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1.
Sensors (Basel) ; 19(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627298

RESUMO

Rather than the internal genome nucleic acids, the biomolecules on the surface of the influenza virus itself should be detected for a more exact and rapid point-of-care yes/no decision for influenza virus-induced infectious diseases. This work demonstrates the ultrasensitive electrical detection of the HA1 domain of hemagglutinin (HA), a representative viral surface protein of the influenza virus, using the top-down complementary metal oxide semiconductor (CMOS) processed silicon nanowire (SiNW) field-effect transistor (FET) configuration. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) was employed as a probe that specifically binds both to the aldehyde self-aligned monolayer on the SiNWs and to HA1 simultaneously. CMP-NANA was serially combined with two kinds of linkers, namely 3-aminopropyltriethoxysilane and glutaraldehyde. The surface functionalization used was verified using the purification of glutathione S-transferase-tagged HA1, contact angle measurement, enzyme-linked immunosorbent assay test, and isoelectric focusing analysis. The proposed functionalized SiNW FET showed high sensitivities of the threshold voltage shift (ΔVT) ~51 mV/pH and the ΔVT = 112 mV (63 mV/decade) with an ultralow detectable range of 1 fM of target protein HA1.


Assuntos
Técnicas Biossensoriais , Hemaglutininas/isolamento & purificação , Infecções por Orthomyxoviridae/diagnóstico , Orthomyxoviridae/isolamento & purificação , Animais , Humanos , Nanofios/química , Orthomyxoviridae/patogenicidade , Sistemas Automatizados de Assistência Junto ao Leito , Silício
2.
BMC Res Notes ; 12(1): 628, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551085

RESUMO

OBJECTIVE: We conducted four cross-sectional studies over 1 year among humans and pigs in three slaughterhouses in Central and Western Kenya (> 350 km apart) to determine infection and exposure to influenza A viruses. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected from participants who reported acute respiratory illness (ARI) defined as fever, cough or running nose. Nasal swabs and blood samples were collected from pigs. Human NP/OP and pig nasal swabs were tested for influenza A virus by real-time reverse transcriptase polymerase chain reaction (PCR) and pig serum was tested for anti-influenza A antibodies by ELISA. RESULTS: A total of 288 participants were sampled, 91.3% of them being male. Fifteen (5.2%) participants had ARI but the nine swabs collected from them were negative for influenza A virus by PCR. Of the 1128 pigs sampled, five (0.4%) nasal swabs tested positive for influenza A/H1N1/pdm09 by PCR whereas 214 of 1082 (19.8%) serum samples tested for Influenza A virus antibodies. There was higher seroprevalence in colder months and among pigs reared as free-range. These findings indicate circulation of influenza A/H1N1/pdm09 among pigs perhaps associated with good adaptation of the virus to the pig population after initial transmission from humans to pigs.


Assuntos
Matadouros , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Doenças dos Suínos/diagnóstico , Adulto , Animais , Anticorpos Antivirais/sangue , Estudos Transversais , Feminino , Geografia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Quênia/epidemiologia , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Pandemias , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Adulto Jovem
3.
BMC Infect Dis ; 19(1): 762, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477028

RESUMO

BACKGROUND: Avian influenza A (H5N6) virus poses a great threat to the human health since it is capable to cross the species barrier and infect humans. Although human infections are believed to largely originate from poultry contaminations, the transmissibility is unclear and only limited information was available on poultry environment contaminations, especially in Fujian Province. METHODS: A total of 4901 environmental samples were collected and tested for Avian Influenza Virus (AIV) from six cities in Fujian Province through the Fujian Influenza Surveillance System from 2013 to 2017. Two patient-related samples were taken from Fujian's first confirmed H5N6 human case and his backyard chicken feces in 2017. Chi-square test or Fisher's exact probability test was used to compare the AIV and the viral subtype positive rates among samples from different Surveillance cities, surveillance sites, sample types, and seasons. Phylogenetic tree analysis and molecular analysis were conducted to track the viral transmission route of the human infection and to map out the evolutions of H5N6 in Fujian. RESULTS: The overall positive rate of the H5 subtype AIVs was 4.24% (208/4903). There were distinctive differences (p < 0.05) in the positive rates in samples from different cities, sample sites, sample types and seasons. The viruses from the patient and his backyard chicken feces shared high homologies (99.9-100%) in all the eight gene segments. Phylogenetic trees also showed that these two H5N6 viruses were closely related to each other, and were classified into the same genetic clade 2.3.4.4 with another six H5N6 isolates from the environmental samples. The patient's H5N6 virus carried genes from H6N6, H5N8 and H5N6 viruses originated from different areas. The R294K or N294S substitution was not detected in the neuraminidase (NA). The S31 N substitution in the matrix2 (M2) gene was detected but only in one strain from the environmental samples. CONCLUSIONS: The H5 subtype of AIVs has started circulating in the poultry environments in Fujian Province. The patient's viral strain originated from the chicken feces in his backyard. Genetic reassortment in H5N6 viruses in Fujian Province was indicated. The H5N6 viruses currently circulating in Fujian Province were still commonly sensitive to Oseltamivir and Zanamivir, but the resistance against Amantadine has emerged.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Aves Domésticas/virologia , Animais , Embrião de Galinha , Galinhas/virologia , China/epidemiologia , Patos/virologia , Meio Ambiente , Microbiologia Ambiental , Genes Virais , Abrigo para Animais/normas , Humanos , Vírus da Influenza A/genética , Influenza Aviária/diagnóstico , Influenza Aviária/epidemiologia , Tipagem Molecular , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Fatores de Risco
4.
Biosens Bioelectron ; 141: 111476, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31272058

RESUMO

The ability of influenza viruses to rapidly evolve has caused significant challenges in viral surveillance, diagnosis, and therapeutic development. Molecular sequencing methods, though powerful tools for monitoring influenza evolution at the genetic level, are not able to fully characterize the antigenic properties of influenza viruses. Understanding influenza virus antigenicity is critical to vaccine development and disease prevention. Traditional immunoassays which have been widely used for evaluating influenza antigenicity have limited throughput. To alleviate these problems, new bioanalytical tools to investigate influenza antigenicity by measuring antibody-antigen binding are an active area of research. Herein, we review immunosensor technologies from the aspects of various sensing principles, while highlighting recent developments in multiplex, label-free detection strategies. Highlighted technologies include electrochemical immunosensors relying on impedimetric detection; these demonstrate simple design and cost effectiveness for mass production. Antibody arrays implemented on an optical interferometric sensor system allow systematic characterization of influenza antigenicity. Quartz microbalance immunosensors are highly sensitive but have yet to be explored for multiplex sensing. Immunosensors made on lateral flow strips have shown promise in rapid diagnosis of influenza subtypes. We anticipate that these and other technologies discussed in the review will facilitate advances in the study of influenza, and other viral pathogens.


Assuntos
Técnicas Biossensoriais/métodos , Influenza Humana/diagnóstico , Orthomyxoviridae/isolamento & purificação , Animais , Antígenos Virais/análise , Antígenos Virais/imunologia , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Influenza Humana/imunologia , Influenza Humana/virologia , Influenzavirus A/imunologia , Influenzavirus A/isolamento & purificação , Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia
5.
Nanoscale ; 11(22): 10819-10827, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31135010

RESUMO

Myxovirus protein A (MxA) is a biomarker that can be used to distinguish between viral and bacterial infections. While MxA lateral flow assays (LFAs) have been successfully used for viral vs. bacterial differential diagnosis for children, the clinically relevant level of MxA for adults has been reported to be 100 times lower, which is too low for traditional LFAs. We present results applying the use of surface enhanced Raman spectroscopy (SERS) to detect MxA. AuAg nanoshells (AuAg NSs) were used to enhance the Raman signal of mercaptobenzoic acid (4-MBA), enabling readout by SERS. The AuAg NSs were conjugated to antibodies for the biomarker of interest, resulting in a "nanotag", that could be used in a dipstick immunoassay for detection. We first optimized the nanotag parameters using anti-human IgG/human IgG as a model antibody/antigen system, and then demonstrated detection of MxA using anti-MxA antibodies. We show that SERS readout of immunoassays for MxA can quantify MxA levels at clinically relevant levels for adult viral infection.


Assuntos
Anticorpos Antivirais/química , Ouro/química , Imunoglobulina G/química , Nanopartículas Metálicas/química , Proteínas de Resistência a Myxovirus/imunologia , Nanoconchas/química , Infecções por Orthomyxoviridae , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/imunologia , Criança , Humanos , Imunoensaio , Orthomyxoviridae , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/imunologia , Papel
6.
Redox Biol ; 22: 101129, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30735910

RESUMO

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.


Assuntos
Infecções por Orthomyxoviridae/etiologia , Infecções por Orthomyxoviridae/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Mucosa Respiratória/enzimologia , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Deleção de Genes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/fisiologia , Camundongos , Camundongos Transgênicos , Infecções por Orthomyxoviridae/diagnóstico , Isomerases de Dissulfetos de Proteínas/metabolismo , Testes de Função Respiratória , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/fisiopatologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Carga Viral
7.
J Vet Diagn Invest ; 31(1): 137-141, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30803412

RESUMO

We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 ( HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Doenças dos Cavalos/diagnóstico , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/veterinária , Infecções por Orthomyxoviridae/veterinária , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Doenças dos Cavalos/virologia , Cavalos , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos
9.
J Vet Diagn Invest ; 31(2): 250-254, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30638140

RESUMO

We investigated, in a cross-sectional study, the prevalence of antibodies against canine influenza A virus (CIV) H3N2 in serum samples collected from dogs and cats using a commercial ELISA and a hemagglutination inhibition (HI) test. Samples were obtained from 519 cats and dogs from 13 states within the United States. Data were analyzed for potential risk factors with positive sera (vs. negative sera) by logistic regression. Odds ratios and their 95% confidence intervals (CIs) were calculated by exponentiation of the regression coefficients. Ten dogs (2.21%; 95% CI: 1.05-3.98%) and 6 cats (8.96%; 95% CI: 3.36-18.48%) tested seropositive for CIV H3N2 by HI. One feline sample (1.49%; 95% CI: 0.04-8.04%) and 16 canine samples (3.53%; 95% CI: 2.01-5.61%) tested seropositive by ELISA for influenza A virus. There were no apparent associations between seropositivity and putative risk factors. All positive animals were from Indiana or Illinois; however, CIV H3N2 seroprevalence was not common in Illinois and Indiana.


Assuntos
Doenças do Gato/epidemiologia , Doenças do Cão/epidemiologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Animais , Anticorpos Antivirais/sangue , Doenças do Gato/diagnóstico , Doenças do Gato/virologia , Gatos , Doenças do Cão/diagnóstico , Doenças do Cão/virologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Prevalência , Estudos Soroepidemiológicos , Estados Unidos/epidemiologia
10.
Transbound Emerg Dis ; 66(1): 268-276, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30179314

RESUMO

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb-based competitive ELISA for the detection of antibodies against IDV. Thirty-one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724-3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid-phase competitive ELISA (IDV-cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase-conjugated MAb as competitor, which had shown wide intratype cross-reactivity and positivity in HI. To evaluate the diagnostic performances of IDV-cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut-off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV-cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ = 0.93; 95%CI -0.899 to 0.961) between HI test and IDV-cELISA. ROC analysis showed that IDV-cELISA was accurate with an area under the curve (AUC) = 0.999, 95% CI 0.993-1.000). A cut-off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1-99.9) and 98.75 (95% CI 97.1-99.6). These results proved excellent diagnostic performances of IDV-cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti-IDV antibodies in different animal species.


Assuntos
Anticorpos Monoclonais/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Orthomyxoviridae/diagnóstico , Thogotovirus/isolamento & purificação , Animais , Animais Domésticos , Animais Selvagens , Ensaio de Imunoadsorção Enzimática/métodos
11.
Influenza Other Respir Viruses ; 13(1): 71-82, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30264926

RESUMO

BACKGROUND: Human- or avian-to-swine transmissions have founded several autonomously circulating influenza A virus (IAV) lineages in swine populations that cause economically important respiratory disease. Little is known on other human influenza virus types, like B (IBV) and C (ICV) in European swine, and of the recently detected novel animal influenza virus type D (IDV). OBJECTIVES: Development of a cost-effective diagnostic tool for large-scale surveillance programmes targeting all four influenza virus types. METHODS: An influenza ABCD tetraplex real-time RT-PCR (RT-qPCR) was developed in the frame of this study. A selection of reference virus strains and more than 4000 porcine samples from a passive IAV surveillance programme in European swine with acute respiratory disease were examined. RESULTS: Two IBV, a single IDV but no ICV infections were identified by tetraplex RT-qPCR. IBV and IDV results were confirmed by conventional RT-PCR and partial sequence analysis. CONCLUSIONS: The tetraplex RT-qPCR proved fit for purpose as a sensitive, specific and high-throughput tool to study influenza virus transmission at the human-animal interface. Complementing close-meshed active virological and serological surveillance is required to better understand the true incidence and prevalence of influenza virus type B, C and D infections in swine.


Assuntos
Monitoramento Epidemiológico/veterinária , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Orthomyxoviridae/isolamento & purificação , Animais , Primers do DNA/genética , Europa (Continente) , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenzavirus C/isolamento & purificação , Infecções por Orthomyxoviridae/diagnóstico , RNA Viral/genética , Sensibilidade e Especificidade , Sus scrofa , Suínos , Doenças dos Suínos/virologia , Thogotovirus/isolamento & purificação
12.
J Fish Dis ; 42(2): 257-267, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30488967

RESUMO

Infectious salmon anaemia (ISA) is a viral disease that affects farmed Atlantic salmon (Salmo salar L.), often leading to mass mortalities. A quick detection of the ISA virus (ISAV) is crucial for decision-making and can prevent the occurrence of future outbreaks. Screening done by Canada's National Aquatic Animal Health Laboratory System (NAAHLS) uses quantitative reverse transcription PCR (RT-qPCR) followed by sequencing of PCR amplicons. As neither technique provides information regarding the infectivity of the virus, suspected virulent strains are subsequently tested using viral isolation. However, this stepwise process can require significant time to deliver results. To speed up this delivery, we have improved on these pre-existing techniques by combining the use of cell culture with RT-qPCR to detect replicative virus in as little as 5 days. Preliminary assays enabled the establishment of a minimal shift in Ct values over time, which is representative of viral replication in cultured cells. Subsequent blind panel analyses allowed the establishment of the optimal sampling days, as well as diagnostic sensitivity (DSe) and specificity (DSp) estimates. This method could be adopted not only by laboratories conducting diagnostic analyses for ISAV, but also for other slow-replicating viral agents that replicate through a budding mechanism.


Assuntos
Doenças dos Peixes/virologia , Isavirus/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Replicação Viral/fisiologia , Animais , Aquicultura/métodos , Linhagem Celular , Células Cultivadas/virologia , Doenças dos Peixes/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Salmo salar
13.
Vector Borne Zoonotic Dis ; 19(3): 212-216, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30183529

RESUMO

Rapid detection of influenza A virus (IAV) at swine exhibitions, where zoonotic transmission has occurred, can allow exhibition officials to quickly implement mitigation strategies and reduce public health risk. While laboratory diagnostic methods using PCR exist, pen-side detection of IAV can reduce lag time between sample collection and results. Portable insulated isothermal PCR (RT-iiPCR) has been used for point-of-care pathogen detection in veterinary medicine. This study compared laboratory methods of real-time reverse transcription PCR (rRT-PCR) to RT-iiPCR to determine the potential effectiveness of RT-iiPCR for detection of IAV in swine in the field. Two methods of extraction (magnetic bead and spin-column) and the two PCR platforms were used in a crossover study design to detect IAV in nasal wipes of 150 individual swine from one exhibition. Magnetic bead extraction is considered the laboratory gold standard while spin-column purification is considered the field-deployable method. IAV RNA was detected in 17 samples using Mag/rRT-PCR (reference assay) and 16 samples using Mag/RT-iiPCR (Sensitivity-S 76.5%), whereas only 14 samples using Spin/rRT-PCR (S 88.2%) and 12 samples using Spin/RT-iiPCR (field method) (S 58.8%) were positive, demonstrating a reduction in detection of viral RNA using column purification. There is moderate agreement (Cohen's kappa = 0.6575) between Mag/rRT-PCR and Spin/RT-iiPCR. There is good agreement between both PCR assays when using the same method of extraction (Mag: Cohen's kappa = 0.8203, Spin: Cohen's kappa = 0.7642). RT-iiPCR requires testing of 10 more samples than the rRT-PCR to detect disease at the 95% confidence level in a population of 300 animals with a disease prevalence of 20%. In conclusion, although there is some reduction in sensitivity, RT-iiPCR used in conjunction with spin-column purification is an acceptable method of IAV in swine detection at exhibitions where it may help reduce lag time and allow for rapid control of an IAV outbreak.


Assuntos
Vírus da Influenza A/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doenças dos Suínos/virologia , Animais , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia
14.
Prev Vet Med ; 161: 33-40, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30466656

RESUMO

Breed-to-wean pig farms play an important role in spreading influenza A virus (IAV) because suckling piglets maintain, diversify and transmit IAV at weaning to other farms. Understanding the nature and extent of which farm factors drive IAV infection in piglets is a prerequisite to reduce the burden of influenza in swine. We evaluated the association between IAV infection in piglets at weaning and farm factors including farm features, herd management practices and gilt- and piglet-specific management procedures performed at the farm. Voluntarily enrolled breed-to-wean farms (n = 83) agreed to share IAV diagnostic testing and farm data from July 2011 through March 2017 including data obtained via the administration of a survey. There were 23% IAV RT-PCR positive samples of the 12,814 samples submitted for IAV testing within 2989 diagnostic submissions with 30% positive submissions. Among all the factors evaluated (n = 24), and considering the season-adjusted multivariable analysis, only sow IAV vaccination and gilt IAV status at entry significantly reduced (p-value<0.05) IAV infections in piglets at weaning. Results from this study indicate that veterinarians and producers could manage these identified factors to reduce the burden of influenza in piglets prior to wean and perhaps, reduce the spread of IAV to other farms and people.


Assuntos
Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/prevenção & controle , Vacinação/veterinária , Criação de Animais Domésticos/métodos , Animais , Bases de Dados Factuais , Feminino , Vírus da Influenza A , Vacinas contra Influenza/uso terapêutico , Estudos Longitudinais , Masculino , Análise Multivariada , Mucosa Nasal/virologia , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas de Plantas , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Fatores de Risco , Inquéritos e Questionários , Suínos , Doenças dos Suínos/diagnóstico , Estados Unidos/epidemiologia , Vacinação/métodos , Desmame
15.
Sci Rep ; 8(1): 14857, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30291257

RESUMO

Influenza is one of the most common causes of virus diseases worldwide. Virus detection requires determination of Influenza RNA in the upper respiratory tract. Efficient screening is not possible in this way. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive and fast monitoring of disease progression. Breath VOC profiles of 14 (3 controls and 11 infected animals) swine were repeatedly analyzed during a complete infection cycle of Influenza A under high safety conditions. Breath VOCs were pre-concentrated by means of needle trap micro-extraction and analysed by gas chromatography mass spectrometry before infection, during virus presence in the nasal cavity, and after recovery. Six VOCs could be related to disease progression: acetaldehyde, propanal, n-propyl acetate, methyl methacrylate, styrene and 1,1-dipropoxypropane. As early as on day four after inoculation, when animals were tested positive for Influenza A, differentiation between control and infected animals was possible. VOC based information on virus infection could enable early detection of Influenza A. As VOC analysis is completely non-invasive it has potential for large scale screening purposes. In a perspective, breath analysis may offer a novel tool for Influenza monitoring in human medicine, animal health control or border protection.


Assuntos
Testes Respiratórios/instrumentação , Vírus da Influenza A/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/diagnóstico , Suínos/virologia , Compostos Orgânicos Voláteis/análise , Animais , Desenho de Equipamento , Infecções por Orthomyxoviridae/diagnóstico , Respiração , Doenças dos Suínos/virologia
16.
Phytother Res ; 32(12): 2560-2567, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30306659

RESUMO

Berberine, a natural isoquinoline alkaloid isolated from the berberis species, has a wide array of biological properties such as anti-inflammatory, antibacterial, antifungal, and antihelminthic effects. We evaluated the antiviral effect of berberine against influenza A/FM1/1/47 (H1N1) in vivo and in vitro. The results showed that berberine strongly suppressed viral replication in A549 cells and in mouse lungs. Meanwhile, berberine relieved pulmonary inflammation and reduced necrosis, inflammatory cell infiltration, and pulmonary edema induced by viral infection in mice when compared with vehicle-treated mice. Berberine suppressed the viral infection-induced up-regulation of TLR7 signaling pathway, such as TLR7, MyD88, and NF-κB (p65), at both the mRNA and protein levels. Furthermore, berberine significantly inhibited the viral infection-induced increase in Th1/Th2 and Th17/Treg ratios as well as the production of inflammatory cytokines. Our data provide new insight into the potential of berberine as a therapeutic agent for viral infection via its antiviral activity.


Assuntos
Antivirais/farmacologia , Berberina/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Animais , Antivirais/uso terapêutico , Berberina/uso terapêutico , Embrião de Galinha , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/diagnóstico , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Pneumonia/diagnóstico , Pneumonia/tratamento farmacológico , Pneumonia/virologia , Prognóstico , Transdução de Sinais/efeitos dos fármacos
17.
J Vet Diagn Invest ; 30(6): 924-928, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30239276

RESUMO

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.


Assuntos
Infecções por Herpesviridae/veterinária , Doenças dos Cavalos/diagnóstico , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Doenças Respiratórias/veterinária , Animais , Primers do DNA , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 4/genética , Herpesvirus Equídeo 4/isolamento & purificação , Doenças dos Cavalos/virologia , Cavalos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Doenças Respiratórias/diagnóstico , Doenças Respiratórias/virologia , Sensibilidade e Especificidade
18.
Methods Mol Biol ; 1836: 59-88, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151569

RESUMO

Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.


Assuntos
Influenza Humana/diagnóstico , Influenza Humana/virologia , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/fisiologia , Carga Viral , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Testes de Hemaglutinação , Humanos , Orthomyxoviridae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Ensaio de Placa Viral
19.
Methods Mol Biol ; 1836: 185-194, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151574

RESUMO

Although several virus families are internalized into their host cells by direct fusion of the viral envelope with the plasma membrane, most viruses, for example, influenza virus, make use of endocytic pathways for productive entry and infection. After endocytosis, the influenza virus escapes from the endocytic compartment to the cytosol. The distribution of the incoming influenza virus could be traced by detection of the viral RNA in the distinct cellular compartments, including endosome, cytosol, and nucleus. To accomplish this work, we developed a subcellular fractionation method based on density gradient ultracentrifugation and detected the viral RNA using quantitative reverse transcription-polymerase chain reaction analysis. This chapter is devoted to the practical methods and precautions for studying endocytic traffic of virus as well as host cellular factors affecting viral endocytosis.


Assuntos
Endocitose , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Fracionamento Celular/métodos , Linhagem Celular Tumoral , Humanos
20.
Methods Mol Biol ; 1836: 401-430, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151585

RESUMO

Animal model systems for human and animal influenza virus infection and transmission have been established to address research questions which cannot be addressed using in vitro models. Several animal models have been established, such as mice, guinea pig, ferret, pig, poultry, nonhuman primates, and others. Each animal model has its own strength and weaknesses, which should be taken into consideration to select the appropriate animal model to use. This chapter will describe standard protocols relevant for in vivo experiment, including procedures required prior to the start of the animal experiment and sample processing. The animal models described in this chapter are mice, guinea pigs, ferrets, pigs, and chickens.


Assuntos
Modelos Animais de Doenças , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/fisiologia , Pesquisa , Aerossóis , Animais , Embrião de Galinha , Furões , Cobaias , Camundongos , Orthomyxoviridae/isolamento & purificação , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/transmissão , Suínos
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