RESUMO
The A(H1N1)pdm09 virus emerged in 2009 and continues to circulate in human populations. Recent A(H1N1)pdm09 viruses, that is, A(H1N1)pdm09 viruses circulating in the post-pandemic era, can cause more or less severe infections than those caused by the initial pandemic viruses. To evaluate the changes in pathogenicity of the A(H1N1)pdm09 viruses during their continued circulation in humans, we compared the nucleotide and amino acid sequences of ten A(H1N1)pdm09 viruses isolated in Japan between 2009 and 2015, and experimentally infected mice with each virus. The severity of infection caused by these Japanese isolates ranged from milder to more severe than that caused by the prototypic pandemic strain A/California/04/2009 (CA04/09); however, specific mutations responsible for their pathogenicity have not yet been identified.
Assuntos
Sequência de Aminoácidos , Sequência de Bases , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/virologia , Animais , Feminino , Humanos , Influenza Humana/epidemiologia , Influenza Humana/virologia , Japão/epidemiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Pandemias , Filogenia , RNA Viral/genética , Índice de Gravidade de Doença , VirulênciaRESUMO
Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi-basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ-HE29-22/2017 (HE29-22) and A/duck/Japan/AQ-HE29-52/2017 (HE29-52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ-HE28-3/2016 (HE28-3)] in mice and ferrets. In mice, HE29-52 was more pathogenic than HE29-22 and HE28-3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Influenza Humana/virologia , Doenças das Aves Domésticas/virologia , Produtos Avícolas/virologia , Animais , Embrião de Galinha , Cães , Patos , Feminino , Furões , Humanos , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Japão/epidemiologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Doenças das Aves Domésticas/epidemiologiaRESUMO
Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.
Assuntos
Furões/virologia , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Animais , Antivirais/farmacologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Galinhas/virologia , Túnica Conjuntiva/patologia , Túnica Conjuntiva/virologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Influenza Aviária/virologia , Pulmão/patologia , Pulmão/virologia , Macaca/virologia , Camundongos , Neuraminidase/efeitos dos fármacos , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Replicação ViralRESUMO
The current live rabies vaccine SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333). This fact generates a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain highly and stably attenuated by multiple mutations. However, the information on attenuating mutations other than that at G333 is very limited. We previously reported that amino acids at positions 273 and 394 in the nucleoprotein (N273/394) (Leu and His, respectively) of fixed rabies virus Ni-CE are responsible for the attenuated phenotype by enhancing interferon (IFN)/chemokine gene expressions in infected neural cells. In this study, we found that amino acid substitutions at N273/394 (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 attenuated by the combination of the above attenuating mutations at G333 and N273/394, and checked its safety. Similar to the ERA-G333, which is attenuated by only the mutation at G333, ERA-N273/394-G333 did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of ERA-N273/394-G333. Further examination revealed that infection with ERA-N273/394-G333 induces IFN-ß and CXCL10 mRNA expressions more strongly than ERA-G333 infection in a neuroblastoma cell line. Importantly, we found that the ERA-N273/394-G333 stain has a lower risk for emergence of a pathogenic revertant than does the ERA-G333. These results indicate that ERA-N273/394-G333 has a potential to be a promising candidate for a live rabies vaccine strain with a high level of safety.
Assuntos
Glicoproteínas/genética , Mutação/genética , Nucleoproteínas/genética , Vacina Antirrábica/genética , Vírus da Raiva/genética , Raiva/genética , Vacinas Atenuadas/genética , Substituição de Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Genes Virais/genética , Humanos , Camundongos , Vacinação/métodos , Células VeroRESUMO
Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-ß promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-ß-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.
Assuntos
Interferons/antagonistas & inibidores , Células Musculares/virologia , Fosfoproteínas/fisiologia , Vírus da Raiva/efeitos dos fármacos , Raiva/virologia , Proteínas Virais/fisiologia , Replicação Viral , Animais , Linhagem Celular , Camundongos , Fosfoproteínas/genética , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Proteínas Virais/genética , Virulência/genéticaRESUMO
UNLABELLED: Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level of Ifn-ß gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-ß promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves. IMPORTANCE: Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.
Assuntos
Fatores Imunológicos/metabolismo , Interferon beta/antagonistas & inibidores , Fosfoproteínas/metabolismo , Isoformas de Proteínas/metabolismo , Vírus da Raiva/patogenicidade , Raiva/patologia , Proteínas Estruturais Virais/metabolismo , Animais , Encéfalo/virologia , Linhagem Celular , Cricetinae , Fatores Imunológicos/genética , Injeções Intramusculares , Camundongos , Chaperonas Moleculares , Músculos/virologia , Fosfoproteínas/genética , Isoformas de Proteínas/genética , Raiva/virologia , Vírus da Raiva/genética , Genética Reversa , Análise de Sobrevida , Proteínas Estruturais Virais/genética , Virulência , Replicação ViralRESUMO
Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.
Assuntos
Charadriiformes/virologia , Parechovirus/classificação , Parechovirus/isolamento & purificação , Infecções por Picornaviridae/veterinária , Animais , Fezes/virologia , Ordem dos Genes , Genoma Viral , Japão , Parechovirus/genética , Filogenia , Infecções por Picornaviridae/virologia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.
Assuntos
Marsupiais/virologia , Rotavirus/isolamento & purificação , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Proteínas do Capsídeo/genética , Fezes/virologia , Feminino , Camundongos , Filogenia , Gravidez , Rotavirus/classificação , Rotavirus/genética , Infecções por Rotavirus/virologiaRESUMO
To obtain complete genome sequences of turkey rotavirus A strains Ty-1 and Ty-3, we sequenced the gene segments that had not been decoded previously. The genotype constellations of the respective strains were determined to be G17-P[38]-I4-R4-C4-M4-A16-N4-T4-E4-H4 and G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H14. Notably, their VP4 and NSP5 genes were classified into novel genotypes.
RESUMO
Previous studies revealed that rotavirus A (RVA) is present in not only the small intestine but also various organs. It was reported that RVA persisted in mesenteric lymph nodes (MLNs) in experimental models. However, there have been no reports focused on RVA in MLNs of animals under natural conditions. In this study, in order to investigate the persistence of the RVA genome in MLNs in cattle under natural conditions, reverse transcription-semi-nested PCR was carried out to detect RVA genomes in the MLNs from 17 calves that had been subjected to autopsy examinations. RVA genomes were detected in MLNs from 10 (Ë60ââ%) of the 17 autopsied calves. MLNs from 170 healthy adult cattle that had been slaughtered were also examined; 15 (â¼10ââ%) of the 170 cattle had RVA genomes in their MLNs, indicating that RNA genomes are found frequently in MLNs of cattle under natural conditions. Genetic analyses revealed that RVAs in MLNs were classified as G and/or P genotypes generally prevalent in bovines. Basically, the strains in intestinal contents were genetically identical to those in MLNs from individual cattle, suggesting that bovine RVAs have the ability to spread from the intestine to MLNs. Furthermore, amongst RVA-positive cattle, six of 10 autopsied calves and 12 of 15 healthy adult cattle were negative for the virus in the intestinal contents, indicating that bovine RVA genomes can persist in MLNs after viral clearance in the digestive tract.
Assuntos
Doenças dos Bovinos/virologia , Genoma Viral , Linfonodos/virologia , Infecções por Rotavirus/veterinária , Rotavirus/genética , Rotavirus/isolamento & purificação , Animais , Bovinos , Feminino , Genótipo , Masculino , Rotavirus/classificação , Infecções por Rotavirus/virologiaRESUMO
A total of 568 normal feces from calves on a beef farm in Fukui Prefecture, Japan, in 2011-2012 were examined by RT-semi-nested PCR for rotavirus A (RVA) VP4 genes. Through partial sequencing and BLAST analyses of 84 VP4-positive specimens, we identified an avian-like RVA strain, N2342, which shares highest nucleotide identity (80.0%) with known avian-like bovine strain 993/83, in one specimen. Phylogenetic analysis also revealed a close genetic relationship between N2342 and avian RVAs, suggesting bird-to-cattle transmission. We observed frequent contact of wild birds with calves in the farm, suggesting that these birds were the source of the virus.
Assuntos
Proteínas do Capsídeo/isolamento & purificação , Doenças dos Bovinos/virologia , Infecções por Rotavirus/veterinária , Rotavirus/classificação , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Bovinos , Regulação Viral da Expressão Gênica/fisiologia , Japão/epidemiologia , Filogenia , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/virologiaRESUMO
Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-ß) gene (Ifn-ß) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.
Assuntos
Células Musculares/virologia , Mioblastos/virologia , Nervos Periféricos/virologia , Fosfoproteínas/metabolismo , Vírus da Raiva/patogenicidade , Raiva/virologia , Proteínas Estruturais Virais/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Western Blotting , Células Cultivadas , Feminino , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Interferons/farmacologia , Camundongos , Chaperonas Moleculares , Células Musculares/metabolismo , Células Musculares/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Neuroblastoma/virologia , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Fosfoproteínas/genética , RNA Mensageiro/genética , Raiva/genética , Raiva/patologia , Vírus da Raiva/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Rabdomiossarcoma/virologia , Proteínas Estruturais Virais/genética , Virulência , Replicação ViralRESUMO
Polymerase chain reaction (PCR) coupled with direct sequencing of the product of the hexon gene was applied to avian adenoviruses (formerly group I-III). The expected sizes of DNA fragments were successfully amplified by PCR from all of the group I-III avian adenoviruses with our designed primers. The resulting PCR product contained diagnostically relevant hexon sequences that could be used to identify the group and type of avian adenovirus.