Small-molecule polymerase inhibitor protects non-human primates from measles and reduces shedding.

Measles virus (MeV) is a highly contagious pathogen that enters the human host via the respiratory route. Besides acute pathologies including fever, cough and the characteristic measles rash, the infection of lymphocytes leads to substantial immunosuppression that can exacerbate the outcome of infections with additional pathogens. Despite the availability of effective vaccine prophylaxis, measles outbreaks continue to occur worldwide. We demonstrate that prophylactic and post-exposure therapeutic treatment with an orally bioavailable small-molecule polymerase inhibitor, ERDRP-0519, prevents measles disease in squirrel monkeys (Saimiri sciureus). Treatment initiation at the onset of clinical signs reduced virus shedding, which may support outbreak control. Results show that this clinical candidate has the potential to alleviate clinical measles and augment measles virus eradication.

Valeriana jatamansi Jones Inhibits Rotavirus-Induced Diarrhea via Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway.

Rotavirus (RV), as the main cause of diarrhea in children under 5 years, contributes to various childhood diseases. Valeriana jatamansi Jones is a traditional Chinese herb and possesses antiviral effects. In this study we investigated the potential mechanisms of V. jatamansi Jones in RV-induced diarrhea. MTT assay was performed to evaluate cell proliferation and the diarrhea mice model was constructed using SA11 infection. Mice were administered V. jatamansi Jones and ribavirin. Diarrhea score was used to evaluate the treatment effect. The enzyme-linked immunosorbent assay was performed to detect the level of cytokines. Western blot and quantitative reverse transcription-PCR were used to determine protein and mRNA levels, respectively. Hematoxylin-eosin staining was applied to detect the pathological change of the small intestine. TdT-mediated dUTP nick-end labeling was conducted to determine the apoptosis rate. The results showed V. jatamansi Jones promoted MA104 proliferation. V. jatamansi Jones downregulated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in protein level, which was consistent with the immunohistochemistry results. Moreover, V. jatamansi Jones combined with ribavirin regulated interleukin-1ß (IL-1ß), interferon γ, IL-6, tumor necrosis factor α, and IL-10, and suppressed secretory immunoglobulin A secretion to remove viruses and inhibit dehydration. V. jatamansi Jones + ribavirin facilitated the apoptosis of small intestine cells. In conclusion, V. jatamansi Jones may inhibit RV-induced diarrhea through PI3K/AKT signaling pathway, and could therefore be a potential therapy for diarrhea.

Hydroxychloroquine prophylaxis and treatment is ineffective in macaque and hamster SARS-CoV-2 disease models.

We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in 2 animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg given daily) did not significantly benefit clinical outcome, nor did it reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment, neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these 2 preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.

Supplemental dietary selenium enhances immune responses conferred by a vaccine against low pathogenicity avian influenza virus.

Selenium is a trace mineral that has antioxidant activities and can influence the immune system. However, antiviral effects of selenium have not been well studies in chickens. Chickens were therefore fed diets supplemented with two levels of two different sources of selenium (organic: selenium enriched yeast; SEY or inorganic: sodium selenite; SS). Chickens in the control groups did not receive supplemental dietary selenium. At 14 and 21 days of age, chickens were vaccinated with an inactivated low pathogenicity avian influenza virus (AIV, subtype H9N2) vaccine and blood samples were collected to determine the level of antibodies using hemagglutination inhibition (HI) and ELISA. At 30 days of age, chickens were also challenged with the same virus and swab samples were collected to assess the amount of virus shedding. Antibody levels, as measured by HI, increased significantly in the chickens that received higher levels of SEY at 16 days post vaccination. ELISA titers for IgM and IgY were higher in selenium supplemented chickens. Comparing to challenged control, virus shedding was lower in organic as well as inorganic selenium treated groups. Therefore, it may be concluded that supplemental dietary selenium could enhance vaccine conferred immunity thereby impacting protection against viral challenge in chickens.

Immune memory induced by intranasal vaccination with a modified-live viral vaccine delivered to colostrum fed neonatal calves.

Bovine respiratory disease (BRD) remains a major health problem despite extensive use of vaccines during the post-weaning period. Apparent vaccine failure is attributed, in part, to primary vaccination during the period of greatest risk for BRD, providing inadequate time for onset of protective immunity. The current study investigated whether intranasal (IN) vaccination of 3-6 week old calves with a modified-live viral (MLV) vaccine induced sufficient immune memory to prevent respiratory disease and accelerate onset of protective immunity 5 months later. Vaccine groups included naïve controls, a single IN vaccination at 3-6 weeks of age, primary IN vaccination at 6 months, and either an IN or subcutaneous (SC) booster vaccination at 6 months (n = 10/group). All calves were challenged with BHV-1 four days after vaccination at 6 months of age. Primary IN vaccination at 6 months did not significantly reduce clinical disease but significantly (P < 0.01) reduced virus shedding. A single IN vaccination at 3-6 weeks of age significantly (P < 0.05) reduced weight loss but did not reduce fever or virus shedding. Both IN and SC booster vaccinations, significantly (P < 0.01) reduced clinical disease but virus shedding was significantly (P < 0.001) reduced only by IN booster vaccination. Reduction in virus shedding was significantly (P < 0.01) greater following booster versus primary IN vaccination at 6 months. All vaccination regimes significantly (P < 0.01) reduced secondary bacterial pneumonia and altered interferon responses relative to naïve controls. Only IN booster vaccination significantly (P < 0.05) increased BHV-1 specific IgA in nasal secretions. These results confirm primary MLV IN vaccination at 3 to 6 weeks of age, when virus neutralizing maternal antibody was present, induced immune memory with a 5 month duration. This immune memory supported rapid onset of protective immunity four days after an IN booster vaccination.

Immunological and pathological effects of vitamin E with Fetomune Plus® on chickens experimentally infected with avian influenza virus H9N2.

Avian influenza virus (AIV) H9N2 infection causes economic losses on poultry farms, and immunostimulants are essential for improving chicken immunity. This study evaluated the immunological and pathological effects of vitamin E with Fetomune Plus® (a commercial product based on a yeast extract and vitamins) on chickens experimentally infected with AIV H9N2. Three groups of white Hy-Line chicks were included. The G1 group was kept as an uninfected untreated control, the G2 group was intranasally infected with the AIV H9N2 strain (0.5 ml of 106 50% egg infectious dose (EID50)), and the G3 group was infected and treated with vitamin E (200 mg/kg of diet) and Fetomune Plus® (1 ml/liter of drinking water) for four weeks. The gene expression of interferon-gamma (IFN-γ), interleukin (IL)-6, and IL-2 was determined at 3, 5 and 7 days post-infection (PI). Virus shedding titers and rates and haemagglutination inhibition (HI) antibody titers were detected. Clinical signs, mortalities and post-mortem lesions were recorded. The birds were weighed, and relative organ weights were calculated. Tissue specimens were taken for histopathological examination and immunohistochemistry (IHC). The expression of IFN-γ in the duodenum revealed a significant increase in G2 compared to G3 at 3 days PI, while the duodenal and splenic expression of IL-6 was significantly increased in G2 compared to G3 at 5 days PI. IL-2 was overexpressed in the duodenum in G3 compared to G2 at 3 and 5 days PI. A significant decrease (P ≤ 0.05) in the virus shedding titer and an increase in the HI titers were detected in G3 compared to G2. The clinical signs and the mortality rate were clearly appeared in G2 than in G3. By IHC, lower H9N2 staining intensity was observed in the examined organs from G3 than in those from G2. In conclusion, as a first report, vitamin E with Fetomune Plus® supplementation for four weeks could improve the immunological and pathological effects of H9N2 infection on chickens.

A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation and gargling for the common cold.

There are no antivirals to treat viral upper respiratory tract infection (URTI). Since numerous viruses cause URTI, antiviral therapy is impractical. As we have evidence of chloride-ion dependent innate antiviral response in epithelial cells, we conducted a pilot, non-blinded, randomised controlled trial of hypertonic saline nasal irrigation and gargling (HSNIG) vs standard care on healthy adults within 48 hours of URTI onset to assess recruitment (primary outcome). Acceptability, symptom duration and viral shedding were secondary outcomes. Participants maintained a symptom diary until well for two days or a maximum of 14 days and collected 5 sequential mid-turbinate swabs to measure viral shedding. The intervention arm prepared hypertonic saline and performed HSNIG. We recruited 68 participants (2.6 participants/week; November 2014-March 2015). A participant declined after randomisation. Another was on antibiotics and hence removed (Intervention:32, Control:34). Follow up data was available from 61 (Intervention:30, Control:31). 87% found HSNIG acceptable, 93% thought HSNIG made a difference to their symptoms. In the intervention arm, duration of illness was lower by 1.9 days (p = 0.01), over-the-counter medications (OTCM) use by 36% (p = 0.004), transmission within household contacts by 35% (p = 0.006) and viral shedding by ≥0.5 log10/day (p = 0.04). We hence need a larger trial to confirm our findings.

Dietary selenium supplementation enhances antiviral immunity in chickens challenged with low pathogenic avian influenza virus subtype H9N2.

Selenium supplementation in poultry feeds has been known to have beneficial effects on the bird health and performance; however antiviral effects of selenium have remained largely unknown. In this study, we have evaluated the effects of supplementation of chicken diets with organic (Selenium Enriched Yeast; SEY) and inorganic selenium (Sodium Selenite; SS) on low pathogenicity avian influenza virus (H9N2) shedding in the cloacal and oropharyngeal swab samples as well as examined the expression of immune related genes. Chickens were fed two doses (High- 0.30 mg/kg of feed; Low- 0.15 mg/kg of feed) of selenium supplementation for 2 weeks followed by low pathogenicity avian influenza virus challenge. Our results showed that the cloacal shedding of virus in all the selenium supplemented groups was significantly lower when compared to the non-supplemented control groups. In addition, the oropharyngeal shedding of virus in chickens fed with organic selenium supplementation was significantly lower than that in the chickens that received either inorganic selenium supplemented feed or controls. Furthermore, the expression of interferon stimulated genes (Viperin, OAS: 2'-5' oligoadenylate synthetase and MDA5: melanoma differentiation-associated gene) in the cecal tonsils was significantly elevated in the selenium treated groups when compared to controls. Additionally, a significantly higher transcription of interferon (IFN)-α, IFN-ß and IFN-γ genes in the cecal tonsils and spleens of chickens receiving SEY-L and SS-H supplemented feed was also observed at post virus challenge time points compared to untreated controls. The results of this study demonstrated that supplementation of chicken diets with selenium, can enhance antiviral defense and thus, may have a beneficial effect in controlling viral infections in poultry.

Effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection - a randomised controlled trial.

Ingestion of probiotics appears to have modest effects on the incidence of viral respiratory infection. The mechanism of these effects is not clear; however, there is evidence from animal models that the probiotic may have an effect on innate immune responses to pathogens. The purpose of this randomised, placebo-controlled study was to determine the effect of administration of Bifidobacterium animalis subspecies lactis Bl-04 on innate and adaptive host responses to experimental rhinovirus challenge. The effect on the response of chemokine (C-X-C motif) ligand 8 (CXCL8) to rhinovirus infection was defined as the primary endpoint for the study. 152 seronegative volunteers who had been supplemented for 28 days, 73 with probiotic and 79 with placebo, were challenged with RV-A39. Supplement or placebo administration was then continued for five days during collection of specimens for assessment of host response, infection, and symptoms. 58 probiotic and 57 placebo-supplemented volunteers met protocol-defined criteria for analysis. Probiotic resulted in higher nasal lavage CXCL8 on day 0 prior to virus challenge (90 vs 58 pg/ml, respectively, P=0.04, ANCOVA). The CXCL8 response to rhinovirus infection in nasal lavage was significantly reduced in the probiotic treated group (P=0.03, ANCOVA). Probiotic was also associated with a reduction in nasal lavage virus titre and the proportion of subjects shedding virus in nasal secretions (76% in the probiotic group, 91% in the placebo group, P=0.04, Fisher Exact test). The administration of probiotic did not influence lower respiratory inflammation (assessed by exhaled nitric oxide), subjective symptom scores, or infection rate. This study demonstrates that ingestion of Bl-04 may have an effect on the baseline state of innate immunity in the nose and on the subsequent response of the human host to rhinovirus infection. Clinicaltrials.gov registry number: NCT01669603.

Taishan Pinus massoniana pollen polysaccharide inhibits subgroup J avian leucosis virus infection by directly blocking virus infection and improving immunity.

Subgroup J avian leucosis virus (ALV-J) generally causes neoplastic diseases, immunosuppression and subsequently increases susceptibility to secondary infection in birds. The spread of ALV-J mainly depends on congenital infection and horizontal contact. Although ALV-J infection causes enormous losses yearly in the poultry industry worldwide, effective measures to control ALV-J remain lacking. In this study, we demonstrated that Taishan Pinus massoniana pollen polysaccharide (TPPPS), a natural polysaccharide extracted from Taishan Pinus massoniana pollen, can significantly inhibit ALV-J replication in vitro by blocking viral adsorption to host cells. Electron microscopy and blocking ELISA tests revealed that TPPPS possibly blocks viral adsorption to host cells by interacting with the glycoprotein 85 protein of ALV-J. Furthermore, we artificially established a congenitally ALV-J-infected chicken model to examine the anti-viral effects of TPPPS in vivo. TPPPS significantly inhibited viral shedding and viral loads in immune organs and largely eliminated the immunosuppression caused by congenital ALV-J infection. Additionally, pre-administration of TPPPS obviously reduced the size and delayed the occurrence of tumors induced by acute oncogenic ALV-J infection. This study revealed the prominent effects and feasible mechanisms of TPPPS in inhibiting ALV-J infection, thereby providing a novel prospect to control ALV-J spread.

Egg yolk IgY antibodies: A therapeutic intervention against group A rotavirus in calves.

Bovine group A rotavirus (RVA) is considered the major cause of diarrhea in intensively reared neonatal calves. Chicken egg yolk antibodies (IgY) are efficient in protecting neonatal calves from RVA diarrhea; however, the value of this intervention in calves once diarrhea has appeared is unclear. The aim of the present study was to evaluate the application of RVA-specific IgY as a passive treatment in those cases. The experimental groups were: G1=RVA-specific IgY treatment; G2=no Ab treatment; and G3=colostrum deprived+no Ab treatment. IgY treatment significantly reduced virus shedding, diarrhea duration and severity compared to G2 and G3 calves. However, it caused a partial suppression of systemic Ab responses to RVA that could be associated with less severe diarrhea. The oral treatment with IgY for 7days was associated with significantly higher antibody secreting cell responses in the calves compared with other groups of animals.

Antiviral effect of sulfated Chuanmingshen violaceum polysaccharide in chickens infected with virulent Newcastle disease virus.

Newcastle disease virus (NDV) belonging to the Paramyxovirinae subfamily is one of the most devastating pathogens in poultry. Although vaccines are widely applied to control the infection, outbreaks of Newcastle disease (ND) repeatedly happen. Currently, there are no alternative control measures available for ND. In the present study, we found that sulfated Chuanmingshen violaceum polysaccharide (sCVPS) were potent inhibitors of NDV in specific pathogen free chickens infected with a virulent strain. With sCVPS treatment, the survival rate increased by almost 20% and virus titers in test organs, including brain, lung, spleen and thymus, were significantly decreased. The sCVPS also exhibited the ability to prevent viral transmission by reducing the amount of virus shed in saliva and feces. Higher concentrations of interferon α and γ in serum were detected in chickens treated with sCVPS, indicating that one of the antiviral mechanisms may be attributed to the property of immunoenhancement. Histopathological examination showed that sCVPS could alleviate the tissue lesions caused by NDV infection. These results suggest that sCVPS are expected to be a new alternative control measure for NDV infection and further studies could be carried out to evaluate the antiviral activity of sCVPS against other paramyxoviruses.

Dietary Enterococcus faecium NCIMB 10415 and zinc oxide stimulate immune reactions to trivalent influenza vaccination in pigs but do not affect virological response upon challenge infection.

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn(high); 50 ppm, Zn(low)). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn(high) and E. faecium groups gained weight after infection while those in the control group (Zn(low)) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn(high)+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn(high) and E. faecium groups at single time points after infection compared to the Zn(low) control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.

Duration of viral shedding of influenza A (H1N1) virus infection treated with oseltamivir and/or traditional Chinese medicine in China: a retrospective analysis.

OBJECTIVE: H1N1 was a new and potentially serious infectious disease, in human, the severity of influenza can vary from mild to severe, thus to find an effective and safety way to control the influenza pandemic is of crucial importance. This retrospective study describes the duration of viral shedding in H1N1 patients that were hospitalized and treated in China. METHODS: Clinical data were collected from May to July, 2009 in China for 963 patients with influenza A (H1N1) virus infection. Patients were treated based on the guidelines issued by the Chinese Ministry of Health. The primary outcome was duration of viral shedding and statistical comparisons were performed. RESULTS: In the patients with body temperature greater than 38.0 degrees C, there were no differences in virus shedding duration among the patients taking oseltamivir within two days, patients undergoing Traditional Chinese Medicine (TCM) therapy or those receiving no drug therapy. In patients with body temperature > or =38.1 degrees C, TCM therapy reduced the viral shedding duration (P < 0.05, vs. oseltamivir therapy). Furthermore, taking oseltamivir two days after onset of symptoms might prolong the virus shedding duration (P < 0.05, vs. taking oseltamivir less than 2 days of onset). CONCLUSION: TCM therapy is effective for reducing the length of virus shedding in patients with body temperature > or =38.0 degrees C. Oseltamivir used for reducing virus shedding duration should be taken within two days of onset.

Effect of ASP2151, a herpesvirus helicase-primase inhibitor, in a guinea pig model of genital herpes.

ASP2151 is a herpesvirus helicase-primase inhibitor with antiviral activity against varicella zoster virus and herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here, we examined the potency and efficacy of ASP2151 against HSV in vitro and in vivo. We found that ASP2151 was more potent in inhibiting the replication of HSV-1 and HSV-2 in Vero cells in the plaque reduction assay and had greater anti-HSV activity in a guinea pig model of genital herpes than did acyclovir and valacyclovir (VACV), respectively. Oral ASP2151 given from the day of infection reduced peak and overall disease scores in a dose-dependent manner, resulting in complete prevention of symptoms at the dose of 30 mg/kg. The 50% effective dose (ED(50)) values for ASP2151 and VACV were 0.37 and 68 mg/kg, respectively, indicating that ASP2151 was 184-fold more potent than VACV. When ASP2151 was administered after the onset of symptoms, the disease course of genital herpes was suppressed more effectively than by VACV, with a significant reduction in disease score observed one day after starting ASP2151 at 30 mg/kg, whereas the therapeutic effect of VACV was only evident three days after treatment at the highest dose tested (300 mg/kg). This indicated that ASP2151 possesses a faster onset of action and wider therapeutic time window than VACV. Further, virus shedding from the genital mucosa was significantly reduced with ASP2151 at 10 and 30 mg/kg but not with VACV, even at 300 mg/kg. Taken together, our present findings demonstrated the superior potency and efficacy of ASP2151 against HSV.

Paracetamol reduces influenza-induced immunopathology in a mouse model of infection without compromising virus clearance or the generation of protective immunity.

BACKGROUND: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. METHODS: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. RESULTS: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. CONCLUSIONS: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection.

Effect of vitamin supplements on HIV shedding in breast milk.

BACKGROUND: Supplementation in lactating HIV-1-infected women with preformed vitamin A and ß-carotene (VA/BC) increases the risk of mother-to-child transmission of HIV through breastfeeding. Identifying a biological mechanism to explain this unexpected finding would lend support to a causal effect. OBJECTIVE: The aim of the study was to evaluate the effect of VA/BC or multivitamin (B complex, vitamin C, and vitamin E) supplementation of HIV-infected women on HIV shedding in breast milk during the first 2 y postpartum. DESIGN: We quantified viral (cell-free) and proviral (cell-associated) HIV loads in breast-milk samples collected ≤15 d after delivery and every 3 mo thereafter from 594 Tanzanian HIV-1-infected women who participated in a randomized trial. Women received 1 of the following 4 daily oral regimens in a 2 × 2 factorial fashion during pregnancy and throughout the first 2 y postpartum: multivitamin, VA/BC, multivitamin including VA/BC, or placebo. RESULTS: The proportion of breast-milk samples with detectable viral load was significantly higher in women who received VA/BC (51.3%) than in women who were not assigned to VA/BC (44.8%; P = 0.02). The effect was apparent ≥6 mo postpartum (relative risk: 1.34; 95% CI: 1.04, 1.73). No associations with proviral load were observed. The multivitamin had no effects. In observational analyses, ß-carotene but not retinol breast-milk concentrations were significantly associated with an increased viral load in milk. CONCLUSIONS: VA/BC supplementation in lactating women increases the HIV load in breast milk. This finding contributes to explaining the adverse effect of VA/BC on mother-to-child transmission. ß-Carotene appears to have an effect on breast-milk viral load, independent of preformed vitamin A. This trial was registered at clinicaltrials.gov as NCT00197756.

In vitro evaluation of neuraminidase inhibitors using the neuraminidase-dependent release assay of hemagglutinin-pseudotyped viruses.

For the treatment of influenza virus infections, neuraminidase inhibitors (NAIs) that prevent the release of virus particles have been effective against most influenza strains. Several neuraminidase (NA) assays are available for the evaluation of NAIs. To understand the NAI functions under physiological conditions, assays mimicking viral particle release should be useful. We have constructed retrovirus-based reporter viruses that are pseudotyped with hemagglutinin (HA) glycoprotein by transfection of producer cells using plasmids expressing retroviral gag-pol, influenza HA, NA, and firefly luciferase genes. Similarly to the life cycle of influenza viruses, the release of pseudotype viruses also requires neuraminidase functions. This requirement was used to develop an assay to evaluate NAI activities by measuring inhibition of pseudotype virus production at different NAI concentrations. The pseudotype virus release assay was used to determine the IC(50) values of Oseltamivir carboxylate, Zanamivir, and the novel phosphonate congeners of Oseltamivir against N1 group neuraminidases and their H274Y Oseltamivir carboxylate-resistant mutants. The deduced IC(50) values obtained using the release assay correlated with those determined using the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (MUNANA) and also correlated with the infectivity results.

Anti-HSV activity of digitoxin and its possible mechanisms.

Herpes simplex virus type 1 (HSV-1) can establish latent infection in the nervous system and usually leads to life-threatening diseases in immunocompromised individuals upon reactivation. Treatment with conventional nucleoside analogue such as acyclovir is effective in most cases, but drug-resistance may arise due to prolonged treatment in immunocompromised individuals. In this study, we identified an in-use medication, digitoxin, which actively inhibited HSV-1 replication with a 50% effective concentration (EC(50)) of 0.05 microM. The 50% cytotoxicity concentration (CC(50)) of digitoxin is 10.66 microM and the derived selective index is 213. Several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also showed anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication and the virus release stage. The observation that digitoxin can inhibit acyclovir-resistant viruses further implicates that digitoxin represents a novel drug class with distinct antiviral mechanisms from traditional drugs.