficient mice. Mercury (Hg), an Se antagonist, is known to aggravate viral infections. The experiments reported here assessed the effect of prior Hg treatment in mice subsequently inoculated with an amyocarditic strain of coxsackievirus. A pilot study showed that under our conditions the maximum tolerated dose of HgCl2 in uninfected mice was 6 mg HgCl2/kg body weight. In the main study, doses of 0, 3 or 6 mg HgCl2/kg body weight were administered intraperitoneally (ip) to 7-wk-old male mice fed a standard chow diet. Two hours later, half the mice were inoculated ip with CVB3/0. Ten days postinoculation, no mortality was observed in mice given only virus. In mice not given virus, 10% injected with 6 mg HgCl2/kg body weight died. On the other hand, 64% of the mice given both virus and 6 mg HgCl2/kg body weight died. Fifteen percent of the hearts from virus-infected mice given 3 mg HgCl2/kg body weight and 33% of the hearts from virus-infected mice given 6 mg HgCl2/kg body weight exhibited a higher incidence of lesions than hearts from mice-given virus alone. Moreover, viral heart titers were elevated in infected mice injected with 6 mg HgCl2/kg body weight compared to infected mice receiving no Hg. Thus, an amyocarditic coxsackievirus given to mice after a nonlethal subacute dose of Hg results in mortality, increased incidence of heart lesions, and elevated viral heart titers. These results demonstrate the important role of toxic elements in determining the severity of viral infections.     

Astragaloside IV exerts antiviral effects against coxsackievirus B3 by upregulating interferon-gamma

Coxsackievirus B3 (CVB3) is a major pathogen for viral myocarditis and dilated cardiomyopathy in children and young adults. The aim of this study was to determine the antiviral effects of astragaloside IV against CVB3, and the underlying mechanism. First, we evaluated antiviral effects of astragaloside IV in vitro by measuring the virus titers of CVB3 in primarily cultured myocardial cells infected with CVB3, and in vivo by assessing the morbidity, mortality, heart-to-body weight ratio (HW/BW), and virus titers in BALB/c mice infected with CVB3. Then, we performed serum pharmacological experiments by testing the effect of sera from SD rats treated with astragaloside IV on proliferation of CVB3 in primarily cultured myocardial cells. Finally, we determined the effect of astragaloside IV on IFN-gamma mRNA expression in the hearts of infected BALB/c mice. We observed that astragaloside IV decreased virus titers of CVB3 in primarily cultured myocardial cells. Morbidity, mortality, HW/BW, and virus titers all decreased, and necrosis and mononuclear cell infiltration were alleviated in CVB3-infected mice treated with astragaloside IV, compared with those infected mice without the treatment. In addition, proliferation of CVB3 was inhibited by the sera of rats treated with astragaloside IV. Moreover, we observed that IFN-gamma mRNA expression was increased in mice treated with astragaloside IV. Therefore, we conclude that astragaloside IV exerts antiviral effects against CVB3 by upregulating expression of IFN-gamma mRNA.     

Polybrominated diphenyl ether exposure suppresses cytokines important in the defence to coxsackievirus B3 infection in mice

Environmental pollutants can adversely affect the immune system. The host defence during infection depends on cytokine signalling and proper function of immune cells. However, no studies have addressed how polybrominated diphenyl ethers (PBDEs) affect cytokine responses. We investigated the combined effects in Balb/c mice of human coxsackievirus B3 (CVB3) infection and exposure to PBDEs (BDE-99 or Bromkal mixture) on 21 serum cytokines. The mice were infected (i.p.) on day 0, orally treated with BDE-99 or Bromkal on day 1 (20 mg/kg bw) and put to death on day 3. CVB3 was quantitatively measured in the liver and pancreas by RT-PCR. The Luminex 200 multi-analyte system was used for cytokine analysis. High numbers of viral copies were found in the liver and pancreas. Infection increased TNF-α, IL-6, MCP-1, IL-12p40, KC and RANTES levels. Notably, PBDE-exposure resulted in a marked decrease, or even lack, of IL-13, MIP-1β, RANTES, IFN-γ and KC levels in non-infected mice. However, the effects of PBDE-exposure on cytokines did not affect viral replication during early CVB3 infection. In conclusion, PBDEs causes a selective block in immune signalling pathways but the consequences of this need to be further studied in different host resistance models of infection.     

Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20 μg YW002 per mouse. When adjusted the amount at 10 μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.     

The comparison of α-bromo-4-chlorocinnamaldehyde and cinnamaldehyde on coxsackie virus B3-induced myocarditis and their mechanisms

Early experiments showed cinnamaldehyde had obvious therapeutic effect on viral myocarditis, but cinnamaldehyde was unstable in vivo. To overcome this limitation, we used cinnamaldehyde as a lead compound to synthesize α-bromo-4-chlorocinnamaldehyde (BCC). In the present study, we compared the therapeutic effects of BCC with cinnamaldehyde on coxsackie virus B3 (CVB3)-induced viral myocarditis (VMC), as well as investigated the possible mechanism. The antiviral and cytotoxic effects in vitro were evaluated on HeLa cells infected by CVB3 and rat cardiomyocytes respectively. Our results showed that IC50 were 0.78±0.13 μM and 48.16±5.79 μM in BCC and cinnamaldehyde-treated cells. 50% toxic concentration (TC) in BCC-treated cells was 22-fold higher than in the cinnamaldehyde group. In vivo BALB/c mice were infected with CVB3 for establishing VMC models. The results demonstrated that BCC reduced the viral titers and cardiac pathological changes in a dose-dependent manner. Myocardial virus titers were significantly lower in the 50 mg/kg BCC-treated group than in cinnamaldehyde groups. In addition, BCC could significantly inhibit the replication of CVB3 mRNA and the secretion of inflammatory cytokines TNF-α, IL-β and IL-6 in CVB3-infected cardiomyocytes. We further observed that BCC suppressed CVB3-induced NF-κB activation, IκB-α degradation and phosphorylation in a concentration-dependent manner, and reduced Toll like receptor (TLR) 4 protein level in hearts. These results suggest that BCC had a promising therapeutic effect on VMC with a highly significant favorable effects and less toxicity than cinnamaldehyde. Furthermore, the effect of BCC on VMC might be through inhibition of inflammatory signaling.     

维拉帕米对CVB_3感染心肌细胞Ca~(2+)内流及CVB_3-RNA含量的影响

观察了维拉帕米(Verapamil,Ver)对感染柯萨奇B_3病毒(CVB_3)的大鼠培养心肌细胞Ca~(2+)内流及CVB_3-RNA复制的影响。结果发现在感染48h后,Ver对感染细胞及正常对照的Ca~(2+)内流均有显著的抑制作用(P<0.01);若在病毒感染同时加入Ver,经48h培养后,细胞中CVB_3-RNA含量显著高于病毒对照组(P<0.05)。提示钙拮抗剂(如Ver)可减少病毒感染引起的心肌Ca~(2+)内流增加,有可能减轻感染细胞的继发性Ca~(2+)损伤;但Ver会促进病毒RNA的复制,提示在急性病毒性心肌炎临床上用Ver治疗心律失常时宜慎重。     

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.     

蛋白酶体抑制剂MG-132对柯萨奇B3病毒性心肌炎小鼠的作用研究

目的：研究泛素蛋白酶体抑制剂MG-132对柯萨奇B3（CVB3）病毒性心肌炎小鼠的作用,探讨泛素蛋白酶体系统在病毒性心肌炎发病学中的作用机制。方法：随机将80只雄性BALB／C小鼠分为4组（,2—20）：正常对照组、心肌炎组、心肌炎＋处理组、正常＋处理组。腹腔接种CVB3诱发急性心肌炎,次日腹腔注射MG-132,0．75mg／kg;连续给药7d,对照组腹腔注射PBS。第8天小鼠取材,观察心脏病理变化,测定心肌CVB3病毒复制及血清肌钙蛋白、脑钠肽水平。结果：MG-132显著减轻心肌炎小鼠心脏病理损伤,显著降低心脏重量／身体重量比值,MG-132干预后第8天小鼠血清肌钙蛋白、脑钠肽水平显著降低,同时荧光定量PCR显示CVB3mR—NA复制水平显著降低。结论：MG-132通过抑制CVB3病毒复制,显著减轻心肌炎小鼠心脏病理损伤,起到保护心肌作用。     

DNA vaccine-mediated immune responses in Coxsackie virus B3-infected mice

DNA immunizations with the major structural protein VP1 of Coxsackie virus B3 (CVB3) have been previously found to protect BALB/c mice from lethal challenge. Here we report that the other CVB3 capsid proteins, VP2, VP3, and VP4, were less effective at preventing CVB3-caused disease. The application of pCMV/VP1 as a vaccine caused decreased myocyte destruction, reduced viral load in the heart tissue, accelerated antibody induction, and an early cytokine expression in heart tissue. In summary, our results indicate that the induction of B cell and/or T cell memory in vaccinated mice prior to challenge is responsible for the protection observed.     

Viral infection and PBDE exposure interact on CYP gene expression and enzyme activities in the mouse liver

In the present study coxsackievirus B3 (CVB3) adapted to Balb/c mice was used to examine whether infection affects xenobiotic-metabolising CYP1A1 and CYP2B gene expression (measured by RT-PCR) and the corresponding enzyme activities of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD), as observed on day 3 of infection. To study the simultaneous effects of xenobiotic exposure, mice were administered the polybrominated diphenyl ether (PBDE) compounds BDE-99 (single congener) and Bromkal 70-5 DE (commercial mixture). Serum thyroxine levels were also measured. High numbers of CVB3 were found in the livers of infected mice but no significant effects of PBDE on virus replication were observed. In infected mice gene expression and CYP activities were decreased in comparison with non-infected mice, especially for CYP2B. PBDE exposure in the non-infected mice was characterised by an increase in both CYP2B and PROD levels/activities, whereas CYP1A levels increased and EROD activity decreased. In general, PBDE exposure in the infected mice did not increase EROD and PROD activities to the same extent as in the non-infected exposed mice. Infected mice exposed to BDE-99 showed significantly higher CYP2B and PROD levels than both the infected non-exposed and Bromkal-exposed groups. T(4) levels were greatly decreased by infection and a tendency of reduced T(4) levels after PBDE exposure could be observed in non-infected mice. In conclusion, infection reduced the detoxifying capacity of the liver and the serum T(4) levels. PBDE exposure can modify these effects. Notably, in the infected mice differences between BDE-99 and Bromkal were observed on CYP2B gene expression and PROD activity.     

Coxsackievirus B3 infection compromises endothelial-dependent vasodilation of coronary resistance arteries

The mechanisms of coronary artery dysfunction in coxsackievirus B3 (CVB3)-mediated viral myocarditis are poorly understood. We used pressure myography of mouse septal coronary arteries to determine the early and late effects of CVB3 infection on vascular function. Male CD-1 mice (age 6-7 weeks) were infected with CVB3 (1.75 x 10(10) pfu, i.p.). Control mice were injected with PBS. Mice were killed at 3, 7, and 42 days post infection, and the ventricular septal artery was dissected and mounted on a pressure myograph. Pressure-induced myogenic tone was similar in CVB3-infected and sham-infected mice at 3 and 7 days post infection. However, at 42 days post infection constriction of septal arteries to pressures equal to or less than 60 mm Hg was enhanced in CVB3-infected mice compared with sham controls. Agonist-induced vasodilation, as assessed by response to acetylcholine (1 nM-3 microM), was unaltered at early time points (days 3 and 7) in CVB3-infected mice. At later time points (day 42), there was a significant decrease in ACh-induced vasodilation in CVB3-infected mice. Bosentan, an ET-1 (ETA and ETB) receptor antagonist, did not completely ameliorate the reduced ACh-induced vasodilation in 42-day infected mice, indicating that ET-1 does not contribute to vascular dysfunction. Smooth muscle function, as measured by constriction to KCl or dilation to sodium nitroprusside, was unchanged in infected mice at early and late time points. Immunohistochemistry and ET-1 immunoassay were then performed to assess ET-1 levels in CVB3- and sham-infected hearts. There were no differences in ET-1 protein localization or levels at 42 days post infection in sham- and CVB3-infected animals. Finally, in situ hybridization and TUNEL staining were performed to assess viral localization and cell death in CVB3-infected hearts. There was no detectable CVB3 or TUNEL positivity in the endothelium of coronary arteries. Therefore, late impairment of endothelial-dependent vasorelaxation of coronary resistance vessels in CVB3-induced myocarditis does not appear to involve altered ET-1 expression but may be secondary to decreased stimulated NO secretion by the endothelium.     

Phyllaemblicin B inhibits Coxsackie virus B3 induced apoptosis and myocarditis

Coxsackie virus B3 (CVB3) is believed to be a major contributor to viral myocarditis since virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. In this study, we investigated the in vitro and in vivo antiviral activities of Phyllaemblicin B, the main ellagitannin compound isolated from Phyllanthus emblica, a Chinese herb medicine, against CVB3. Herein we report that Phyllaemblicin B inhibited CVB3-mediated cytopathic effects on HeLa cells with an IC₅₀ value of 7.75 ± 0.15 μg/mL. In an in vivo assay, treatment with 12 mg kg⁻¹ d⁻¹ Phyllaemblicin B reduced cardiac CVB3 titers, decreased the activities of LDH and CK in murine serum, and alleviated pathological damages of cardiac muscle in myocarditic mice. Moreover, Phyllaemblicin B clearly inhibited CVB3-associated apoptosis effects both in vitro and in vivo. These results show that Phyllaemblicin B exerts significant antiviral activities against CVB3. Therefore, Phyllaemblicin B may represent a potential therapeutic agent for viral myocarditis.     

Co-expression of interleukin-2 to increase the efficacy of DNA vaccine-mediated protection in coxsackievirus B3-infected mice

DNA immunizations with the major structural protein VP1 of coxsackievirus B3 (CVB3) have been previously found to protect mice from a lethal challenge with CVB3. The function of this vaccination procedure is mainly based on accelerated antibody induction with an early cytokine expression and increased virus-specific cytotoxic activity of spleen cells causing decreased myocyte destruction and reduced viral replication. Here, we report that the co-expression of the immune-stimulatory interleukin-2 (IL-2) can increase the efficacy of the inoculated DNA vaccine depending on the route of administration and the mouse strain used.     

卡维地洛和美托洛尔对病毒性心肌炎小鼠IL-12与IFN-γ表达水平的影响

目的：对比研究卡维地洛和美托洛尔对柯萨奇B3（CVB3）病毒性心肌炎小鼠的作用。方法：随机将110只雄性BALB/C小鼠分为4组：正常对照组（n=20）,心肌炎组（n=30）,卡维地洛组（n=30）与美托洛尔组（n=30）。后3组经腹腔接种CVB3诱发急性心肌炎,感染24h后卡维地洛组与美托洛尔组分别每日灌胃给予卡维地洛10mg/kg、美托洛尔30mg/kg,直至第14天末,分别于接种第7天和第14天随机从各组抽取若干只小鼠取血后处死并留取心脏等标本。结果：卡维地洛组显著减轻心脏病理损伤,显著降低心脏重量/身体重量（HW/BW）比值,但美托洛尔组与心肌炎组比较无统计学差异;卡维地洛干预后第7天小鼠心肌IFN-γ与IL-12含量显著升高,MDA含量显著降低,但美托洛尔组与心肌炎组比较无统计学差异;干预后第14天卡维地洛组SOD含量显著增加、MDA含量显著降低,美托洛尔组SOD和MDA含量与心肌炎组比较无统计学差异。结论：卡维地洛通过增加心肌IFN-γ与IL-12表达以及抗氧化损伤的作用,减轻心肌炎小鼠的心肌损害,起到保护性作用,而美托洛尔无类似作用。     

Oral pseudomonas aeruginosa immunization enhances survival in mice subsequently burned and infected with P. aeruginosa

Mice fed 0 serotype-specific strains of P. aeruginosa for two weeks, had increased titers of IgM but not IgG antibodies to the strains fed. Immunized mice, burned and infected with P. aeruginosa, showed significant 0 serotype-specific enhanced survival. Survival of mice fed several 0 serotype-specific strains simultaneously increased when these mice were burned and infected with P. aeruginosa homologous to those fed except when a high exotoxin A producing strain was used. Mice fed purified exotoxin A showed an increased LD₅₀ when injected with graded toxin doses. Feeding both 0 serotype-specific P. aeruginosa plus exotocin A increased survival even with burn and infection using the high toxin producing strain. We conclude that feeding P. aeruginosa antigens provides successful immunization which avoids the effects of parental adminstration.     

参麦注射液对感染柯萨奇B3病毒心肌细胞的影响

目的在细胞水平探讨参麦注射液（SMI）对实验性病毒性心肌炎的治疗作用。方法采用原代培养的SD大鼠心肌细胞感染柯萨奇B3病毒（CVB3）造成实验性病毒性心肌炎细胞模型;设立正常对照组、模型组、SMI高剂量（10g／L）组、SMI中剂量（5g／L）组及SMI低剂量（2．5g／L）组,观察心肌细胞感染CVB,后第3天和第5天的搏动频率、细胞病变、细胞超微结构、上清液中乳酸脱氢酶（LDH）的活性和CVB,滴度。结果感染CVB,后心肌细胞的搏动频率明显减慢;第5天模型组有半数细胞搏动停止,细胞病变明显,线粒体肿胀且形态不完整,内质网扩张,部分肌原纤维损坏,上清液中LDH的活性显著升高;SMI各剂量组心肌细胞均维持搏动,且高剂量组细胞病变程度、LDH活性和CVB,滴度均明显低于模型组,除部分肌原纤维损坏外,线粒体形态完整,内质网未见扩张。结论SMI对感染CVB,的培养心肌细胞搏动功能具有保护作用,并能降低心肌细胞上清液中CVB3滴度和减轻CVB,对心肌细胞的损伤。     

牛磺酸对培养大鼠心肌细胞感染 Coxsackie B_3 病毒的影响

目的：观察牛磺酸对病毒性心肌炎模型的保护作用。方法：取新生ＳＤ大鼠心室肌制备培养搏动心肌细胞，１８ｈ后接种１００ＴＣＩＤ５０的ＣｏｘｓａｃｋｉｅＢ３病毒（ＣＶＢ３）做为实验性病毒性心肌炎模型。结果：牛磺酸能剂量依赖性地减少感染ＣＶＢ３后心肌细胞乳酸脱氢酶、天冬氨酸氨基转移酶的释放，降低上清液的病毒滴度，对搏动停止、细胞病变及超微结构均有保护作用，并发现感染ＣＶＢ３后心肌细胞内牛磺酸含量下降。结论：临床应用牛磺酸治疗病毒性心肌炎有实验依据。     

C3H/HeN mouse model for the evaluation of antiviral agents for the treatment of Venezuelan equine encephalitis virus infection

The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4+/-0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-12, MCP-1, IFNgamma, TNFalpha, MIP-1alpha, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-alpha B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.     

Coxsackievirus B3 infection and PBDE exposure causes organ-specific effects on CYP-gene expression in the mouse

Common viral infections have been shown to change the tissue distribution of xenobiotics, including polybrominated diphenyl ethers (PBDEs). In previous studies, it has been shown that CYP2B gene expression is induced after PBDE exposure whereas coxsackievirus B3 (CBV3) infection suppresses the expression of CYP-gene expression in the liver. In the present study, CVB3 adapted to Balb/c mice was used to study the combined effects of infection and exposure to pure BDE-99 or the commercial mixture Bromkal on CYP1A1 and CYP2B expression in the lungs and pancreas on day 3 of the infection. The quantitative gene expression of virus, CYP1A1 and CYP2B was measured by real-time polymerase chain reaction (RT-PCR). PBDE exposure in the non-infected mice tended to increase CYP2B expression in the lungs but not in the pancreas. Infection in both non-exposed and PBDE-exposed mice increased CYP2B expression in the lungs but was non-detectable in the pancreas. In the non-infected mice PBDE exposure left the CYP1A1 expression unaltered in both the lungs and pancreas. Infection in both non-exposed and PBDE-exposed mice tended to decrease the gene expression of CYP1A1 in the lungs but to induce it in the pancreas. A correlation between the amount of virus and the gene expression of CYP2B was found in the lungs. However, no effects of PBDE on virus replication were observed in any organ. In conclusion, viral infection affects CYP-gene expression differently in the pancreas and lungs whereas PBDE-induced effects were not obvious. The organ-specific change in gene expression could explain a changed tissue distribution of xenobiotics during infection.     

Peramivir (BCX-1812, RWJ-270201): potential new therapy for influenza

The cyclopentane peramivir (BCX-1812, RWJ-270201) is a highly selective inhibitor of influenza A and B virus neuraminidases and a potent inhibitor of influenza A and B virus replication in cell culture. The in vitro potency appears to be greater than either zanamivir or oseltamivir carboxylate based on the generally lower EC₅₀ values seen using peramivir in studies run in parallel with each compound. In mice infected with influenza A or B viruses, oral treatment with peramivir was highly effective in preventing death, signs of the disease and in lowering lung virus titres. Similar effects were seen in influenza A virus-infected ferrets. Efficacy was seen in mice when therapy began after virus exposure. Peramivir is non-toxic in mice and rats at doses of ≥ 1000 mg/kg/day and ferrets tolerated doses of ≥ 100 mg/kg/day. Doses of 100 mg/kg/day do not appear to affect murine immune parameters. A pharmacokinetic study of this compound in influenza virus-infected mice indicates once-, twice- or thrice-daily oral dosing was equal in efficacy; once-daily dosing has been recommended in clinical trials of influenza therapy. Treatment of influenza virus infections in cyclophosphamide-immunosuppressed mice was effective in inhibiting the infection; an infection induced in severe combined immunodeficient mice was only weakly affected. Development of viral resistance to peramivir can occur by serial cell culture passage of the virus in the presence of the compound but the resistant virus was less virulent than the wild type virus. Viruses with neuraminidase mutations are not necessarily all cross-resistant to peramivir, zanamivir and oseltamivir carboxylate. In Phase I studies, peramivir was well-tolerated, with single or multiple oral doses up to 800 mg/kg/day evaluated. In clinical trials with patients experimentally infected with influenza A or B viruses, oral treatment with peramivir significantly reduced nasal wash virus titres with no adverse effects. Phase III clinical trials are underway.