RESUMEN
During influenza A virus (IAV) entry, the hemagglutinin (HA) protein is triggered by endosomal low pH to undergo irreversible structural changes that mediate membrane fusion. HA proteins from different isolates vary in the pH at which they become activated in endosomes or become irreversible inactivated if exposed to extracellular acid. Little is known about extracellular pH in the upper respiratory tracts of mammals, how pH may shift during IAV infection, and its impact on replication of viruses that vary in HA activation pH. Here, we inoculated DBA/2J mice intranasally with A/TN/1-560/2009 (H1N1) (activation pH 5.5) or a mutant containing the destabilizing mutation HA1-Y17H (pH 6.0). We measured the kinetics of extracellular pH during infection using an optical pH-sensitive microsensor probe placed in the naris, nasal sinus, soft palate, and trachea. We also measured intracellular pH of single-cell suspensions of live, primary lung epithelial cells with various wavelength pH-sensitive dyes localized to cell membranes, cytosol, endosomes, secretory vesicles, microtubules, and lysosomes. Infection with either virus decreased extracellular pH and increased intracellular pH. Peak host immune responses were observed at 2 days post infection (DPI) and peak pH changes at 5 DPI. Extracellular and intracellular pH returned to baseline by 7 DPI in mice infected with HA1-Y17H and was restored later in wildtype-infected. Overall, IAV infection altered respiratory tract pH, which in turn modulated replication efficiency. This suggests a virus-host pH feedback loop that may select for IAV strains containing HA proteins of optimal pH stability, which may be approximately pH 5.5 in mice but may differ in other species.
Asunto(s)
Inmunidad/fisiología , Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/fisiopatología , Sistema Respiratorio/virología , Animales , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Ratones , Sistema Respiratorio/fisiopatología , Internalización del Virus , Replicación ViralRESUMEN
Influenza D virus (IDV), a novel orthomyxovirus, is currently emerging in cattle worldwide. It shares >50% sequence similarity with the human influenza C virus (HICV). Two clades of IDV are currently co-circulating in cattle herds in the U.S. New assays specific for each lineage are needed for accurate surveillance. Also, differential diagnosis between zoonotic human influenza C virus and the two clades of IDV are important to assess the zoonotic potential of IDV. We developed an enzyme-linked immunosorbent assay (ELISA) based on two different epitopes HEF and NP and four peptides, and fluorescent focus neutralization assay to differentiate between IDV bovine and swine clades. Calf sera were obtained, and bovine samples underwent surveillance. Our results highlight the importance of position 215 with 212 in determining the heterogeneity between the two lineages. We needed IFA and FFN for tissue culture-based analysis and a BSL2 facility for analyzing virus interactions. Unfortunately, these are not available in many veterinary centers. Hence, our second aim was to develop an iELISA using specific epitopes to detect two lineages of IDVs simultaneously. Epitope-iELISA accurately detects neutralizing and non-neutralizing antibodies against the IDV in non-BSL2 laboratories and veterinary clinics and is cost-effective and sensitive. To differentiate between IDVs and HICVs, whole antigen blocking, polypeptides, and single-peptide ELISAs were developed. A panel of ferret sera against both viruses was used. Results suggested that both IDV and ICV had a common ancestor, and IDV poses a zoonotic risk to individuals with prior or current exposure to cattle. IDV peptides IANAGVK (286-292 aa), KTDSGR (423-428 aa), and RTLTPAT (448-455 aa) could differentiate between the two viruses, whereas peptide AESSVNPGAKPQV (203-215 aa) detected the presence of IDV in human sera but could not deny that it could be ICV, because the only two conserved influenza C peptides shared 52% sequence similarity with IDV and cross-reacted with IDV. However, blocking ELISAs differentiated between the two viruses. Diagnostic tools and assays to differentiate between ICV and IDV are required for serological and epidemiological analysis to clarify the complexity and evolution and eliminate misdiagnosis between ICV and IDV in human samples.
RESUMEN
Passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (PEDV). Here, we investigated the immunogenicity of an orf virus (ORFV) vector expressing the full-length spike (S) protein of PEDV (ORFV-PEDV-S) in pregnant gilts and its ability to confer passive immunity and protection in piglets. Three doses of ORFV-PEDV-S were given to two groups of PEDV-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. One of the two groups immunized with the ORFV-PEDV-S recombinant virus was also exposed to live PEDV orally on day 31 post-immunization (pi). Antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. The protective efficacy of ORFV-PEDV-S was evaluated after challenge of the piglets with PEDV. PEDV-specific IgG, IgA and neutralizing antibody (NA) responses were detected in ORFV-PEDV-S-immunized and ORFV-PEDV-S-immunized/PEDV-exposed gilts. PEDV NA, IgG and IgA were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. Piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after PEDV challenge in comparison to control piglets. Piglets born to gilts that received ORFV-PEDV-S and were exposed to live PEDV showed stronger NA responses and lower clinical scores when compared to piglets born to gilts immunized with ORFV-PEDV-S alone. These results demonstrate the potential of ORFV as a vaccine delivery platform capable of eliciting passive immunity against PEDV.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/prevención & control , Inmunidad Materno-Adquirida , Virus del Orf/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Glicoproteína de la Espiga del Coronavirus/administración & dosificación , Enfermedades de los Porcinos/prevención & control , Animales , Animales Recién Nacidos , Anticuerpos Neutralizantes/sangre , Calostro , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/química , Vectores Genéticos/inmunología , Inmunización Pasiva/métodos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Leche , Virus del Orf/genética , Virus de la Diarrea Epidémica Porcina/patogenicidad , Embarazo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virologíaRESUMEN
For decades, hemagglutinin (HA) protein structure and its refolding mechanism have served as a paradigm for understanding protein-mediated membrane fusion. HA trimers are in a high-energy state and are functionally activated by low pH. Over the past decade, HA stability (or the pH at which irreversible conformational changes are triggered) has emerged as an important determinant in influenza virus host range, infectivity, transmissibility, and human pandemic potential. Here, we review HA protein structure, assays to measure its stability, measured HA stability values, residues and mutations that regulate its stability, the effect of HA stability on interspecies adaptation and transmissibility, and mechanistic insights into this process. Most importantly, HA stabilization appears to be necessary for adapting emerging influenza viruses to humans.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/química , Mutación , Animales , Hurones/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Concentración de Iones de Hidrógeno , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/transmisión , Gripe Humana/virología , Fusión de Membrana , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Pandemias , Estabilidad Proteica , Estructura Terciaria de Proteína , Internalización del VirusRESUMEN
The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids [aa] 744-759, 747-774 and/or 756-771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371-1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499-600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Epítopos de Linfocito B/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos Inmunodominantes/inmunología , Pruebas de NeutralizaciónRESUMEN
The present study aimed to investigate the protective effect of glycyrrhizin (locally isolated and purified from licorice root) against duck hepatitis virus through the assessment of some hematological and biochemical parameters. One hundred and sixty white Pekin ducklings-one day old-were randomly divided into four equal groups. Group (1) was kept as normal control. Group (2) was inoculated I/P with 10 mg glycyrrhizin/kg BW, three times per week for four weeks. Group (3) was inoculated I/M with 0.5 ml of live attenuated DHV vaccine. Group (4) was inoculated with both glycyrrhizin (10 mg/kg BW I/P, three times per week for four weeks) and live attenuated DHV vaccine (0.5 ml, I/M). Then, all groups of treatment were challenged using virulent DHV except for 20 ducklings from the normal control group which were continued to be kept as negative control. The results revealed that duck hepatitis virus (DHV) caused macrocytic hypochromic anemia, leukopenia, hypoproteinemia, hypoalbuminemia, hyperglycemia, hypercholesterolemia, and marked elevation of liver enzymes and renal parameters. In conclusion, glycyrrhizin injected alone or in combination with DHV vaccine protected or ameliorated the deteriorating effects induced by DHV vaccine and/or duck hepatitis virus infection by improvement of erythrogram and leukogram, as well as liver and kidney functions.
RESUMEN
This study was conducted to investigate the effect of glycyrrhizin as an immune stimulant against duck hepatitis virus (DHV). In vitro study was carried out to determine cytotoxic and antiviral effects of glycyrrhizin in VERO cells. In vivo study was performed on 40 one-day-old White Pekin ducklings. -and the birds weres divided into 4 groups: control, glycyrrhizin treated, vaccinated with live attenuated DHV vaccine and glycyrrhizin treated and vaccinated; to investigate the changes in immunity and challenge test. Blood samples were collected from each duckling for evaluation of cellular and humeral immunity. The in vitro results revealed that glycyrrhizin had antiviral and no toxic effects till 106 dilutions. Higher antibody titer was observed from the 5th week till the end of experiment in glycyrrhizin and vaccinated group. Treatment with glycyrrhizin alone or with DHV vaccine demonstrated a pronounced lymphocytic proliferation response after 4 days post-inoculation till the end of experiment, while vaccinated group revealed a pronounced proliferation response after 24 days post-inoculation. Treatment with glycyrrhizin alone or combination with DHV vaccine revealed good immune stimulant and antiviral effect against DHV.