RESUMO
We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.
Assuntos
Coinfecção , Vírus da Influenza B , Influenza Humana , Filogenia , Vírus Reordenados , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/classificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza B/classificação , Humanos , Criança , Influenza Humana/virologia , Coinfecção/virologia , Genoma Viral , Masculino , Proteínas Virais/genéticaRESUMO
The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability.
Assuntos
Variação Antigênica/imunologia , Antígenos Virais , Gammainfluenzavirus , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Cães , Gammainfluenzavirus/genética , Gammainfluenzavirus/imunologia , Células Madin Darby de Rim CaninoRESUMO
Influenza C virus is a pathogen that causes acute respiratory illness in children and results in the hospitalization of infants. The antigenicity of the hemagglutinin esterase (HE) glycoprotein is highly stable, and it is not yet known whether antigenic changes contribute to the worldwide transmission and the occurrence of outbreaks of influenza C virus. Here, we performed antigenic analysis of 84 influenza C viruses isolated in Yamagata, Japan, during a 4-year period from 2015 to 2018 and analyzed sequence data for strains of the virus from Japan and many other parts of the world. Antigenic and phylogenetic analyses revealed that 83 strains belonged to the C/Sao Paulo lineage, and two sublineage strains, the Aichi99 sublineage and Victoria2012 sublineage, cocirculated between 2016 and 2018. Aichi99 sublineage strains exhibiting decreased reactivity with the monoclonal antibody YA3 became predominant after 2016, and these strains possessed the K190N mutation. Residue 190 is located in the 190-loop on the top side of the HE protein within a region that is known to show variation that does not impair the biological activity of the protein. The Aichi99 sublineage strains possessing the K190N mutation were detected after 2012 in Europe, Australia, the USA, and Asia as well as Japan. These observations suggest that antigenic variants with K190N mutations have circulated extensively around the world and caused outbreaks in Japan between 2016 and 2018. Our study indicated that the 190-loop is an important antigenic region, and the results suggested that changes in the 190-loop have contributed to the extensive transmission of the virus.
Assuntos
Variação Antigênica/genética , Antígenos Virais/genética , Gammainfluenzavirus/genética , Influenza Humana/virologia , Sequência de Aminoácidos , Ásia , Austrália , Surtos de Doenças , Europa (Continente) , Testes de Inibição da Hemaglutinação/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas Virais/genética , Humanos , Japão , Filogenia , Análise de Sequência de DNA/métodos , Proteínas Virais de Fusão/genéticaRESUMO
We mapped the hemagglutinin-esterase (HE) antigenic epitopes of the influenza C virus on the three-dimensional (3D) structure of the HE glycoprotein using 246 escape mutants that were selected by a panel of nine anti-HE monoclonal antibodies (MAbs), including seven of the C/Ann Arbor/1/50 virus and two of the C/Yamagata/15/2004 virus. The frequency of variant selection in the presence of anti-HE MAbs was very low, with frequencies ranging from 10-4.62 to 10-7.58 for the C/Ann Arbor/1/50 virus and from 10-7.11 to 10-9.25 for the C/Yamagata/15/2004 virus. Sequencing of mutant HE genes revealed 25 amino acid substitutions at 16 positions in three antigenic sites: A-1, A-2, and A-3, and a newly designated Y-1 site. In the 3D structure, the A-1 site was widely located around the receptor-binding site, the A-2 site was near the receptor-destroying enzyme site, and the Y-1 site was located in the loop on the topside of HE. The hemagglutination inhibition reactions of the MAbs with influenza C viruses, circulating between 1947 and 2016, were consistent with the antigenic-site amino acid changes. We also found some amino acid variations in the antigenic site of recently circulating strains with antigenic changes, suggesting that viruses that have the potential to alter antigenicity continue to circulate in humans.
Assuntos
Variação Antigênica , Epitopos/química , Gammainfluenzavirus/genética , Hemaglutininas Virais/química , Proteínas Virais de Fusão/química , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Sítios de Ligação , Epitopos/genética , Testes de Inibição da Hemaglutinação , Hemaglutininas Virais/genética , Gammainfluenzavirus/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas Virais de Fusão/genéticaRESUMO
Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.
Assuntos
Anticorpos Neutralizantes/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H3N2/química , Influenza Humana/sangue , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site.IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of the influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo We previously demonstrated that the influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike that of the influenza A virus M2 protein, plays a role in virus replication.
Assuntos
Gammainfluenzavirus/fisiologia , Influenza Humana/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas da Matriz Viral/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular Tumoral , Cães , Humanos , Influenza Humana/genética , Células Madin Darby de Rim Canino , Mutação , Fosforilação/genética , Proteínas da Matriz Viral/genéticaAssuntos
Coronavirus Humano 229E/crescimento & desenvolvimento , Coronavirus Humano 229E/isolamento & purificação , Células Epiteliais/virologia , Peptidil Dipeptidase A/biossíntese , Serina Endopeptidases/biossíntese , Cultura de Vírus/métodos , Enzima de Conversão de Angiotensina 2 , Criança , Pré-Escolar , Expressão Gênica , Células HeLa , Humanos , Projetos Piloto , Sensibilidade e Especificidade , Carga ViralRESUMO
Since influenza C virus was first isolated in 1947, the virus has been only occasionally isolated by cell culture; there are only four strains for which complete genome sequences are registered. Here, we analyzed a total of 106 complete genomes, ranging from the first isolate from 1947 to recent isolates from 2014, to determine the genetic lineages of influenza C virus, the reassortment events, and the rates of nucleotide substitution. The results showed that there are six lineages, named C/Taylor, C/Mississippi, C/Aichi, C/Yamagata, C/Kanagawa, and C/Sao Paulo. They contain both antigenic and genetic lineages of the hemagglutinin-esterase (HE) gene, and the internal genes PB2, PB1, P3, NP, M, and NS are divided into two major lineages, a C/Mississippi/80-related lineage and a C/Yamagata/81-related lineage. Reassortment events were found over the entire period of 68 years. Several outbreaks of influenza C virus between 1990 and 2014 in Japan consisted of reassortant viruses, suggesting that the genomic constellation is related to influenza C virus epidemics. The nucleotide sequences were highly homologous to each other. The minimum percent identity between viruses ranged from 91.1% for the HE gene to 96.1% for the M gene, and the rate of nucleotide substitution for the HE gene was the highest, at 5.20 × 10(-4) substitutions/site/year. These results indicate that reassortment is an important factor that increases the genetic diversity of influenza C virus, resulting in its ability to prevail in humans. IMPORTANCE Influenza C virus is a pathogen that causes acute respiratory illness in children and results in hospitalization of infants. We previously demonstrated (Y. Matsuzaki et al., J Clin Virol 61:87-93, 2014, http://dx.doi.org/10.1016/j.jcv.2014.06.017) that periodic epidemics of this virus occurred in Japan between 1996 and 2014 and that replacement of the dominant antigenic group occurred every several years as a result of selection by herd immunity. However, the antigenicity of the HE glycoprotein is highly stable, and antigenic drift has not occurred for at least 30 years. Here, we analyzed a total of 106 complete genomes spanning 68 years for the first time, and we found that influenza C viruses are circulating worldwide while undergoing reassortment as well as selection by herd immunity, resulting in an increased ability to prevail in humans. The results presented in this study contribute to the understanding of the evolution, including reassortment events, underlying influenza C virus epidemics.
Assuntos
Evolução Molecular , Gammainfluenzavirus/classificação , Gammainfluenzavirus/genética , Variação Genética , Influenza Humana/virologia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Biologia Computacional , Surtos de Doenças , Genótipo , Saúde Global , Humanos , Influenza Humana/epidemiologia , Gammainfluenzavirus/isolamento & purificação , Vírus Reordenados/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
CM2 is an integral membrane protein encoded by the influenza C virus M gene. To examine the effects of the cytoplasmic tail of CM2 on its biochemical properties, deletion and substitution mutations were introduced into CM2 cytoplasmic tail at residues 47-115, and the expressed CM2 mutants were investigated. Although the cytoplasmic tail is not essential for the oligomerization of CM2, it may affect the degree of oligomerization. The residues 47-48, 67-69, 73-90 and 113-115 were all required for the proper expression of CM2. Pulse-chase experiments suggest that residues 47-48, 67-69, 73-75 and 79-87 stabilize CM2, thereby affecting CM2 expression. The C-terminal region at residues 61-115 is not essential for CM2 transport to the cell surface, and a 14-amino-acid, but not an 11-amino-acid, cytoplasmic tail is sufficient for the cell surface expression of CM2. These results suggest that either certain amino acid sequences or the length of the CM2 cytoplasmic tail are necessary for the proper conformational maturation, stability, expression level and intracellular transport of CM2.
RESUMO
UNLABELLED: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain. IMPORTANCE: The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Animais , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , RNA Viral/genética , Seleção Genética , Análise de Sequência de DNA , Cultura de VírusRESUMO
BACKGROUND: Although influenza C virus is widely distributed throughout the world, epidemiological information, based on long-term surveillance, has not yet been acquired. OBJECTIVES: To clarify the epidemiological features of influenza C virus infection, and to examine whether the prevalence of the antibodies against the influenza C virus is associated with the epidemics. STUDY DESIGN: Between 1996 and 2013, 36,973 respiratory specimens were collected from two pediatric outpatient clinics in Yamagata, Japan. The specimens were examined for the presence of influenza C virus using cell culture methods. Isolated viruses were antigenically analyzed. The differences in seropositivity, with respect to the different antigenic groups, were examined using serum samples collected in 2001 and 2011 by a hemagglutination inhibition assay. RESULTS: Influenza C viruses were isolated from 190 specimens during an 18-year period. Most influenza C viruses were isolated from winter to early summer in even-numbered years, and the frequency of virus isolation per year ranged from 0.43% to 1.73%. An antigenic analysis revealed that the dominant antigenic groups were the C/Yamagata/26/81 from 1996 to 2000, the C/Kanagawa/1/76 in 2002 and 2004, and the C/Sao Paulo/378/82 from 2006 to 2012. When compared to the other antigenic groups, the seroprevalence of the C/Sao Paulo/378/82 group was lower in 2001 for individuals older than 5 years and was higher in 2011 in individuals younger than 40 years. CONCLUSIONS: The results from our study suggest that epidemics of influenza C virus infection periodically occur and the replacement of the dominant antigenic group may be caused by immune selection within older children and/or adults in the community.
Assuntos
Antígenos Virais/genética , Epidemias , Gammainfluenzavirus/classificação , Gammainfluenzavirus/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Adolescente , Adulto , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Lactente , Recém-Nascido , Gammainfluenzavirus/genética , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral/genética , RNA Viral/isolamento & purificação , Seleção Genética , Análise de Sequência de DNA , Cultura de Vírus , Adulto JovemRESUMO
UNLABELLED: Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract. IMPORTANCE: Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.
Assuntos
Infecções por Enterovirus/diagnóstico , Enterovirus/fisiologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Aglutinação , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Códon , Enterovirus/classificação , Eritrócitos/metabolismo , Testes de Inibição da Hemaglutinação , Humanos , Dados de Sequência Molecular , Neuraminidase/metabolismo , Testes de Neutralização , Oligossacarídeos/metabolismo , Seleção Genética , Alinhamento de SequênciaRESUMO
Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.
Assuntos
Esterases/metabolismo , Gammainfluenzavirus/metabolismo , Hemaglutininas Virais/metabolismo , Temperatura , Proteínas Virais de Fusão/metabolismo , Animais , Células COS , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Gammainfluenzavirus/fisiologia , Replicação Viral/fisiologiaRESUMO
BACKGROUND: The epidemiological and clinical impacts of influenza C virus infection may have been underestimated by conventional viral culture screening alone. OBJECTIVE: To evaluate a newly developed real-time polymerase chain reaction (PCR) assay as a tool for diagnosing influenza C virus infection. STUDY DESIGN: The primers and probe for real-time PCR were designed to amplify the conserved region of the nucleoprotein gene based on the aligned sequences of nine isolates from 1967 to 2010. Respiratory specimens from children collected between January 2010 and August 2010 were examined for the presence of influenza C virus by cell culture and real-time PCR. Specimens that were positive for the virus using real-time PCR were further examined using an infectivity assay with embryonated hen's eggs. RESULTS: Of the 1203 specimens examined, 34 (2.8%) tested positive for the influenza C virus by cell culture and 51 (4.2%) tested positive by real-time PCR. The mean viral load and infectivity titer in specimens that tested positive using cell culture were 3.97×10(8)copies/ml and 5.43×10(5)EID(50)/ml, respectively, and those in specimens that were negative using cell culture were 2.18×10(6)copies/ml and 3.67×10(2)EID(50)/ml, respectively. In the clinical specimens with viral loads less than 10(5)copies/ml, it was not possible to isolate the virus using embryonated hen's eggs. The copy number-to-EID(50) ratio of the clinical specimens was much higher, ranging from 32 to 278,000, than those of culture fluid, ranging from 2.3 to 13.5. CONCLUSION: The real-time PCR assay described here can be used as a sensitive method for diagnosing influenza C virus infection.
Assuntos
Gammainfluenzavirus/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Cultura de Vírus/métodos , Adolescente , Animais , Embrião de Galinha , Pré-Escolar , Primers do DNA/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.
Assuntos
Gammainfluenzavirus/crescimento & desenvolvimento , Gammainfluenzavirus/fisiologia , Lipoilação , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular Tumoral , Galinhas/metabolismo , Galinhas/virologia , Ovos/virologia , Humanos , Gammainfluenzavirus/genética , RNA Viral/metabolismo , Recombinação Genética , Proteínas da Matriz Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/metabolismo , Replicação ViralRESUMO
CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.
Assuntos
Gammainfluenzavirus/fisiologia , Proteínas da Matriz Viral/fisiologia , Montagem de Vírus , Desenvelopamento do Vírus , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Gammainfluenzavirus/genética , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Matriz Viral/deficiência , VirossomosRESUMO
The purpose of this study was to evaluate the frequency of human metapneumovirus (hMPV) infection in adults and to determine the association between the levels of serum antibody titers and the susceptibility to reinfection. Serum samples collected at the periodic occupational medical checkup for employees of a hospital were subjected to an ELISA test. Of the 289 subjects, 288 (99.7%) had hMPV antibody titers that were more than 1:100 in May 2006. The percentage of subjects with a titer of ≥ 3,200 was significantly higher in adults aged 40-65 years old (30.2-31.5%) compared to young adults 20-39 years old (13.6%) (P < 0.05). To investigate the longitudinal course of the hMPV antibody titer, a total of 649 serum samples collected from 59 subjects who had participated in all biannual medical checkups between 2001 and 2006 were tested. We found that ten serum pairs showed a greater than fourfold increase in hMPV antibody titers. Additionally, the 5-year reinfection rate was estimated at 16.9% (10 of 59 subjects). The baseline titer before the fourfold increase ranged from 1:100 to 1:3,200, and the titer returned to baseline levels 2 or 3 years after the fourfold increase. The antibody titer of the person with the baseline titer of 1:100 showed a greater than fourfold increase twice within a year. Sixty to 80% of adults had an ELISA titer of 1:800 to 1:1,600, suggesting that such an antibody titer is not enough to protect from hMPV infection and that reinfection could occur among adults.
Assuntos
Anticorpos Antivirais/sangue , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/prevenção & controle , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunidade Humoral , Masculino , Metapneumovirus/patogenicidade , Pessoa de Meia-Idade , Testes de Neutralização , Infecções por Paramyxoviridae/virologia , Recidiva , Estudos Soroepidemiológicos , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir. RESULTS: Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356). CONCLUSION: Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Oseltamivir/uso terapêutico , Zanamivir/uso terapêutico , Adolescente , Substituição de Aminoácidos/genética , Animais , Antivirais/farmacologia , Linhagem Celular , Pré-Escolar , Cães , Humanos , Lactente , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Japão , Epidemiologia Molecular , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neuraminidase/genética , Oseltamivir/farmacologia , Filogenia , Análise de Sequência de DNA , Resultado do Tratamento , Proteínas Virais/genética , VirulênciaRESUMO
Pre-mRNAs of the influenza A virus M and NS genes are poorly spliced in virus-infected cells. By contrast, in influenza C virus-infected cells, the predominant transcript from the M gene is spliced mRNA. The present study was performed to investigate the mechanism by which influenza C virus M gene-specific mRNA (M mRNA) is readily spliced. The ratio of M1 encoded by a spliced M mRNA to CM2 encoded by an unspliced M mRNA in influenza C virus-infected cells was about 10 times larger than that in M gene-transfected cells, suggesting that a viral protein(s) other than M gene translational products facilitates viral mRNA splicing. RNase protection assays showed that the splicing of M mRNA in infected cells was much higher than that in M gene-transfected cells. The unspliced and spliced mRNAs of the influenza C virus NS gene encode two nonstructural (NS) proteins, NS1(C/NS1) and NS2(C/NS2), respectively. The introduction of premature translational termination into the NS gene, which blocked the synthesis of the C/NS1 and C/NS2 proteins, drastically reduced the splicing of NS mRNA, raising the possibility that C/NS1 or C/NS2 enhances viral mRNA splicing. The splicing of influenza C virus M mRNA was increased by coexpression of C/NS1, whereas it was reduced by coexpression of the influenza A virus NS1 protein (A/NS1). The splicing of influenza A virus M mRNA was also increased by coexpression of C/NS1, though it was inhibited by that of A/NS1. These results suggest that influenza C virus NS1, but not A/NS1, can upregulate viral mRNA splicing.
Assuntos
Gammainfluenzavirus/genética , Gammainfluenzavirus/metabolismo , Splicing de RNA/genética , Splicing de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Primers do DNA/genética , Genes Virais , Humanos , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Regulação para Cima , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
To apply serological testing for human metapneumovirus (hMPV) to large-scale sera samples, an enzyme-linked immunosorbent assay (ELISA) was developed in which purified virions were used as the antigen. The ELISA was evaluated using 102 human sera specimens from patients aged 0-59 years. There was a positive association between the ELISA results and neutralization test titers, with the correlation coefficients being greater in children <6 years old (rho=0.899, P<0.0001), which is consistent with a primary infection, than in persons >or=6 years old (rho=0.523, P<0.0001). Fifty sera samples were subjected to radioimmunoprecipitation to measure the quantity of antibodies to the fusion protein (RIP-F) and the nucleoprotein (RIP-N). The results showed significant associations between the ELISA titers and the amount of RIP-F as determined by radioimmunoprecipitation in children <6 years old (rho=0.804, P=0.0083) and in persons >or=6 years old (rho=0.577, P=0.0009). The correlation between the ELISA titer and the amount of RIP-N determined by radioimmunoprecipitation was not significant in persons >or=6 years old (rho=0.417, P=0.0829), although this correlation was significant in children <6 years old (rho=0.764, P=0.0137). The ELISA titer correlated with the amount of antibodies to the F protein, but not to the N protein. This whole virus-based ELISA will be useful for the diagnosis of hMPV infection in clinical laboratories and is also useful for the large-scale investigations, such as seroprevalence among residents of a particular region.