RESUMO
During an influenza A(H7N7) virus outbreak among poultry in Italy during August-September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.
Assuntos
Vírus da Influenza A Subtipo H7N7/patogenicidade , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Animais , Galinhas , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Vírus da Influenza A Subtipo H7N7/genética , Influenza Aviária/epidemiologia , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Filogenia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Ecological interactions between wild aquatic birds and outdoor-housed poultry can enhance spillover events of avian influenza viruses (AIVs) from wild reservoirs to domestic birds, thus increasing the related zoonotic risk to occupationally exposed workers. To assess serological evidence of AIV infection in workers operating in Northern Italy at the wildfowl/poultry interface or directly exposed to wildfowl, serum samples were collected between April 2005 and November 2006 from 57 bird-exposed workers (BEWs) and from 7 unexposed controls (Cs), planning three sample collections from each individual. Concurrently, AIV surveillance of 3587 reared birds identified 4 AIVs belonging to H10N7, H4N6 and H2N2 subtypes while serological analysis by hemagglutination inhibition (HI) assay showed recent infections caused by H1, H2, H4, H6, H10, H11, H12, and H13 subtypes. Human sera were analyzed for specific antibodies against AIVs belonging to antigenic subtypes from H1 to H14 by using HI and virus microneutralization (MN) assays as a screening and a confirmatory test, respectively. Overall, antibodies specific to AIV-H3, AIV-H6, AIV-H8, and AIV-H9 were found in three poultry workers (PWs) and seropositivity to AIV-11, AIV-H13-still detectable in October 2017-in one wildlife professional (WP). Furthermore, seropositivity to AIV-H2, accounting for previous exposure to the "extinct" H2N2 human influenza viruses, was found in both BEWs and Cs groups. These data further emphasize the occupational risk posed by zoonotic AIV strains and show the possible occurrence of long-lived antibody-based immunity following AIV infections in humans.
RESUMO
A modified-live vaccine against the respiratory form of bovine coronavirus (BCoV) infection was developed by progressive attenuation of a respiratory strain (438/06-TN). The vaccine was found to be safe as four colostrum-deprived newborn calves remained healthy after oronasal administration of ten doses of the vaccine. The immunogenicity of the vaccine was assessed by intramuscular injection of one vaccine dose to 30 BCoV-antibody negative 2-3-month-old calves. At 30 days post-vaccination, all vaccinated calves displayed high antibody titres against BCoV. Sequence analysis of the S gene of wild-type and cell-adapted 438/06-TN strain detected 10 nucleotide changes, 9 of which were non-synonymous.
Assuntos
Anticorpos Antivirais/sangue , Doenças dos Bovinos/prevenção & controle , Infecções por Coronavirus/veterinária , Coronavirus Bovino/imunologia , Vacinas Atenuadas , Vacinas Virais , Animais , Bovinos , Doenças dos Bovinos/virologia , Linhagem Celular , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Coronavirus Bovino/genética , Coronavirus Bovino/isolamento & purificação , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Infecções Respiratórias/prevenção & controle , Infecções Respiratórias/veterinária , Infecções Respiratórias/virologia , Análise de Sequência de DNA , Glicoproteína da Espícula de Coronavírus , Vacinação/veterinária , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH4Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents.
Assuntos
Antivirais/farmacologia , Cloroquina/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Cloreto de Amônio/metabolismo , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/fisiologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A/fisiologia , Lisossomos/metabolismo , Aves Domésticas , RNA Viral/biossíntese , Fatores de Tempo , Replicação Viral/efeitos dos fármacosRESUMO
In December 2005, equine influenza virus infection was confirmed as the cause of clinical respiratory disease in vaccinated horses in Apulia, Italy. The infected horses had been vaccinated with a vaccine that contained strains representatives from both the European (A/eq/Suffolk/89) and American (A/eq/Newmarket/1/93) H3N8 influenza virus lineages, and the H7N7 strain A/eq/Praga/56. Genetic characterization of the hemagglutinin (HA) and neuraminidase (NA) genes of the virus from the outbreak, indicated that the isolate (A/eq/Bari/2005) was an H3N8 strain closely related to recent representatives (Kentucky/5/02-like) of the American sub-lineage Florida, that was introduced in Italy through movement of infected horses from a large outbreak described in 2003 in United Kingdom. Strain A/eq/Bari/2005 displayed 9 amino acid changes in the HA1 subunit protein with respect to the reference American strain A/eq/Newmarket/1/93 contained in the vaccine. Four changes were localized in the antigenic regions C-D and likely accounted for the vaccine failure.
Assuntos
Surtos de Doenças/veterinária , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Vírus da Influenza A Subtipo H3N8/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/veterinária , Doenças Respiratórias/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral/química , DNA Viral/genética , Hemaglutininas/química , Hemaglutininas/genética , Cavalos , Vírus da Influenza A Subtipo H3N8/genética , Itália/epidemiologia , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/genética , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Filogenia , Reação em Cadeia da Polimerase/veterinária , Doenças Respiratórias/epidemiologia , Doenças Respiratórias/virologia , Alinhamento de Sequência , Vacinação/veterináriaRESUMO
Canine distemper virus is the etiological agent of a severe disease in dogs and many other carnivores. Clinical diagnosis of canine distemper is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Accordingly, a laboratory confirmation is required for suspected cases. In this study a real-time RT-PCR assay was developed for detection and quantitation of canine distemper virus. The assay exhibited high specificity as all the negative controls (no-template-controls and samples from healthy sero-negative dogs) and other canine pathogens were not misdetected. Up to 1 x 10(2) copies of RNA were detected by the TaqMan assay, thus revealing a high sensitivity. Quantitative TaqMan was validated on clinical samples, including various tissues and organs collected from dogs naturally infected by canine distemper virus. Urines, tonsil, conjunctival swabs and whole blood were found to contain high virus loads and therefore proved to be suitable targets for detection of canine distemper virus RNA.
Assuntos
Vírus da Cinomose Canina/isolamento & purificação , Cinomose/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estruturas Animais/virologia , Animais , Líquidos Corporais/virologia , Cinomose/virologia , Vírus da Cinomose Canina/genética , Cães , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC. METHODS: RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1-H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. RESULTS: The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 x 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10-100 times higher than conventional RT-PCR. CONCLUSION: The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.
Assuntos
Doenças das Aves/virologia , Primers do DNA , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase/métodos , Animais , AvesRESUMO
Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.
Assuntos
Infecções por Coronavirus/veterinária , Coronavirus Canino/isolamento & purificação , Diarreia/veterinária , Doenças do Cão/diagnóstico , RNA Viral/análise , Animais , Infecções por Coronavirus/diagnóstico , Coronavirus Canino/genética , Diarreia/diagnóstico , Cães , Fezes/virologia , Fluorescência , Itália , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
A case of feline infectious peritonitis (FIP) in an 11-month-old European shorthair cat is reported. The infected cat displayed loss of weight, respiratory distress, ascitis, anemia and died within 15 days after the first appearance of clinical signs. Lesions typical of a mixed form (effusive and non-effusive) of FIP were observed and by RT-PCR a feline coronavirus (FCoV) type I strain was detected in several tissues. The RT-PCR results were confirmed by sequence analysis of the amplified products. Phylogeny carried out on fragments of the M and S genes showed that the FCoV strain segregates with typical type I FCoVs.
Assuntos
Doenças do Gato/virologia , Coronavirus Felino/classificação , Peritonite Infecciosa Felina/virologia , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Sequência de Bases , Gatos , Coronavirus Felino/genética , Coronavirus Felino/isolamento & purificação , DNA Viral/análise , Evolução Fatal , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Following the emergence of the A(H1N1)pdm09 in humans, this novel influenza virus was reverse transmitted from infected people to swine population worldwide. In this study we investigated the molecular evolution of A(H1N1)pdm09 virus identified in pigs reared in a single herd. Nasal swabs taken from pigs showing respiratory distress were tested for influenza type A and A(H1N1)pdm09 by real-time RT-PCR assays. Virus isolation from positive samples was attempted by inoculation of nasal swabs samples into specific pathogen free embryonated chicken eggs (ECE) and complete genome sequencing was performed on virus strains after replication on ECE or from original swab sample. The molecular analysis of hemagglutinin (HA) showed, in four of the swine influenza viruses under study, a unique significant amino acid change, represented by a two-amino acid insertion at the HA receptor binding site. Phylogenetic analysis of HA, neuraminidase, and concatenated internal genes revealed a very similar topology, with viruses under study forming a separate cluster, branching outside the A(H1N1)pdm09 isolates recognized until 2014. The emergence of this new cluster of A(H1N1)pdm09 in swine raises further concerns about whether A(H1N1)pdm09 with new molecular characteristics will become established in pigs and potentially transmitted to humans.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Neuraminidase/genética , Substituição de Aminoácidos/genética , Animais , Evolução Molecular , Genoma Viral , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Pandemias , Filogenia , Suínos/virologiaRESUMO
Detection of avian influenza virus (AIV) in poultry meat is hampered by the lack of an efficient analytical method able to extract and concentrate viral RNA prior to PCR. In this study we developed a method for extracting and detecting AIV from poultry meat by a previously standardized 1-step real-time reverse transcriptase PCR (RRT-PCR) assay. In addition, a new process control, represented by feline calicivirus (FCV), was included in the original protocol, to evaluate all analytical steps from sample preparation to the detection phase. The detection limit was below 1×10(-1) TCID50 of AIV per sample, and the quantification limit corresponded to 1×10(1) TCID50 of AIV per sample. Moreover, the addition of 1×10(2) TCID50/sample of FCV did not affect the quantification and detection limit of the reaction. These results show that the developed assay is suitable for detecting small amounts of AIV in poultry meat. In addition, the developed biopreparedness protocol can be applied to detect AIV in legal or illegal imported broiler chicken meat. The availability of a rapid and sensitive diagnostic method based on molecular identification of AIV in poultry meat provides an important tool in the prevention of AIV circulation.
Assuntos
Vírus da Influenza A/isolamento & purificação , Carne/virologia , RNA Viral/análise , Virologia/métodos , Animais , Galinhas/virologia , Vírus da Influenza A/genética , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Pigs play a key epidemiologic role in the ecology of influenza A viruses (IAVs) emerging from animal hosts and transmitted to humans. Between 2008 and 2010, we investigated the health risk of occupational exposure to swine influenza viruses (SIVs) in Italy, during the emergence and spread of the 2009 H1N1 pandemic (H1N1pdm) virus. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples from 123 swine workers (SWs) and 379 control subjects (Cs), not exposed to pig herds, were tested by haemagglutination inhibition (HI) assay against selected SIVs belonging to H1N1 (swH1N1), H1N2 (swH1N2) and H3N2 (swH3N2) subtypes circulating in the study area. Potential cross-reactivity between swine and human IAVs was evaluated by testing sera against recent, pandemic and seasonal, human influenza viruses (H1N1 and H3N2 antigenic subtypes). Samples tested against swH1N1 and H1N1pdm viruses were categorized into sera collected before (n. 84 SWs; n. 234 Cs) and after (n. 39 SWs; n. 145 Cs) the pandemic peak. HI-antibody titers ≥10 were considered positive. In both pre-pandemic and post-pandemic peak subperiods, SWs showed significantly higher swH1N1 seroprevalences when compared with Cs (52.4% vs. 4.7% and 59% vs. 9.7%, respectively). Comparable HI results were obtained against H1N1pdm antigen (58.3% vs. 7.7% and 59% vs. 31.7%, respectively). No differences were found between HI seroreactivity detected in SWs and Cs against swH1N2 (33.3% vs. 40.4%) and swH3N2 (51.2 vs. 55.4%) viruses. These findings indicate the occurrence of swH1N1 transmission from pigs to Italian SWs. CONCLUSION/SIGNIFICANCE: A significant increase of H1N1pdm seroprevalences occurred in the post-pandemic peak subperiod in the Cs (p<0.001) whereas SWs showed no differences between the two subperiods, suggesting a possible occurrence of cross-protective immunity related to previous swH1N1 infections. These data underline the importance of risk assessment and occupational health surveillance activities aimed at early detection and control of SIVs with pandemic potential in humans.
Assuntos
Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Imunidade/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Exposição Ocupacional/estatística & dados numéricos , Pandemias/estatística & dados numéricos , Suínos/virologia , Adolescente , Adulto , Idoso , Animais , Antígenos Virais/imunologia , Feminino , Humanos , Vírus da Influenza A Subtipo H1N2/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Estações do Ano , Adulto JovemAssuntos
Antivirais/uso terapêutico , Cloroquina/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1 , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Antimaláricos/uso terapêutico , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidroxicloroquina/uso terapêutico , Resultado do Tratamento , Carga Viral , Replicação Viral/efeitos dos fármacosRESUMO
Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Vírus Reordenados/isolamento & purificação , Doenças dos Suínos/virologia , Suínos/virologia , Animais , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N2/classificação , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H3N2/genética , Itália/epidemiologia , Pulmão/virologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Vírus Reordenados/classificação , Vírus Reordenados/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de RNA , Doenças dos Suínos/epidemiologiaRESUMO
The first outbreak of the pandemic H1N1 virus in a swine breeder farm in Italy in November 2009 was reported. Clinical signs observed in sows included fever, depression, anorexia and agalactia, while in piglets diarrhoea and weight loss. The morbidity in sows was approximately 30% and the accumulated mortality rate was similar with those usually reported in piggeries (<10%). Virus was isolated from piglets (A/Sw/It/290271/09) and the sequencing of the whole genome was then performed. Comparison with all (H1N1)v sequences available in GenBank shows A/Sw/It/290271/09 three unique amino-acid (aa) changes in PB2 (S405T), PB1 (K386R) and PA (K256Q), not yet associated to any well characterized phenotype markers of Influenza viruses. All eight aa at positions representing the so-called species specific swine-human signatures, found in both swine and in the pandemic H1N1v, are also present. The M2 protein displays the C55F and the PA protein the S409N substitutions, both corresponding to enhanced transmission phenotype markers. Phylogenetic analysis showed that the virus was genetically related to the pandemic H1N1 virus. In addition, serological samples were collected from 40 sows, of which 20 resulted positive to the pandemic H1N1 virus by HI test proving a virus circulation in the farm.
RESUMO
Wild aquatic birds in the Orders Anseriformes and Charadriiformes are the main reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic viruses. High viral loads in feces of infected birds permit a fecal-oral route of transmission. Numerous studies have reported the isolation of avian influenza viruses (AIVs) from surface water at aquatic bird habitats. These isolations indicate aquatic environments have an important role in the transmission of AIV among wild aquatic birds. However, the progressive dilution of infectious feces in water could decrease the likelihood of virus/host interactions. To evaluate whether alternate mechanisms facilitate AIV transmission in aquatic bird populations, we investigated whether the preen oil gland secretions by which all aquatic birds make their feathers waterproof could support a natural mechanism that concentrates AIVs from water onto birds' bodies, thus, representing a possible source of infection by preening activity. We consistently detected both viral RNA and infectious AIVs on swabs of preened feathers of 345 wild mallards by using reverse transcription-polymerase chain reaction (RT-PCR) and virus-isolation (VI) assays. Additionally, in two laboratory experiments using a quantitative real-time (qR) RT-PCR assay, we demonstrated that feather samples (n = 5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces. The data presented herein provide information that expands our understanding of AIV ecology in the wild bird reservoir system.
Assuntos
Patos/virologia , Asseio Animal , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/transmissão , Animais , Vírus da Influenza A/genética , Influenza Aviária/virologia , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To investigate the molecular adaptation of influenza viruses during natural interspecies transmission, we performed a phenotypic and genotypic analysis of a low-pathogenic duck H7N3 influenza virus after experimental passages in turkey and quail. Results obtained showed differences in the HA receptor-binding and in NA enzyme activities in viruses recovered after passages in quail, compared to those obtained from passages in turkey. Sequencing of the HA, NA and genes of internal proteins of the viruses obtained from quail and turkey, identified several amino acid substitutions in comparison with the progenitor virus. Of note, in the quail-adapted viruses the emergence of a 23-amino acid deletion in the stalk of the NA and the introduction of a glycosylation site in the HA were a reminiscence of changes typically observed in nature confirming a potential role of the quail in the adaptation of wild birds viruses to domestic poultry.
Assuntos
Vírus da Influenza A Subtipo H7N3/fisiologia , Vírus da Influenza A Subtipo H7N3/patogenicidade , Influenza Aviária/virologia , Adaptação Biológica/genética , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Coturnix , Patos , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Vírus da Influenza A Subtipo H7N3/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neuraminidase/genética , Neuraminidase/fisiologia , Oligossacarídeos/química , Oligossacarídeos/fisiologia , Receptores Virais/fisiologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Perus , Virulência/genética , Virulência/fisiologiaRESUMO
The sequence of the full-length gene encoding for the main capsid protein VP2 of 58 canine parvovirus (CPV) type 2c strains, along with recent CPV-2a/2b strains, was determined and analysed in comparison with reference CPV isolates. The CPV-2c strains displayed a low genetic variability and shared amino acid changes already detected in recent CPV-2a/2b isolates, with a phylogenetic clustering accounting for their geographical distribution. Analysis of the selection pressure driving CPV evolution confirmed that the VP2 gene is under purifying selection. The emergence and global spread of the new CPV variant provides an interesting model to better understand virus evolution.
Assuntos
Proteínas do Capsídeo/genética , Doenças do Cão/virologia , Infecções por Parvoviridae/veterinária , Parvovirus Canino/classificação , Parvovirus Canino/genética , Parvovirus Canino/isolamento & purificação , Animais , Proteínas do Capsídeo/química , Cães , Variação Genética , Dados de Sequência Molecular , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/virologia , FilogeniaRESUMO
Avian influenza infections by high and low pathogenicity H7 influenza viruses have caused several outbreaks in European poultry in recent years, also resulting in human infections. Although in some cases the source of H7 strains from domestic poultry was shown to be the viruses circulating in the wild bird reservoir, a thorough characterization of the entire genome of H7 viruses from both wild and domestic Eurasian birds, and their evolutionary relationships, has not been conducted. In our study, we have analysed low pathogenicity H7 influenza strains isolated from wild and domestic ducks in Italy and southern China and compared them with those from reared terrestrial poultry such as chicken and turkey. Phylogenetic analysis demonstrated that the H7 haemagglutinin genes were all closely related to each other, whereas the remaining genes could be divided into two or more phylogenetic groups. Almost each year different H7 reassortant viruses were identified and in at least two different years more than one H7 genotype co-circulated. A recent precursor in wild waterfowl was identified for most of the gene segments of terrestrial poultry viruses. Our data suggest that reassortment allows avian influenza viruses, in their natural reservoir, to increase their genetic diversity. In turn this might help avian influenza viruses colonize a wider range of hosts, including domestic poultry.
Assuntos
Doenças das Aves/virologia , Vírus da Influenza A Subtipo H7N7/genética , Influenza Aviária/genética , Doenças das Aves Domésticas/virologia , Animais , Antígenos Virais/análise , Ásia , Sequência de Bases , Aves , DNA Viral/genética , Europa (Continente) , Genes Virais , Humanos , Vírus da Influenza A Subtipo H7N7/classificação , Dados de Sequência Molecular , Filogenia , Aves Domésticas , RNA Viral/genéticaRESUMO
Two epidemics of avian influenza due to H5 and H7 highly pathogenic viruses occurred in poultry in Italy in 1997/98 and 1999/2000, respectively. The circulation of these serotypes in wild aquatic birds was investigated examining 638 cloacal swabs and 621 sera collected from 150 gulls, 162 coots, and 326 ducks trapped in Italian wetlands from 1998 to 2000. Seroprevalences against influenza A viruses, detected by a double-antibody sandwich-blocking enzyme-linked immunosorbent assay (ELISA), were 11% in gulls, 16% in coots, and 45% in ducks. Among the Anatidae group, duck species wintering in Mediterranean areas showed significantly higher values than ducks wintering in South-Saharan areas of Africa. In order to detect H5 and H7 antibodies, the haemagglutination-inhibition assay and two competitive ELISA tests (H5-ELISA and H7-ELISA) using monoclonal antibodies specific for H5 and H7 subtypes were performed. None of the aquatic bird species were found seropositive for H7 subtype, whereas H5-positive sera were found by both the haemagglutination-inhibition and ELISA assays in ducks only. The highest H5 seroprevalences were detected by H5-ELISA; overall, 5% (10/201) of duck species wintering in Mediterranean areas tested positive by this assay, with annual seroprevalences ranging from 2% (2/123) to 12% (6/51). In the present study, only five viruses belonging to H1N1, H11N6, and H2N3 subtypes were isolated from ducks. However, the H5 seroconversion observed in one mallard duck at the beginning of 1998 indicates that H5 virus circulation also occurred in the study area.