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BACKGROUND: Unprecedented anti-epidemic measures and the widespread use of vaccines against COVID-19 have reduced the rate of hospitalization and mortality from the disease, but have not stopped the SARS-CoV-2 pandemic spread. The development of live vaccines against COVID-19, capable of providing the formation of a long-term humoral and cellular immune response and cross-protection against new SARS-CoV-2 variants of concern, is relevant. Previously at the I.I. Mechnikov Research Institute of Vaccines and Sera SARS-CoV-2 cold-adapted (ca, cold-adapted) variants were obtained. This work is aimed to search for methodological approaches that allow in vitro screening studies to assess the attenuation (att) phenotype of ca SARS-CoV-2 variants. MATERIALS AND METHODS: The SARS-CoV-2 laboratory strain Dubrovka and its variants were cultured in Vero and Calu-3 cells. Quantitation of the virus was carried out by titration in Vero cells and by real-time RT-PCR. The attenuation (att) phenotype of SARS-CoV-2 variants was determined on an animal model of COVID-19 on Syrian hamsters. RESULTS: In experiments on Syrian hamsters, the presence of the att phenotype in the ca variants of the virus was established. Animals infected with virus ca variants had significantly less weight lost, had less viral load in the lungs and brain and less pronounced pathological changes in the lungs compared to infection with the virulent strain. In vitro experiments on Vero and Calu-3 cells revealed probable attenuation markers of the virus ca variants for syrian hamsters: (1) ability to reproduce at low temperature (ca phenotype); (2) inability to reproduce at 39 C (ts phenotype); (3) changes in the species and tissue specificity of the virus. CONCLUSIONS: The developed methodological approaches to the identification of SARS-CoV-2 attenuation markers are a valuable tool for monitoring the stability of the phenotype of candidate vaccine strains.

The World Health Organization (WHO) recommends antivirals as an additional line of defense against influenza. One of such drugs is rimantadine. However, most of the circulating strains of influenza A viruses are resistant to this drug. Thus, a search for analogs effective against rimantadine-resistant viruses is of the utmost importance. Here, we examined the efficiency of two adamantane azaheterocyclic rimantadine derivatives on a mouse model of pneumonia caused by the rimantadine-resistant influenza A virus /California/04/2009 (H1N1). BALB/c mice inoculated with the virus were treated with two doses (15 mg and 20 mg/kg a day) of tested analogs via oral administration for 5 days starting 4 hours before the infection. The efficacy was assessed by survival rate, mean day to death, weight loss, and viral titer in the lungs. Oral treatment with both compounds in both doses protected 60100% of the animals, significantly increased the survival rate, and abolished weight loss. The treatments also inhibited virus titer in the lungs in comparison to the control group. This treatment was more effective compared to rimantadine at the same scheme and dosage. Moreover, the study of the sensitivity of the virus isolated from the lungs of the treated mice and grown in MDCK cells showed that no resistance had emerged during the 5 days of treatment with both compounds.

Hepatitis B virus (HBV) is a partially double-stranded DNA virus that specifically targets hepatocytes. It is considered a major health issue due to its high prevalence and the life-threatening consequences of chronic infection, including liver cirrhosis and hepatocellular carcinoma. Despite widespread vaccination against HBV, millions of people live with chronic HBV infection. Existing antiviral therapies fail to achieve full HBV elimination, so most patients with the disease require lifelong treatment. The search for new antiviral therapy strategies is hindered by the limited availability of in vitro HBV infection models that are able to support the full HBV life cycle. Therefore, the development and optimization of cellular models are crucial to the search for drugs effective against HBV. In this study, we optimized an in vitro HBV infection model consisting of two cell lines: HepAD38 cells, which are able to produce infectious HBV; and HepG2-NTCP cells, which are susceptible to HBV infection. We showed that prolonged production of HBV in the "donor" cells and HBV inoculation of the "acceptor" cells simultaneously with seeding improves the established procedure. This modified protocol was proven effective in experiments involving compounds with known activity against HBV, suggesting its utility for future high-throughput screening. Keywords: HBV; HBV in vitro models; HepG2-NTCP; HepAD38.

Background The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. Objectives To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. Methods The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. Results Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin–Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. Conclusions Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.

An analysis of coronavirus infection in Russia and evaluation of different AVT regimens effectiveness.The study involved a retrospective analysis of 1082 patient records with laboratory-confirmed COVID-19 in 17 regions of Russia. The number of men and women was equal, mean age 48.718.1 (median 50). Patients with moderate COVID-19 (85%) versus mild COVID-19 (15%) were characterized by higher age (median 54 vs 21 years; p0.001), higher body mass index (27.8 vs 23.4; p0.001), prevalence of chronic diseases (75.3% vs 8.5%; p0.001), including circulatory system diseases (37.8%). Moderate COVID-19 characterized higher intoxication (10.86.1 vs 4.22.7 days; p0.001) and catarrhal symptoms duration (10.25.4 vs 6.14.1 days; p0.001).During hospitalization 92% of the patients received AVT, 77% antibiotics, and 16% corticosteroids. Umifenovir therapy resulted in a significant reduction of intoxication (8.75.5 vs 11.75.5 days; p0.001) and catarrhal symptoms duration (8.85.1 vs 12.04.9 days; p0.001) compared to the group without AVT. The usage of INF reduced intoxication symptoms compared with the group without AVT (8.97.5 vs 11.75.5; p0.05). Therapy with hydroxychloroquine, imidazolylethanamide pentandioic acid, and lopinavir + ritonavir combination did not affect the course of COVID-19. Most of adverse reactions were related to antibiotics.Umifenovir therapy and inclusion of interferon in AVT regimens was associated improvement in the clinical manifestation of the disease among patients.Обоснование. В течение 2020 г. в Российской Федерации был рекомендован к применению ряд этиотропных препаратов, однако вопрос противовирусной терапии (ПВТ) COVID-19 остается открытым. Цель. Фармакоэпидемиологический анализ коронавирусной инфекции COVID-19 на территории РФ с февраля по ноябрь 2020 г. и оценка эффективности разных схем ПВТ. Материалы и методы. Анализируемая популяция включала 1082 пациента с лабораторно подтвержденным диагнозом COVID-19, проходивших стационарное лечение в лечебных учреждениях в 17 регионах РФ. Средний возраст составил 48,718,1 года (медиана возраста 50 лет), равное число мужчин и женщин. Среднетяжелая форма COVID-19 (85% пациентов) по сравнению с легкой (15%) характеризовалась большим возрастом пациентов (медиана возраста 54 vs 21 год; p0,001), индексом массы тела (27,8 vs 23,4; p0,001), частотой хронических заболеваний в анамнезе (75,3% vs 8,5%; p0,001), в основном заболевания системы кровообращения (37,8%). Клиническая картина среднетяжелой формы COVID-19 отличалась более длительной продолжительностью симптомов интоксикации (10,86,1 vs 4,22,7 дня; p0,001), катаральных симптомов (10,25,4 vs 6,14,1 дня; p0,001). Результаты. Во время лечения в стационаре 92% пациентов получали ПВТ, 77% антибактериальные препараты, 16% кортикостероиды (дексаметазон). Терапия умифеновиром приводила к значимому снижению продолжительности интоксикационного (8,75,5 vs 11,75,5 дня; p0,001) и катарального (8,85,1 vs 12,04,9 дня; p0,001) синдромов в сравнении с группой без ПВТ. Применение интерферона снижало продолжительность симптомов интоксикации в сравнении с группой без ПВТ (8,97,5 vs 11,75,5 дня; p0,05). Терапия гидроксихлорохином, имидазолилэтанамид пентандиовой кислотой и комбинацией лопинавир + ритонавир не влияла на течение COVID-19. Нежелательные реакции в основном были связаны с приемом антибиотиков. Заключение. Умифеновир снижал продолжительность интоксикационных и катаральных симптомов. Включение интерферона в схемы ПВТ также приводит к улучшению клинической картины.

The enzymatic depolymerization of fucoidans from brown algae allowed the production of their standardized derivatives with different biological activities. This work aimed to compare the antiviral activities of native (FeF) and modified with enzyme (FeHMP) fucoidans fromF. evanescens. The cytotoxicity and antiviral activities of the FeF and FeHMP against herpes viruses (HSV-1, HSV-2), enterovirus (ECHO-1), and human immunodeficiency virus (HIV-1) in Vero and human MT-4 cell lines were examined by methylthiazolyltetrazolium bromide (MTT) and cytopathic effect (CPE) reduction assays, respectively. The efficacy of fucoidans in vivo was evaluated in the outbred mice model of vaginitis caused by HSV-2. We have shown that both FeF and FeHMP significantly inhibited virus-induced CPE in vitro and were more effective against HSV. FeF exhibited antiviral activity against HSV-2 with a selective index (SI) > 40, and FeHMP with SI ˃ 20, when they were added before virus infection or at the early stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that after intraperitoneal administration (10 mg/kg), both FeF and FeHMP protected mice from lethal intravaginal HSV-2 infection to approximately the same degree (44–56%). Thus, FeF and FeHMP have comparable potency against several DNA and RNA viruses, allowing us to consider the studied fucoidans as promising broad-spectrum antivirals. The study showed that standardized fucoidan with a regular structure as well as native fucoidan have comparable potency against a range of DNA- and RNA-containing viruses associated with severe human pathology.

Antiviral drugs can play a significant role in the control of influenza. Umifenovir (Arbidol) is licensed and widely used in Russia for the prophylaxis and/or treatment of influenza. We evaluated the susceptibility to umifenovir of reference influenza A and B viruses and influenza A viruses isolated from patients in the ARBITR clinical trial in 2012-2014 seasons. Using an MDCK cell-based enzyme-linked immunosorbent assay (ELISA), we showed that the replication of antigenically dominant human influenza A and B viruses was efficiently inhibited by umifenovir. The wild-type А/Perth/265/2009 (H1N1)pdm09, A/Fukui/45/2004 (H3N2), and B/Perth/211/2001 viruses and their oseltamivir-resistant counterparts were susceptible to umifenovir among in vitro laboratory assays. All 18 clinical isolates of influenza A viruses obtained before and during therapy were susceptible to umifenovir with 50% effective concentration (EC 50 ) ranging from 8.4 ± 1.1 to 17.4 ± 5.4 µM. No molecular markers of umifenovir resistance were identified in influenza viruses isolate d from patients at 3, 5, and 7 days after initiation of therapy. None of the viruses isolated before and during umifenovir therapy displayed reduced susceptibility to neuraminidase (NA) inhibitors. Thus, umifenovir is effective against influenza viruses circulating in 2012-2014 seasons, and therapy did not lead to the emergence of drug-resistant variants.

An influenza virus infection primes the host for secondary bacterial infections. We previously have shown that immunity towards the influenza hemagglutinin (HA) alone reduces disease and mortality caused by pneumococcal infections after an influenza infections in mice. Whether this protective effect may be potentiated by supplementing the HA preparation with the influenza neuraminidase (NA) remains underexplored. To address this question, we employed HIV-1 Gag VLPs pseudo-typed with the influenza HA or NA from PR8 as platform to investigate immunity towards these to antigens, exclusively or in combination, in a lethal BALB/c mouse model of pneumococcal infection after vaccine-matched or -mismatched influenza virus challenge. A cocktail of vaccine-matched HA-Gag and NA-Gag VLPs fully protected from weight loss, mortality and viral replication and significantly reduced the bacterial burden in the lungs of infected animals. More importantly, immunization with this immunogen cocktail also protected 60% of animals from mortality associated with secondary bacterial infection using a heterologous H1N1 influenza virus and mice were significantly protected from weight loss and pulmonary pathogen replication in the absence of hemagglutination-inhibition and neuraminidase–inhibition antibodies. Our results indicate that influenza vaccination may profoundly influence the outcome of a sequential influenza-pneumococcal infection scenario even when the viral invader is antigenically distant to the vaccine strain. Work was supported by the RSF-FWF grant 18-45-05002 (FWF I 3490-B30)

4-Substituted 2,4-dioxobutanoic acids inhibit influenza virus cap-dependent endonuclease (CEN) activity. Baloxavir marboxil, 4, is approved for treating influenza virus infections. We describe here the synthesis and biological evaluation of active compounds, 5a-5g, and their precursors (6a, 6b, 6d, and 6e) with flexible bulky hydrophobic groups instead of the rigid polyheterocyclic moieties. In silico docking confirmed the ability of 5a-5g to bind to the active site of influenza A CEN (PDB code: 6FS6) like baloxavir acid, 3. These novel compounds inhibited polymerase complex activity, inhibited virus replication in cells, prevented death in a lethal influenza A virus mouse challenge model, and dramatically lowered viral lung titers. 5a and 5e potently inhibited different influenza genera in vitro. Precursors 6a and 6d demonstrated impressive mouse oral bioavailability with 6a, providing effective in vivo protection. Thus, these novel compounds are potent CEN inhibitors with in vitro and in vivo activity comparable to baloxavir.

The enzymatic depolymerization of fucoidans from brown algae allowed the production of their standardized derivatives with different biological activities. This work aimed to compare the antiviral activities of native (FeF) and modified with enzyme (FeHMP) fucoidans from F. evanescens. The cytotoxicity and antiviral activities of the FeF and FeHMP against herpes viruses (HSV-1, HSV-2), enterovirus (ECHO-1), and human immunodeficiency virus (HIV-1) in Vero and human MT-4 cell lines were examined by methylthiazolyltetrazolium bromide (MTT) and cytopathic effect (CPE) reduction assays, respectively. The efficacy of fucoidans in vivo was evaluated in the outbred mice model of vaginitis caused by HSV-2. We have shown that both FeF and FeHMP significantly inhibited virus-induced CPE in vitro and were more effective against HSV. FeF exhibited antiviral activity against HSV-2 with a selective index (SI) &gt, 40, and FeHMP with SI ˃ 20, when they were added before virus infection or at the early stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that after intraperitoneal administration (10 mg/kg), both FeF and FeHMP protected mice from lethal intravaginal HSV-2 infection to approximately the same degree (44&ndash, 56%). Thus, FeF and FeHMP have comparable potency against several DNA and RNA viruses, allowing us to consider the studied fucoidans as promising broad-spectrum antivirals.

Free radical reactions play an important role in biological functions of living systems. The balance between oxidants and antioxidants is necessary for the normal homeostasis of cells and organisms. Experimental works demonstrate the role of oxidative stress that is caused by influenza virus as well as the toxic effects of some antiviral drugs. Therefore, antiviral drugs should be characterized by its pro- and antioxidant activity, because it can affect its therapeutic efficiency. The aim of the study was to quantify the antioxidant capacity and propose the mechanism of the antioxidant effect of the antiviral drug Umifenovir (Arbidol&reg, ). The kinetic chemiluminescence with the 2,2'-azobis (2-amidinopropane) dihydrochloride + luminol system was used to quantify the antioxidant capacity of Umifenovir relative to the standard compound Trolox. With computer simulation, the reaction scheme and rate constants were proposed. The antioxidant capacity of 0.9 &mu, M Umifenovir (maximum concentration of Umifenovir in blood after oral administration of 200 mg) was as high as 1.65 &plusmn, 0.18 &mu, M of Trolox. Thus, the total antioxidant capacity of Umifenovir is comparable to the antioxidant capacity of Trolox. Unlike Trolox, Umifenovir reacts with free radicals in two stages. For Trolox, the free radical scavenging rate constant was k = 2000 nM&minus, 1 min.&minus, 1, for Umifenovir k1 = 300 nM&minus, 1min.&minus, 1, k2 = 4 nM&minus, 1. Slower kinetics of Umifenovir provides the prolonged antioxidant effect when compared to Trolox. This phenomenon can make a serious contribution to the compensation of oxidative stress that is caused by a viral disease and the therapeutic effect of the drug.

An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21–36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.

Background: Secondary bacterial pneumonia is an important cause of influenza-associated death in the case of lacking anti-influenza immunity, such as in the event of a vaccine mismatch. Aim: We studied the protective efficacy and antigenicity of influenza HA-VLPs in a lethal murine model of postinfluenza S.pneumoniae pneumonia imitating the situation of a vaccine match or mismatch. Methods: BALB/c mice received two vaccinations with insect-cell expressed influenza HA-VLPs (0,05 or 0,01 µg HA/mouse). At day 21 post vaccination, mice were infected with a homologous A/PR/8/34/RG (H1N1) or heterologous NIBRG-121xp (H1N1) (A/PR/8/34 reassortant with HA and NA from A/California/4/2009) influenza virus followed by S. pneumoniae infection 7 days post influenza. Antigenicity was estimated by HAI-assays, protective efficacy was assessed by morbidity and mortality and viral and bacterial murine pulmonary titers. Results: Mice receiving a single low dose of 0.05 µg HA-VLPs were fully protected from mortality as well as viral and bacterial replication in the lung when a vaccine-matched influenza virus was used for infection before S.pneumoniae challenge. When using a heterologous influenza virus before bacterial superinfection the survival rate dropped to 25% and bacterial pulmonary titers were only slightly lower than in non-vaccinated mice. Conclusions: Postinfluenza S.pneumoniae complications may be limited by a potent anti-influenza HA immunity. In this respect, VLPs are an attractive vaccine platform and tool to study the role of influenza-specific immunity in the prevention of postinfluenza bacterial pneumonia. This work was supported by the RSF-FWF grant No 18-45-05002

Antiviral drugs can play a significant role in the control of influenza. Umifenovir (Arbidol) is licensed and widely used in Russia for the prophylaxis and/or treatment of influenza. We evaluated the susceptibility to umifenovir of reference influenza A and B viruses and influenza A viruses isolated from patients in the ARBITR clinical trial in 2012-2014 seasons. Using an MDCK cell-based enzyme-linked immunosorbent assay (ELISA), we showed that the replication of antigenically dominant human influenza A and B viruses was efficiently inhibited by umifenovir. The wild-type А/Perth/265/2009 (H1N1)pdm09, A/Fukui/45/2004 (H3N2), and B/Perth/211/2001 viruses and their oseltamivir-resistant counterparts were susceptible to umifenovir among in vitro laboratory assays. All 18 clinical isolates of influenza A viruses obtained before and during therapy were susceptible to umifenovir with 50% effective concentration (EC

Pneumonia often occurs as secondary infection post influenza disease and accounts for a large proportion of the morbidity and mortality associated with seasonal and pandemic influenza outbreaks. The antiviral drug triazavirine is licensed in Russia for the treatment and prophylaxis of acute respiratory infections, including influenza A and B viruses. In the present study, we investigated the efficacy of triazavirine in a mouse model of secondary Staphylococcus aureus pneumonia following A/California/04/2009 (H1N1)pdm09 influenza virus infection. We also performed a study of the efficacy of triazavirine against the A/California/04/2009 (H1N1)pdm09 lethal influenza infection in mice. In this model, triazavirine at the dose of 25 mg/kg/day significantly enhanced the survival of animals (60% compared to 20%) and the mean survival time to death, prevented weight loss, and reduced viral titer in the lungs of mice infected with influenza virus. At doses of 50 and 100 mg/kg/day, triazavirine was highly effective in the treatment of the secondary bacterial pneumonia following influenza infection in mice. At these doses, triazavirine protected 67-75% of animals against death, increased the mean survival time to death by twofold, and reduced the virus titer by 2.2-3.0 log10TCID50/ml compared to the mice in the control group. These findings suggest the possible benefit of triazavirine treatment in reducing post influenza pneumonia incidence in humans.

The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 μM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.

In 1997, an H5N1 avian influenza A/Hong Kong/156/97 virus transmitted directly to humans and killed six of the 18 people infected. In 1999, another avian A/Hong/1074/99 (H9N2) virus caused influenza in two children. In such cases in which vaccines are unavailable, antiviral drugs are crucial for prophylaxis and therapy. Here we demonstrate the efficacy of the neuraminidase inhibitor GS4104 (oseltamivir phosphate) against these H5N1 and H9N2 viruses. GS4071 (the active metabolite of oseltamivir) inhibited viral replication in MDCK cells (EC(50) values, 7.5-12 microM) and neuraminidase activity (IC(50) values, 7.0-15 nM). When orally administered at doses of 1 and 10 mg/kg per day, GS4104 prevented death of mice infected with A/Hong Kong/156/97 (H5N1), mouse-adapted A/Quail/Hong Kong/G1/97 (H9N2), or human A/Hong Kong/1074/99 (H9N2) viruses and reduced virus titers in the lungs and prevented the spread of virus to the brain of mice infected with A/Hong Kong/156/97 (H5N1) and mouse-adapted A/Quail/Hong Kong/G1/97 (H9N2) viruses. When therapy was delayed until 36 h after exposure to the H5N1 virus, GS4104 was still effective and significantly increased the number of survivors as compared with control. Oral administration of GS4104 (0.1 mg/kg per day) in combination with rimantadine (1 mg/kg per day) reduced the number of deaths of mice infected with 100 MLD(50) of H9N2 virus and prevented the deaths of mice infected with 5 MLD(50) of virus. Thus, GS4104 is efficacious in treating infections caused by H5N1 and H9N2 influenza viruses in mice.

The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection.

In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97 (A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian H9N2 influenza virus infected two children in Hong Kong. These viruses and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the H5N1 virus, these H6N1 and H9N2 viruses may have the potential for interspecies transmission. We demonstrate that these H6N1 and H9N2 viruses are pathogenic in mice but that their pathogenicities are less than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs, but only A/HK/156/97 (H5N1) was found in the brain. After three passages (P 3 ) in mouse lungs, the pathogenicity of the viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P 3 ) and A/Quail/HK/G1/97 (H9N2) (P 3 ) viruses being found in the brain. The neuraminidase inhibitor zanamivir inhibited viral replication in Madin-Darby canine kidney cells in virus yield assays (50% effective concentration, 8.5 to 14.0 μM) and inhibited viral neuraminidase activity (50% inhibitory concentration, 5 to 10 nM). Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of body weight) completely protected infected mice from death. At a dose of 10 mg/kg, zanamivir completely protected mice from infection with H9N2 viruses and increased the mean survival day and the number of survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses tested, significantly reduced the virus titers in the lungs and completely blocked the spread of virus to the brain. Thus, zanamivir is efficacious in treating avian influenza viruses that can be transmitted to mammals.

In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized., published_or_final_version