RESUMO
The present study was aimed to investigate the role of cannibalism in transmission of H5N1 avian influenza virus to house crows (Corvus splendens). Four crows were intranasally inoculated with 108.0 EID50 (A/crow/India/01CA249/2021) H5N1 highly pathogenic avian influenza (HPAI) virus and were observed for 14 days for any overt signs of illness. Two of the infected crows showed signs of wing paralysis, incoordination, and torticollis. For cannibalism experiment, two crows showing clinical signs were euthanized on 14th day post-infection (dpi) and were kept in the isolator and four naïve healthy crows were introduced along with the euthanized crows. The viscera from the infected carcasses were eaten by all the four crows. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. All four crows showed clinical signs viz., dullness, reluctance to move with ruffled feathers on 6th day post cannibalism along with neurological signs including incoordination and paralysis of the wings. All the crows gradually recovered after showing clinical signs and were euthanized on 21st day of observation period. Virus excretion was observed from 3rd to 11th day post cannibalism through both oropharyngeal and cloacal routes with maximum shedding through oropharyngeal route. The virus was isolated from lungs and trachea of one the infected crows at 21st day after euthanasia. All the four crows seroconverted against H5N1 virus infection at 14th day post cannibalism. Our study confirms the transmission of H5N1 virus in crows through cannibalism and highlights how H5N1 virus might circulate in a crow colony once they become infected.
Assuntos
Corvos , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Aviária , Animais , Paralisia , Ingestão de AlimentosRESUMO
The global spread of H5N1 highly pathogenic avian influenza virus (HPAIV) in poultry has caused great economic loss to the poultry farmers and industry with significant pandemic threat. The current study involved production of recombinant HA1 protein of clade 2.3.2.1a H5N1 HPAIV (rH5HA1) in E.coli and evaluation of its protective efficacy in chickens. Purification under denaturing conditions and refolding by dialysis against buffers containing decreasing concentrations of urea was found to preserve the biological activity of the expressed recombinant protein as assessed by hemagglutination assay, Western blot and ELISA. The Montanide ISA 71 VGA adjuvanted rH5HA1 protein was used for immunization of chickens. Humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. We evaluated the protective efficacy of rH5HA1 protein in immunized chickens by challenging them with homologous (2.3.2.1a) and heterologous (2.3.2.1c) clades of H5N1 HPAIV. In both the groups, the HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge. The virus shedding ratio in oro-pharyngeal swabs did not differ significantly between both the groups except on 7 days post challenge and during the entire experimental period in cloacal swabs. These results indicate that rH5HA1 was able to induce homologous and cross protective immune response in chickens and could be a potential vaccine candidate used for combating the global spread of H5N1 HPAIV threat. To our knowledge, this is the first study to report immunogenicity and protective efficacy of prokaryotic recombinant H5HA1 protein in chicken.
Assuntos
Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza , Influenza Aviária , Animais , Galinhas , Escherichia coli/genética , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/genética , Óleo Mineral , Proteínas Recombinantes/genética , Diálise RenalRESUMO
The present experiment was conducted to study the role of cytokine, chemokine and TLRs responses of H9N2-PB2 reassortant H5N1 virus as compared to non-reassortant H5N1 virus isolated from crows in BALB/c mice. Two groups (12 mice each) of 6-8 weeks old BALB/c mice were intranasally inoculated with 106 EID50/ml of viruses A/crow/India/03CA04/2015 (H9N2-PB2 reassortant H5N1) and A/crow/India/02CA01/2012 (non-reassortant H5N1). At each interval, brain, lung and spleen were collected and relative quantification of cytokines, chemokines and TLRs was done by qPCR. The H9N2-PB2 reassortant H5N1 infected mice brain, the transcripts of TLR7 were significantly higher than other cytokines at 3dpi and KC was significantly upregulated at 7dpi. In non-reassortant H5N1 infected mice brain showed, TLR 7 and IFNα upregulation at 3dpi and IFNγ and TLR7 upregulation at 7dpi. The H9N2-PB2 reassortant H5N1 infected mice lung revealed, IL2 and TLR7 significant upregulation at 3dpi and in non-reassortant H5N1 infected mice, IL6 was significantly upregulated. At 7dpi in H9N2-PB2 reassortant H5N1 virus infected group mice, IL1 and TLR 3 were significantly upregulated in lungs and in non-reassortant group mice, IL1 and TLR7 were significantly upregulated. At 3dpi in H9N2-PB2 reassortant H5N1 virus infected mice spleen, IL4, IFNα, IFNß were significantly downregulated and TLR7 transcript was significantly upregulated. In non-reassortant group mice, IL6, IFNα, IFNß and TLR 3 were significantly upregulated. At 7dpi in H9N2-PB2 reassortant H5N1 virus infected mice spleen, IFNα, IFNß and TLR7 were significantly lower than other cytokines and in non-reassortant group mice, IFNα and IFNß were significantly downregulated. This study concludes that dysregulation of cytokines in lungs and brain might have contributed to the pathogenesis of both the viruses in mice.
Assuntos
Corvos , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Animais , Galinhas , Citocinas , Vírus da Influenza A Subtipo H9N2/genética , Camundongos , Camundongos Endogâmicos BALB C , Vírus Reordenados/genéticaRESUMO
Swine influenza virus (SIV) belongs to family Orthomyxoviridae and can cause acute respiratory infection in pigs. Several pandemic H1N1 human fatal influenza cases were reported in India. Though pigs are predisposed to both avian and human influenza virus infections with the potential to generate novel reassortants, there are only a few reports of SIV in Indian pigs. We conducted a serological survey to assess the status of H1N1 infection in pigs of various states in India, between 2009 and 2016. Based on Haemagglutination inhibition (HI) assay, seroprevalence rate of H1N1 virus ranged between 5.2% (2009) and 36.3% (2011). Widespread prevalence of antibody was observed in eastern Uttar Pradesh from 6.2 to 37.5% during the study period. Co-circulation of seasonal H1N1 virus along with pandemic H1N1 virus was indicated by the presence of specific antibodies against seasonal H1N1 virus in eastern part of Uttar Pradesh. Seroprevalence rate in pigs and influenza infection trend in human shows the possible spill over transmission of influenza to pigs from human. Hence, besides serological surveillance, continuous and systematic molecular surveillance should be implemented in pig population to reduce/quantify the risk and emergence of pandemic influenza.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Infecções por Orthomyxoviridae , Doenças dos Suínos , Animais , Anticorpos Antivirais , Humanos , Índia/epidemiologia , Influenza Humana/epidemiologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Prevalência , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
In this study, we assessed the pathogenicity of two H5N1 viruses isolated from crows in mice. Eighteen 6-8 weeks BALB/c mice each were intranasally inoculated with 106 EID50/ml of H5N1 viruses A/crow/India/03CA04/2015 (H9N2-PB2 reassortant H5N1) and A/crow/India/02CA01/2012 (Non-reassortant H5N1). The infected mice showed dullness, weight loss and ruffled fur coat. Histopathological examination of lungs showed severe congestion, haemorrhage, thrombus, fibrinous exudate in perivascular area, interstitial septal thickening, bronchiolitis and alveolitis leading to severe pneumonic changes and these lesions were less pronounced in reassortant virus infected mice. Viral replication was demonstrated in nasal mucosa, lungs, trachea and brain in both the groups. Brain, lung, nasal mucosa and trachea showed significantly higher viral RNA copies and presence of antigen in immunohistochemistry in both the groups. This study concludes that both the crow viruses caused morbidity and mortality in mice and the viruses were phenotypically highly virulent in mice. The H5N1 viruses isolated from synanthropes pose a serious public health concern and should be monitored continuously for their human spill-over.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Infecções por Orthomyxoviridae/virologia , Animais , Biópsia , Corvos , Suscetibilidade a Doenças , Histocitoquímica , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , RNA Viral , Vírus Reordenados/genética , Carga Viral , Replicação ViralRESUMO
Herein, the induction of TLRs and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza (HPAI) H5N1 virus was studied. Four groups (1-4) of chickens inoculated with 106 EID50 of H9N2 virus were challenged with 106 EID50 of H5N1 virus on days 1, 3, 7 and 14 post H9N2 inoculation, respectively. In groups (1-4) TLRs and cytokines induction was studied in chicken PBMCs on day 3 post H5N1 challenge. In H5N1 control group TLRs (1, 2, 5 and 7) cytokines (IFNα, IFNß, IFNγ, IL1ß, IL2, IL4, IL8 and TGF ß3) were down regulated. In group 1 down regulation of cytokines and TLRs was similar to H5N1 control birds. Down regulation of TLRs and cytokines in H5N1 control and group 1 resulted death of all the chickens. In group 2, up-regulation of TLRs (3, 7 and 15) and induction of TNFα, IFNα, IFNß, IFNγ aided virus clearance leading to survival of all the chickens. In group 3 significant up-regulation of TLRs (3, 4 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL4, IL6, IL8, IL10 and TGF ß3) was detected. In group 4 significant up-regulation of TLRs (2, 3, 7 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL2, IL6, IL8 and IL10) was detected. In groups 3 and 4 simultaneous and significant induction of pro-inflammatory, antiviral and anti-inflammatory cytokine resulted cytokine dysregulation leading to death of (2/6) and (3/6) chickens respectively. Hence, the study revealed TLRs and cytokines role in modulating the H5N1 infection outcome in chickens pre-exposed to H9N2 virus.
Assuntos
Citocinas/sangue , Interações Hospedeiro-Patógeno/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/imunologia , RNA Mensageiro/metabolismo , Receptores Toll-Like/sangue , Animais , Galinhas , Citocinas/biossíntese , Modelos Animais de Doenças , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Imunidade Celular , Imunidade Inata , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/virologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Regulação para CimaRESUMO
Highly pathogenic avian influenza (H5N8) viruses were detected in waterfowl at 2 zoos in India in October 2016. Both viruses were different 7:1 reassortants of H5N8 viruses isolated in May 2016 from wild birds in the Russian Federation and China, suggesting virus spread during southward winter migration of birds.
Assuntos
Animais de Zoológico , Vírus da Influenza A Subtipo H5N8/genética , Influenza Aviária/virologia , Vírus Reordenados , Animais , Aves , Índia/epidemiologia , Influenza Aviária/epidemiologia , FilogeniaRESUMO
Highly pathogenic avian influenza (HPAI) is a major health concern worldwide. In this study, we focused on antigenic analysis of HPAI H5N1 viruses isolated from poultry in India between 2006 and 2015 comprising 25 isolates from four phylogenetic clades 2.2 (1 isolate), 2.2.2.1 (1 isolate), 2.3.2.1a (17 isolates) and 2.3.2.1c (6 isolates). Seven H5N1 isolates from all four clades were selected for production of chicken antiserum, and antigenic analysis was carried out by hemagglutination inhibition (HI) assay. HI data indicated antigenic divergence (6-21 fold reduction in cross-reactivity) between the two recently emerged clades 2.3.2.1a and 2.3.2.1c. These two clades are highly divergent (21-128 fold reduction in HI titre) from the earlier clades 2.2 /2.2.2.1 isolated in India. However, a maximum of 2-fold and 4-fold reduction in cross-reactivity was observed within the isolates of homologous clades 2.3.2.1c and 2.3.2.1a, respectively. The molecular basis of inter-clade antigenic divergence was examined in the haemagglutinin (HA) antigenic sites of the H5N1 virus. Amino acid changes at 8 HA antigenic sites were observed between clades 2.3.2.1a and 2.3.2.1c, whereas 20-23 substitutions were observed between clades 2.3.2.1a/2.3.2.1c and 2.2/2.2.2.1. Therefore, a systematic analysis of antigenic drift of the contemporary field isolates is a pre-requisite for determining the suitable strain(s) for vaccine candidature.
Assuntos
Antígenos Virais/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Substituição de Aminoácidos , Animais , Antígenos Virais/imunologia , Galinhas , Patos , Variação Genética , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Índia/epidemiologia , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/imunologia , Influenza Aviária/patologia , Influenza Aviária/virologia , Filogenia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Perus , VirulênciaRESUMO
Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating.
Assuntos
Proteção Cruzada , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/prevenção & controle , Vírus Reordenados/imunologia , Proteínas Virais/genética , Animais , Galinhas , Cloaca/virologia , Índia , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/imunologia , Orofaringe/virologia , Análise de Sobrevida , Carga Viral , Eliminação de Partículas ViraisRESUMO
The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.
Assuntos
Anticorpos Antivirais/sangue , Infecções por Henipavirus/sangue , Infecções por Henipavirus/veterinária , Vírus Nipah , Proteínas do Nucleocapsídeo/química , Doenças dos Suínos/sangue , Suínos/sangue , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Suínos/virologiaRESUMO
African swine fever (ASF), considered as the most dreadful swine disease due to its very high mortality, emerged in India in 2020. The complete genome analysis of ASF viruses isolated during the first outbreaks in India showed a few unique non-synonymous mutations in MGF 369-11L, MGF 505-4R, K205R and B263R genes. Frame shifts in the protein coding sequences were observed in DP60R, ASFV-G_ACD 00190, MGF 110-10-L-MGF110-14L fusion, MGF 360-14L and I267L genes of Indian ASF viruses as compared to ASFV/Georgia/2007. Complete genome based phylogenetic analysis of p72-genotype-II viruses showed the clustering of Indian isolates with ASFV/Wuhan/2019 in a separate clade. Phylogenetic analysis of concatenated sequences of 14 open reading frames (ORF) having single nucleotide polymorphisms (SNP) showed distinct grouping of Indian ASFVs with other Asian ASFVs. This is the first complete genome characterization of ASF viruses isolated from domestic pigs in India. The results indicate that number of Tandem Repeat Sequence (TRS) in the intergenic region between I73R and I329L genes, and the 14 ORFs with SNP reported in this study could be the genetic determinants to differentiate the closely related p72-genotype II viruses circulating in Asia.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Febre Suína Africana/epidemiologia , Animais , DNA Intergênico , DNA Viral/genética , Surtos de Doenças/veterinária , Genótipo , Filogenia , Análise de Sequência de DNA/veterinária , Sus scrofa , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
This study aimed to investigate the potential of H9N2 avian influenza virus to cause disease and intra-species transmission in house crows (Corvus splendens). A group of six crows were intranasally inoculated with 106.0 EID50 of H9N2 virus (A/chicken/India/07OR17/2021), and 24 h post-inoculation six naïve crows were co-housed with infected crows. Crows were observed for 14 days for any overt signs of illness. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. No apparent clinical signs were observed in either infected or in-contact crows. Virus excretion was observed only in infected birds up to 9 days post-infection (dpi) through both oropharyngeal and cloacal routes. All six infected crows seroconverted to H9N2 virus at 14 dpi, whereas all in-contact crows remained negative to H9N2 virus antibodies. No virus could be isolated from tissues viz., lung, liver, kidney, pancreas, small intestine and large intestine. Although crows became infected with the H9N2 virus, transmission of the virus was inefficient to the in-contact group. However, virus excretion through oral and cloacal swabs from infected crows suggests a potential threat for inter-species transmission, including humans. Crows, being a common synanthrope species, might have some role in influenza virus transmission to poultry and humans, which needs to be explored further.
RESUMO
African swine fever (ASF) is the most dreaded disease of pigs, which can cause mortality of up to 100%. Following disease outbreaks with high mortality in pigs in two states of north-east India, namely Arunachal Pradesh and Assam in early 2020, we confirmed the first occurrence of African swine fever (ASF) in domestic pigs in India by real-time PCR, virus isolation and nucleotide sequencing. Genetic analyses in three independent genomic regions (B646L gene encoding the p72 protein, E183L gene encoding the p54 protein and the central variable region (CVR) of B602L gene) showed that the Indian ASF viruses are similar to the post-2007-p72-genotype II viruses reported from Asia and Europe, suggesting the transboundary expansion of ongoing ASF outbreaks in the region.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Febre Suína Africana/epidemiologia , Vírus da Febre Suína Africana/genética , Animais , Surtos de Doenças/veterinária , Genótipo , Filogenia , Análise de Sequência de DNA/veterinária , Sus scrofa , SuínosRESUMO
We investigated the molecular epidemiology of foot-and-mouth disease virus (FMDV) serotype Asia 1, which caused outbreaks of disease in Asia during 2003-2007. Since 2004, the region affected by outbreaks of this serotype has increased from disease-endemic countries in southern Asia (Afghanistan, India, Iran, Nepal, Pakistan) northward to encompass Kyrgyzstan, Tajikistan, Uzbekistan, several regions of the People's Republic of China, Mongolia, Eastern Russia, and North Korea. Phylogenetic analysis of complete virus capsid protein 1 (VP1) gene sequences demonstrated that the FMDV isolates responsible for these outbreaks belonged to 6 groups within the Asia 1 serotype. Some contemporary strains were genetically closely related to isolates collected historically from the region as far back as 25 years ago. Our analyses also indicated that some viruses have spread large distances between countries in Asia within a short time.
Assuntos
Vírus da Febre Aftosa/genética , Febre Aftosa/epidemiologia , Afeganistão/epidemiologia , Animais , Ásia/epidemiologia , China/epidemiologia , DNA Viral/genética , Surtos de Doenças , Vírus da Febre Aftosa/classificação , Geografia , Humanos , Índia/epidemiologia , Nepal/epidemiologia , Paquistão/epidemiologia , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SorotipagemRESUMO
Ducks are the "Trojan Horses" for Asian H5N1 avian influenza viruses (AIV) and attain carrier status without displaying overt infection. These birds help in the spread of the virus among the poultry and human population through direct or indirect contact. Preen oil is the secretion of preen gland of water birds such as ducks. In a process called preening, the water birds spread preen oil across their feather and body. Preen oil has been known to play a significant role in the accumulation of various pathogens including Highly Pathogenic Avian Influenza (HPAI) from water onto feathers. However, the studies are scarce on the role of preen oil in the survivability of HPAIV. We conducted a simulative study to analyse the effect of preen oil on the survivability of the HPAI virus (H5N1) on duck feathers. Duck feather samples along with relevant controls were spiked with the H5N1 virus at two different initial concentrations (104 EID50 and 106 EID50 ), stored at 37°C, 25°C and 10°C temperatures and tested at regular intervals for percent infectivity by egg culture method and qRT-PCR. The infectivity and viral load were significantly higher in naturally preened duck feathers in comparison to the three preen oil deficit controls at both low and high initial concentrations of virus (104 EID50 and 106 EID50 ). Maximum persistence was seen at 10°C in naturally preened duck feathers spiked with 106 EID50 concentration of viruses. It was also seen that depletion of preen oil from duck feathers reduced the persistence of the virus. These results demonstrate that preen oil plays a significant role in survivability and protection of HPAIV on duck feathers. This study herein will present new avenues in understanding one of the epidemiological niches of HPAIV.
Assuntos
Patos/virologia , Plumas/virologia , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/virologia , Animais , Aves , Asseio Animal , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Temperatura , Carga ViralRESUMO
This study deals with a comparative analysis of complete genome sequences of twenty-one serotype Asia 1 foot-and-mouth disease (FMD) field viruses isolated over a period of two decades from India, two vaccine strains and seven exotic sequences. The Indian viruses could be grouped in to three distinct lineages at the entire coding region, evolving independently probably under differential selection pressure as evident from the lineage-specific signatures identified. This comparison revealed 80% of amino acids at the polyprotein region to be invariant. Twenty-one residues in L, 3A and P1 region were identified to be under positive selection of which some are antigenically critical. Analysis at functionally crucial motifs, receptor contact residues, polyprotein cleavage sites and at putative T-cell epitopes expands the knowledge beyond other serotypes. Antigenic site II in betaB-betaC loop of VP2 was highly unstable suggesting its exposure to extreme immune pressure. A single cross-over at the L proteinase region in an isolate from buffalo, also featuring an extensive deletion at the 5' untranslated region (UTR), reflects the role of intraserotypic genetic recombination in natural evolution. The likely biological relevance of deletions/insertions observed at UTRs, VP1 and 3A could not be deduced. Altogether, a substantial amount of data raised on full length genomes of type Asia 1 virus adds value to the FMD virus genomics.
Assuntos
Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/genética , Febre Aftosa/epidemiologia , Febre Aftosa/virologia , Genoma Viral , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Sequência de Bases , Análise por Conglomerados , Sequência Conservada , Evolução Molecular , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Genótipo , Índia/epidemiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Filogenia , RNA Viral/genética , Recombinação Genética , Seleção Genética , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência , Sorotipagem , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The recent reports of human infection due to H6 subtype avian influenza viruses (AIV), which are prevalent in terrestrial poultry, indicate evolution of the virus to a possible pandemic strain. Here, we report antigenic and genetic characterization of two H6N2 viruses isolated from apparently healthy domestic ducks in Kerala and Assam, India during 2014 and 2015, respectively. Hemagglutination inhibition assay revealed antigenic divergence between the two isolates, which was corroborated by amino acid differences at 55 positions (15.98%) between their hemagglutinin (HA) 1.The sequence analyses indicated that both the viruses are avian origin with avian receptor specificity, low pathogenic to poultry and sensitive to oseltamivir. However, Kerala14 had V27I mutation marker for amantadine resistance in M2. The Assam15 virus had an additional N-linked glycosylation on HA2 (position 557) compared to Kerala14 virus. Phylogenetic analysis of the HA gene revealed that both the viruses belonged to distinct lineages (Eurasian and Asia II). Phylogeny of neuraminidase and internal gene segments revealed that both the viruses are novel reassortants and are genetically distinct with different gene constellations. The results suggest independent introductions of the two H6N2 viruses into India and migratory wild birds in the Central Asian flyway might be the source of H6N2 viruses in ducks in India. Therefore, continued AIV surveillance in poultry and wild birds is essential for early detection of emergence of novel strains with pandemic potential and control of their spread.
Assuntos
Patos/virologia , Vírus da Influenza A/genética , Influenza Aviária/virologia , Vírus Reordenados/genética , Animais , Índia , FilogeniaRESUMO
Years of molecular epidemiological surveillance has revealed co-circulation of two antigenically divergent genotypes of foot and mouth disease virus serotype A in India. Genotype differentiating RT-PCR and sandwich ELISA were developed as fast, cost-effective and user-friendly alternatives to 1D region based phylogeny for detection and differentiation of genotype VI and VII. The RT-PCR assay targeting 1D region was found to be more sensitive and authentic in distinguishing genotypes than sandwich ELISA. These assays promise to be reliable tools in the epidemiological investigation of foot and mouth disease in the country.
Assuntos
Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Antígenos Virais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Febre Aftosa/epidemiologia , Febre Aftosa/imunologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/imunologia , Genótipo , Índia , Filogenia , SorotipagemRESUMO
Detection of foot-and-mouth disease virus (FMDV) from clinical specimens by conventional sandwich enzyme-linked immunosorbent assay (ELISA) and virus isolation in cell culture is often compromised owing to limited sensitivity and inactivation during transit, respectively. A RT-PCR (oligoprobing) ELISA in both solid and aqueous phase hybridization formats targeting an across serotype conserved site at 3C-3D region was developed and its effectiveness was compared with that of the known targets at the IRES region. A non-isotopic RNA dot hybridization assay with colorimetric detection targeting both the IRES and the 3D region were also validated, which is capable of handling high throughput samples with ease. RT-PCR (oligoprobing) ELISA and dot hybridization assay showed 1000- and 10-fold greater sensitivity than the sandwich ELISA, respectively. Robustness of these diagnostic methods was explored by examining on sandwich ELISA-negative clinical samples. Both the assays developed in the present study were able to detect viral genomes in samples undetectable by conventional ELISA, thereby demonstrating 'proof of sensitivity'. Although the potential of these assays for providing definitive diagnosis in carrier hosts and in species where clinical disease is inapparent remains to be examined, nevertheless these assays can be adapted for comprehensive surveillance of foot-and-mouth disease in India.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Genoma Viral , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteases Virais 3C , Animais , Antígenos Virais/genética , Colorimetria , Cisteína Endopeptidases/genética , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos , RNA Viral/genética , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética , Proteínas Virais/genéticaRESUMO
Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health.