Microbiology Independent Research Journal (MIR Journal)
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Effect of triazavirine on the outcome of a lethal influenza infection and secondary bacterial pneumonia following influenza in mice
Get PDFPneumonia 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.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
Influenza vaccines manufacturing in continuous cell lines: problems and solutions
Get PDFIn order to decrease the morbidity and mortality caused by seasonal influenza outbreaks, several hundred million vaccine doses are produced worldwide each year. The predominant substrate for the production of the influenza vaccine today is fertilized hen’s eggs. The substitution of the technology based on living organisms by the cell culture-based process offers many advantages, including easier scalability and reduced dependence on the availability of eggs. The African green monkey kidney and Madin Darby canine kidney cell lines support the efficient growth of influenza viruses of different subtypes and, therefore, are considered to be the two most promising alternative substrates for the production of the human influenza vaccine. However, the pH of endosomes in both of these cell lines is higher than the pH essential for triggering a conformational change of the hemagglutinin (HA) of human influenza viruses, which enables the viral-cellular membrane fusion. This mismatch gives rise to mutations in the HA that lead to an increase of the optimum pH of HA conformational change. As of a result of these mismatches, the HA, and consequently the whole virus, has reduced stability to low pH and elevated temperatures. The production of a vaccine from less stable virus will lead to an elevated HA content in the low pH conformation that can affect the safety, potency, infectivity, and protective efficacy of the final inactivated and live attenuated influenza vaccines. The main limitations of the cell line-based influenza vaccine technology and the possibilities to preserve the viral stability over the course of influenza vaccine production are discussed in the review.In order to decrease the morbidity and mortality caused by seasonal influenza outbreaks, several hundred million vaccine doses are produced worldwide each year. The predominant substrate for the production of the influenza vaccine today is fertilized hen’s eggs. The substitution of the technology based on living organisms by the cell culture-based process offers many advantages, including easier scalability and reduced dependence on the availability of eggs. The African green monkey kidney and Madin Darby canine kidney cell lines support the efficient growth of influenza viruses of different subtypes and, therefore, are considered to be the two most promising alternative substrates for the production of the human influenza vaccine. However, the pH of endosomes in both of these cell lines is higher than the pH essential for triggering a conformational change of the hemagglutinin (HA) of human influenza viruses, which enables the viral-cellular membrane fusion. This mismatch gives rise to mutations in the HA that lead to an increase of the optimum pH of HA conformational change. As of a result of these mismatches, the HA, and consequently the whole virus, has reduced stability to low pH and elevated temperatures. The production of a vaccine from less stable virus will lead to an elevated HA content in the low pH conformation that can affect the safety, potency, infectivity, and protective efficacy of the final inactivated and live attenuated influenza vaccines. The main limitations of the cell line-based influenza vaccine technology and the possibilities to preserve the viral stability over the course of influenza vaccine production are discussed in the review
Применение микрочипов для идентификации происхождения генов вирусов гриппа диких птиц
Get PDFForty-two strains of avian influenza viruses were isolated from the wild waterfowl’s feces in the city of Moscow. These viruses as well as reference strains and some experimental reassortants were analyzed by microarrays. The used microarrays contained 176 probes to the different segments of influenza virus genome. The microarray allows to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons and substitution N66S in the reading frame of the viral protein PB1-F2; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of H3N1, H3N6, H3N8, H4N6, H1N1, H5N3 and H11N9 subtypes were identified from the group of wild bird’s isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos, were infectious and immunogenic for mice, but did not induce any clinical symptoms.В черте города Москвы из фекалий диких водоплавающих птиц изолировали 42 штамма вируса гриппа птиц и проанализировали их на микрочипах «Биогрипп», которые содержат 176 зондов к различным участкам генома вирусов гриппа. Микрочип позволяет определять: 1) субтип поверхностных белков гемагглютинина и нейраминидазы; 2) структуру С-концевой последовательности вирусного белка NS1, влияющую на степень ингибирования транскрипции клеточных хозяйских генов, в том числе ответственных за синтез интерферона; 3) наличие стоп-кодонов и мутацию N66S в рамке считывания вирусного белка PB1-F2, 4) наличие полиосновного сайта протеолитического расщепления гемагглютинина. Среди изолятов от диких птиц идентифицированы вирусы гриппа субтипов H3N1, H3N6, H3N8, H4N6, H1N1, H5N3 и H11N9. Все они содержали последовательность ESEV на С-конце белка NS1, полноразмерную рамку считывания для белка PB1-F2. Замена N66S в PB1-F2 обнаружена у шести штаммов. Однако такие маркеры патогенности, как последовательность ESEV (лиганд PDZ-домена) в вирусном белке NS1 и замена N66S PB1-F2 в контексте генома вирусов гриппа диких уток, не делали вирус патогенным для мышей. Все изоляты были высокоурожайны в куриных эмбрионах, инфекционны и иммуногенны для мышей, но не вызывали у этих животных клинических симптомов заболевания
The use of microarrays for the identification of the origin of genes of avian influenza viruses in wild birds
Get PDFForty-two strains of avian influenza viruses were isolated from the wild waterfowls’ feces in the city of Moscow. These viruses, as well as reference strains and some experimental reassortants, were analyzed by microarrays. The microarrays contained 176 probes to the different segments of influenza virus genome. The microarray helps to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons in the reading frame of PB1-F2 as well as the N66S substitution in the PB1-F2 viral protein; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of the H3N1, H3N6, H3N8, H4N6, H1N1, H5N3, and H11N9 subtypes were identified from the group of wild birds’ isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of the ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos and were infectious and immunogenic for mice, but did not induce any clinical symptoms.Forty-two strains of avian influenza viruses were isolated from the wild waterfowls’ feces in the city of Moscow. These viruses, as well as reference strains and some experimental reassortants, were analyzed by microarrays. The microarrays contained 176 probes to the different segments of influenza virus genome. The microarray helps to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons in the reading frame of PB1-F2 as well as the N66S substitution in the PB1-F2 viral protein; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of the H3N1, H3N6, H3N8, H4N6, H1N1, H5N3, and H11N9 subtypes were identified from the group of wild birds’ isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of the ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos and were infectious and immunogenic for mice, but did not induce any clinical symptoms
Разработка холодоадаптированного реассортантного вируса гриппа А/H6N1 на основе донора аттенуации A/Ленинград/134/17/57 (H2N2) и его генотипирование методом анализа кривых плавления высокого разрешения (HRM-анализ)
Get PDFThe cold-adapted reassortant influenza virus А/17/herring gull/Sarma/2006/887 (H6N1) was developed in chicken embryos by genetic reassortment based on the A/Leningrad/134/17/57 (H2N2) master donor virus. The genome composition of the obtained reassortant was analyzed by means of real time PCR with the high resolution melting (HRM-analysis) using an intercalating fluorescent dye EvaGreen. Analysis of the DNA copies melting curves of RNA gene fragments (PB2, PB1, PA, NP, M, NS) showed that the reassortant influenza virus А/17/herring gull/2006/887(H6N1) contained the internal proteins coding genes from the master donor virus and the surface antigens coding genes from the A/herring gull/Sarma/51c/2006 (H6N1) avian influenza virus. The study of phenotypic properties showed that the А/17/herring gull/Sarma/2006/887 (H6N1) is temperature sensitive (ts) and coldadapted (ca) in chicken embryos and attenuated in mice when administered intranasally. This reassortant can be recommended as a live influenza vaccine candidate for humans.Холодоадаптированный реассортантный штамм вируса гриппа А/17/серебристая чайка/Сарма/2006/887 (H6N1) получен на основе донора аттенуации А/Ленинград/134/17/57 (H2N2) методом генетической реассортации в куриных эмбрионах. Структура генома реассортанта была проанализирована методом ПЦР в реальном времени с последующим анализом кривых плавления высокого разрешения (high resolution melting, HRM-анализ) при использовании интеркалирующего флуоресцентного красителя EvaGreen. Анализ кривых плавления ДНК копий РНК фрагментов (PB2, PB1, PA, NP, М, NS) показал, что реассортантный вирус гриппа А/17/серебристая чайка/Сарма/2006/887 (H6N1) унаследовал гены внутренних и неструктурных белков от донора аттенуации. По антигенным свойствам реассортант соответствовал вирусу гриппа птиц А/серебристая чайка/Сарма/51с/2006 (H6N1). Изучение фенотипических свойств реассортанта показало, что вирус А/17/ серебристая чайка/Сарма/2006/887 (H6N1) характеризуется температурочувствительностью и холодоадаптированностью в куриных эмбрионах и аттенуирован для мышей при интраназальном введении. Данный реассортант может быть рекомендован кандидатом в вакцинные штаммы для живой гриппозной вакцины для людей
Influence of single amino acid substitutions in the hemagglutinin on the antigenic and receptor-binding properties of influenza virus B/Florida/04/2006 of Yamagata-like evolutionary lineage
Get PDFInfluenza A and B viruses use sialylated oligosaccharide chains expressed on the surface of a host cell as the cell entry receptors. The type of the bond between sialic acid (SA) and the neighboring galactose residue (Gal) is one of the main characteristics that define the type of receptor. Influenza viruses recognize SAα2-3Gal- or SAα2-6Gal-structures on the surface of the cells. Influenza A viruses of avian origin bind α2-3-sialylated glycans, while the human strains bind preferentially α2-6-sialylated ones. However, the receptor-binding specificity of influenza B viruses has not been characterized sufficiently so far. In this study, we selected the escape mutants of influenza B/Florida/04/2006 strain (Yamagata-like lineage) using monoclonal antibodies (mAb) to hemagglutinin (HA). The analysis of the amino acid sequences of mAb-induced escape mutants revealed the single amino acid substitutions 40Tyr→His, 85His→Tyr, 202Asn→Lys and 242Ser→Arg in 10F4-, 8Н11-, 8Н3- and 9А3-induced HA variants, correspondingly. It was shown that the single amino acid substitutions 202Asn→Lys and 242Ser→Arg alter the receptor-binding specificity of the influenza B virus. These findings are important for the understanding of the influence of individual amino acid residues in HA on the receptor-binding properties of influenza B Yamagata-like lineage viruses and allow us to predict the possible ways of their evolution.Influenza A and B viruses use sialylated oligosaccharide chains expressed on the surface of a host cell as the cell entry receptors. The type of the bond between sialic acid (SA) and the neighboring galactose residue (Gal) is one of the main characteristics that define the type of receptor. Influenza viruses recognize SAα2-3Gal- or SAα2-6Gal-structures on the surface of the cells. Influenza A viruses of avian origin bind α2-3-sialylated glycans, while the human strains bind preferentially α2-6-sialylated ones. However, the receptor-binding specificity of influenza B viruses has not been characterized sufficiently so far. In this study, we selected the escape mutants of influenza B/Florida/04/2006 strain (Yamagata-like lineage) using monoclonal antibodies (mAb) to hemagglutinin (HA). The analysis of the amino acid sequences of mAb-induced escape mutants revealed the single amino acid substitutions 40Tyr→His, 85His→Tyr, 202Asn→Lys and 242Ser→Arg in 10F4-, 8Н11-, 8Н3- and 9А3-induced HA variants, correspondingly. It was shown that the single amino acid substitutions 202Asn→Lys and 242Ser→Arg alter the receptor-binding specificity of the influenza B virus. These findings are important for the understanding of the influence of individual amino acid residues in HA on the receptor-binding properties of influenza B Yamagata-like lineage viruses and allow us to predict the possible ways of their evolution
Влияние единичных аминокислотных замен в гемагглютинине вируса гриппа В/Флорида/04/2006 ямагатской эволюционной линии на антигенные и рецепторсвязывающие свойства
Get PDFInfluenza A and B viruses use sialylated oligosaccharide chains expressed on the surface of a host cell as the cell entry receptors. Type of the bond between sialic acid (SA) and neighboring galactose residue (Gal) is one of the main characteristics that define the type of receptor. Influenza viruses recognize SAα2-3Gal- or SAα2-6Gal-structures on the surface of the cells. The Yamagata-like virus strains are predominantly bound to α2,6-sialylated glycans, while Victoria-like strains are bound to both α2,3- and α2,6-sialylated glycans. However, the receptor-binding specificity of influenza B viruses has not been characterized enough. In this study, we selected escape mutants of influenza B/Florida/04/2006 strain (Yamagata-like lineage) using monoclonal antibodies (mAb) to hemagglutinin (HA). Analysis of the amino acid sequences of mAb-induced escape-mutants revealed the single amino acid substitutions 40Tyr→His, 85His→Tyr, 202Asn→Lys and 242Ser→Arg in 10F4-, 8Н11-, 8Н3- and 9А3-induced HA variants, correspondingly. It was shown that the single amino acid substitutions 202Asn→Lys and 242Ser→Arg alter the receptor-binding specificity of the influenza B virus. These findings are important for the understanding of the influence of individual amino acid residues in HA on the receptor-binding properties of influenza B Yamagata-like lineage viruses and allow us to predict the possible ways of their evolution.Известно, что рецептором для проникновения в клетку хозяина для вирусов гриппа A и В служат углеводные цепи, терминированные остатками нейраминовой кислоты. Тип связи между сиаловой кислотой (sialic acid, SA) и соседним остатком галактозы (Gal) является одной из главных характеристик, определяющих тип рецептора. Вирусы гриппа узнают на поверхности клетки SAα2-3Gal- или SAα2-6Gal-структуры. Птичьи изоляты вирусов гриппа A связываются с SAα2-3- сиалированными цепями, тогда как вирусы гриппа А человека – с SAα2-6Gal. Рецепторсвязывающая специфичность вирусов гриппа B изучена мало, однако известно, что вирусы разновидности Ямагата преимущественно узнают олигосахариды, терминированные SAα2-6Gal, тогда как вирусы генетической линии Виктория узнают оба типа сиалозидов. Используя четыре вируснейтрализующих моноклональных антитела (монАТ): 10F4, 8Н11, 8Н3 и 9А3, – мы получили эскейп-мутанты вируса гриппа В/Флорида/04/2006 ямагатской линии. При анализе последовательности гемагглютинина (HA) выявлено, что НА эскейп-мутантов, индуцированных монАТ 10F4, 8Н11, 8Н3 и 9А3, несут следующие единичные аминокислотные замены: 40Tyr→His, 85His→Tyr, 202Asn→Lys и 242Ser→Arg соответственно. Показано, что замены 202Asn→Lys и 242Ser→Arg приводят к изменению рецепторсвязывающей специфичности вируса. Полученные данные имеют важное значение для понимания роли отдельных аминокислотных остатков HA в формировании рецепторсвязывающих свойств вирусов гриппа В ямагатской эволюционной линии, что позволяет прогнозировать возможные пути эволюции этих вирусов
Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line
Get PDFThe biological basis for the restricted immunogenicity of some live attenuated influenza vaccine strains generated on the backbone of the cold adapted (ca) A/Singapore/1/1957/ca (H2N2) influenza A virus master strain and produced in the Vero cells was investigated. According to our previous results the vaccine candidate made from A/Hong Kong/1035/1998 (H1N1) Vero-derived virus did not provoke a measurable antibody titers following the intranasal immunization of humans. We report here that the hemagglutinin (HA) of A/Hong Kong/1035/1998 virus contained the mutation 10Ile→Val in the HA2 subunit, that increased the pH threshold of HA conformational change (pH of activation) by 0.3 pH units and therefore might be responsible for the lack of immune response in humans. Similar effect was shown for the reassortant made from the Vero-derived A/Switzerland/5389/1995 (H1N1) (5389wt) virus which had the HA2 mutation 3Phe→Leu leading to the lack of immune response in mice. Another factor compromising the immunogenicity of a vaccine candidate is the incompatibility of epidemic virus HA with the M gene of the master strain. In mice the 6/2 A/Switzerland/5389/1995 reassortant induced antibodies that were directed predominantly to the HA2 subunit and were detectable by ELISA but not by a hemagglutination inhibition (HAI) test. In contrast, the 5/3 reassortant, bearing the HA, neuraminidase (NA), and M genes from the epidemic virus induced an equivalent amount of antibodies against the HA1 and HA2 subunits detected by HAI and ELISA. By comparing the sensitivity of the viruses to amantadine, we showed that the M2 ion channel of the master strain had lower activity than that of the A/Switzerland/5389/1995. These data suggest that M2 of the master strain was not sufficiently active to keep the pH of the transGolgi network high enough to prevent the conformational change of the acid sensitive HA to the low pH form. Overall, the adaptation mutations in the HA of the vaccine candidate that increase the pH of HA activation as well as the incompatibility of HA and M genes must be taken into consideration when constructing the reassortant strains for the live attenuated vaccine.The biological basis for the restricted immunogenicity of some live attenuated influenza vaccine strains generated on the backbone of the cold adapted (ca) A/Singapore/1/1957/ca (H2N2) influenza A virus master strain and produced in the Vero cells was investigated. According to our previous results the vaccine candidate made from A/Hong Kong/1035/1998 (H1N1) Vero-derived virus did not provoke a measurable antibody titers following the intranasal immunization of humans. We report here that the hemagglutinin (HA) of A/Hong Kong/1035/1998 virus contained the mutation 10Ile→Val in the HA2 subunit, that increased the pH threshold of HA conformational change (pH of activation) by 0.3 pH units and therefore might be responsible for the lack of immune response in humans. Similar effect was shown for the reassortant made from the Vero-derived A/Switzerland/5389/1995 (H1N1) (5389wt) virus which had the HA2 mutation 3Phe→Leu leading to the lack of immune response in mice. Another factor compromising the immunogenicity of a vaccine candidate is the incompatibility of epidemic virus HA with the M gene of the master strain. In mice the 6/2 A/Switzerland/5389/1995 reassortant induced antibodies that were directed predominantly to the HA2 subunit and were detectable by ELISA but not by a hemagglutination inhibition (HAI) test. In contrast, the 5/3 reassortant, bearing the HA, neuraminidase (NA), and M genes from the epidemic virus induced an equivalent amount of antibodies against the HA1 and HA2 subunits detected by HAI and ELISA. By comparing the sensitivity of the viruses to amantadine, we showed that the M2 ion channel of the master strain had lower activity than that of the A/Switzerland/5389/1995. These data suggest that M2 of the master strain was not sufficiently active to keep the pH of the transGolgi network high enough to prevent the conformational change of the acid sensitive HA to the low pH form.Overall, the adaptation mutations in the HA of the vaccine candidate that increase the pH of HA activation as well as the incompatibility of HA and M genes must be taken into consideration when constructing the reassortant strains for the live attenuated vaccine