Sulfated glycans containing NeuAcα2-3Gal facilitate the propagation of human H1N1 influenza A viruses in eggs.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galß1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galß1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

Comparative biology of two genetically closely related Newcastle disease virus strains with strongly contrasting pathogenicity.

A lentogenic strain of Newcastle disease virus (NDV) with an intracerebral pathogenicity index (ICPI) of 0.36 was derived by the passage of a mesogenic NDV isolate with an original ICPI of 1.04. Animal experiments showed that the original strain caused much severer clinical signs and mortality than the derived strain in chickens. To elucidate the molecular reason for this virulence change, the complete viral genomes of the original and derived strains were sequenced. Molecular analysis showed that both viruses contained the same fusion (F) protein with a cleavage site (Fcs) motif that is usually associated with velogenic viruses. Molecular comparison revealed five amino acid (aa) differences in nucleoprotein (NP) (aa 426), hemagglutinin-neuraminidase (HN) (aas 215 and 430), and large protein (L) (aas 1694 and 1767), accompanied by the changes of relevant biological activities in membrane fusion and replication. Thus, we believe that the virulence changes may induced by these mutations. Our findings make a foundation for more in-depth investigations of the molecular mechanism underlying virulence.

N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses.

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Isolation and complete genome analysis of a novel duck picornavirus in China.

A novel duck picornavirus, designated as duck/AH15/CHN/2015, was isolated and identified from Cherry Valley ducks with short beak and dwarfism syndrome in 2015 in Anhui province of China. Duck/AH15/CHN/2015 has the highest degree of amino acid sequence identity (approximately 43 %) with duck hepatitis A viruses (DHAV) Complete genome analysis revealed that duck/AH15/CHN/2015 possesses a typical picornavirus-like genomic organization, 5' UTR-L-P1 (VP0-VP3-VP1)-P2 (2A1-2A2- 2B-2C)-P3 (3A-3B-3C-3D)-3'UTR-poly (A). The 5'UTR contains a potential type IV internal ribosome entry site, while a conserved "barbell"-like structure is found at the 3'UTR, which is similar to DHAV. Compared to the closest related DHAVs, two unrelated 2A proteins were predicted in duck/AH15/CHN/2015, while three unrelated 2A proteins were presented in DHAVs. Based on the amino acid identity comparison and phylogenetic analysis of P1, 2C, and 3CD (3C and 3D), duck/AH15/CHN/2015 was closely related to but distinct from DHAVs, and it was proposed to be a member of a novel species in the genus Avihepatovirus of the family Picornaviridae.

Genomic Diversity and Evolution of Quasispecies in Newcastle Disease Virus Infections.

Newcastle disease virus (NDV) infections are well known to harbour quasispecies, due to the error-prone nature of the RNA polymerase. Quasispecies variants in the fusion cleavage site of the virus are known to significantly change its virulence. However, little is known about the genomic patterns of diversity and selection in NDV viral swarms. We analyse deep sequencing data from in vitro and in vivo NDV infections to uncover the genomic patterns of diversity and the signatures of selection within NDV swarms. Variants in viruses from in vitro samples are mostly localised in non-coding regions and 3' and 5' untranslated regions (3'UTRs or 5'UTRs), while in vivo samples contain an order of magnitude more variants. We find different patterns of genomic divergence and diversity among NDV genotypes, as well as differences in the genomic distribution of intra-host variants among in vitro and in vivo infections of the same strain. The frequency spectrum shows clear signatures of intra-host purifying selection in vivo on the matrix protein (M) coding gene and positive or diversifying selection on nucleocapsid (NP) and haemagglutinin-neuraminidase (HN). The comparison between within-host polymorphisms and phylogenetic divergence reveals complex patterns of selective pressure on the NDV genome at between- and within-host level. The M sequence is strongly constrained both between and within hosts, fusion protein (F) coding gene is under intra-host positive selection, and NP and HN show contrasting patterns: HN RNA sequence is positively selected between hosts while its protein sequence is positively selected within hosts, and NP is under intra-host positive selection at the RNA level and negative selection at the protein level.

Isolation and Propagation of Coronaviruses in Embryonated Eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.

Antiviral activity of Apigenin against buffalopox: Novel mechanistic insights and drug-resistance considerations.

We describe herein that Apigenin, which is a dietary flavonoid, exerts a strong in vitro and in ovo antiviral efficacy against buffalopox virus (BPXV). Apigenin treatment was shown to inhibit synthesis of viral DNA, mRNA and proteins, without affecting other steps of viral life cycle such as attachment, entry and budding. Although the major mode of antiviral action of Apigenin was shown to be mediated via targeting certain cellular factors, a modest inhibitory effect of Apigenin was also observed directly on viral polymerase. We also evaluated the selection of drug-resistant virus variants under long-term selection pressure of Apigenin. Wherein Apigenin-resistant mutants were not observed up to ~ P20 (passage 20), a significant resistance was observed to the antiviral action of Apigenin at ~ P30. However, a high degree resistance could not be observed even up to P60. To the best of our knowledge, this is the first report describing in vitro and in ovo antiviral efficacy of Apigenin against poxvirus infection. The study also provides mechanistic insights on the antiviral activity of Apigenin and selection of potential Apigenin-resistant mutants upon long-term culture.

Effects of an H7 Highly Pathogenic and Related Low Pathogenic Avian Influenza Virus on Chicken Egg Production, Viability, and Virus Contamination of Egg Contents and Surfaces.

Both highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) can cause decreases or even cessation of egg production in chickens and turkeys. Production of abnormal eggs (deformed, thin-shelled, soft-shelled) can also be caused by AIV infection. Additionally, egg surfaces and contents may also be contaminated with virus. Because data quantifying these effects are lacking, white Plymouth Rock hens were inoculated with HP or LP AIV while in production. No decreases in egg production or abnormal eggs were observed with LPAIV-infected hens. No lesions or viral antigen staining in ovary and oviduct were observed in LPAIV-infected hens 3 days postchallenge. LPAIV RNA was detected on eggs collected from 12 hr to 11 days postinoculation (PI) and was on or in 6.4% (15/234) of the eggs. Titer equivalents of LPAIV ranged from 1.3-2.5 log10 50% egg infectious doses (EID50). No virus was detected in embryo tissue from eggs laid by LPAIV-infected hens. In contrast, egg production by HPAIV-inoculated hens decreased at 72 hr PI and 18.4% (16/87) of the eggs were abnormal. However, viability was similar to that of the sham inoculates. HPAIV RNA was detected in or on 11.1% (9/81) of the eggs from 36 hr through 96 hr PI, when the hens were euthanatized. HPAIV RNA was detected on 6.2% of eggshells, in 4.2% of albumin/yolk samples, and in 8.3% of embryo tissue. Forty percent of the abnormal eggs were positive for HPAIV RNA. Titer equivalents on or in HPAIV-contaminated eggs ranges from 1.0-4.0 log10 EID50. Lesions and viral antigen staining were present in the ovary and all sections of the oviduct of infected hens 3 days postchallenge. These data will inform models using production-based triggers for LPAIV monitoring and for risk assessments to determine the disposition of eggs from flocks infected with LPAIV or HPAIV.

Efectos de un virus de la influenza aviar altamente patógeno y un virus de baja patogenicidad H7 relacionados en la producción de huevos de gallina, la viabilidad y con la contaminación viral del contenido y las superficies del huevo. Tanto el virus de la influenza aviar (AIV) altamente patógeno (HP) como el de baja patogenicidad (LP) pueden causar una disminución o incluso el cese de la producción de huevos en pollos y pavos. La producción de huevos anormales (con cascaron deforme o delgado, o con cascarón blando) también puede ser causada por la infección por el virus de la influenza aviar. Además, las superficies y el contenido del huevo también pueden estar contaminados con virus. Debido a la falta de datos que cuantifiquen estos efectos, gallinas Plymouth Rock blancas fueron inoculadas con virus de alta o baja patogenicidad mientras estaban en producción. No se observaron disminuciones en la producción de huevos o huevos anormales en las gallinas infectadas con el virus de influenza aviar de baja patogenicidad. No se observaron lesiones ni la presencia del antígeno viral en el ovario y oviducto de gallinas infectadas con el virus de baja patogenicidad a los tres días después del desafío. El ARN del virus de baja patogenicidad se detectó en los huevos recolectados de 12 horas a 11 días después de la inoculación y estaba sobre o dentro del 6.4% (15/234) de los huevos. Los títulos equivalentes del virus de baja patogenicidad variaron de 1.3 a 2.5 log10 dosis infecciosas 50% para embrión de huevo (EID50). No se detectó virus en el tejido embrionario de los huevos puestos por gallinas infectadas con el virus de alta patogenicidad. En contraste, la producción de huevos por gallinas inoculadas con el virus de alta disminuyó a las 72 horas después de la inoculación y el 18.4% (16/87) de los huevos fueron anormales. Sin embargo, la viabilidad fue similar a la de los controles negativos inoculados. Se detectó ARN del virus de alta patogenicidad en el 11.1% (9/ 81) de los huevos desde las 36 horas hasta 96 horas después de la inoculación, cuando las gallinas se sacrificaron. Se detectó ARN del virus de alta patogenicidad en el 6.2% de los cascarones de huevo, en el 4.2% de las muestras de albúmina/yema y en el 8.3% del tejido embrionario. Cuarenta por ciento de los huevos anormales fueron positivos para ARN del virus de alta patogenicidad. Los títulos equivalentes sobre o por dentro de los huevos contaminados con influenza aviar de alta patogenicidad variaron de 1.0­4.0 log10 EID50. Las lesiones y la presencia del antígeno viral estaban presentes en el ovario y en todas las secciones del oviducto de las gallinas infectadas tres días después del desafío. Estos datos servirán como fuente de información para los modelos que utilizan disparadores basados en la producción para el monitoreo del virus de influenza aviar de baja patogenicidad y para las evaluaciones de riesgos para determinar la eliminación de los huevos de las parvadas infectadas con virus de influenza aviar de baja o de alta patogenicidad.

An Embryonated Egg Transmission Model for Epizootic Hemorrhagic Disease Virus.

Epizootic hemorrhagic disease virus (EHDV) is a vector-borne orbivirus of ruminants; in North America there are three serotypes (EHDV-1, -2, and -6) and these primarily affect white-tailed deer (Odocoilus virginianus). EHDV is vectored by biting midges, Culicoides spp. Embryonated chicken eggs (ECE) have recently been used as an experimental host to investigate the vector competence of Australian Culicoides spp. for bluetongue serotype virus 1 and 23. In this study, we evaluated the use of the ECE model to determine its applicability for evaluating vector competence related to transmission of North American EHDV serotypes. We demonstrated that all three North American EHDV serotypes were able to replicate in ECEs and be transmitted from infected ECEs to Culicoides sonorensis Wirth & Jones. In addition, we were able to complete the transmission cycle from infected C. sonorensis to uninfected ECEs for EHDV-1 and -2.

Methods to prevent PCR amplification of DNA from non-viable virus were not successful for infectious laryngotracheitis virus.

Molecular-based testing of poultry dust has been used as a fast, sensitive and specific way to monitor viruses in chicken flocks but it provides no information on viral viability. Differentiation of viable and nonviable virus would expand the usefulness of PCR-based detection. This study tested three treatments (1. DNAse, 2. propidium monoazide [PMA], 3. immunomagnetic separation [IMS]) applied to dust or virus stock prior to nucleic acid extraction for their ability to exclude nonviable virus from PCR amplification. Infectious laryngotracheitis virus (ILTV) was used as a model. These treatments assume loss of viral viability due to damage to the capsid or to denaturation of epitope proteins. DNAse and PMA assess the integrity of the capsid to penetration by enzyme or intercalating dye, while IMS assesses the integrity of epitope proteins. Treatments were evaluated for their ability to reduce PCR signal, measured as ILTV log10 genomic copies (ILTV GC), of heat and chemically inactivated ILTV in poultry dust and virus stock. Compared to untreated dust samples, there was an overall reduction of 1.7 ILTV GC after IMS treatment (p<0.01), and a reduction of 2.0 ILTV GC after PMA treatment (p<0.0001). DNAse treatment did not reduce ILTV GC in dust (p = 0.68). Compared to untreated virus stocks, there was an overall reduction of 0.5 ILTV GC after DNAse treatment (p = 0.04), a reduction of 1.8 ILTV GC after IMS treatment (p<0.001) and a reduction of 1.4 ILTV GC after PMA treatment (p<0.0001). None of the treatments completely suppressed the detection of inactivated ILTV GC. In conclusion, treatments that use capsid integrity or protein epitope denaturation as markers to assess ILTV infectivity are unsuitable to accurately estimate proportions of viable virus in poultry dust and virus stocks.

Regulatory mechanism of microRNA-155 in chicken embryo fibroblasts in response to reticuloendotheliosis virus infection.

Reticuloendotheliosis virus (REV) infection of multiple avian species can lead to a number of diseases such as runting syndrome, immunosuppression and oncogenesis, causing major economic losses. MicroRNAs play important roles in post-transcriptional regulation, effectively inhibiting protein synthesis, and participating in many biological processes in cells, including proliferation, differentiation, apoptosis, lipometabolism, virus infection and replication, and tumorigenesis. Based on our previous high-throughput sequencing results, we explore the regulatory mechanisms of microRNA-155(miR-155) in chicken embryo fibroblasts (CEFs) in response to REV infection. Our results revealed expression of miR-155 in CEFs after REV infection upregulated in a time- and dose-dependent manner, indicating miR-155 plays a role in REV infection in CEFs indeed. After transfected with miR-155-mimic and miR-155-inhibitor, we found overexpression of miR-155 targeted caspase-6 and FOXO3a to inhibit apoptosis and accelerate cell cycle, thus improving viability of REV-infected CEFs. This result also verified the protective role of miR-155 in the viability of CEFs in the presence of REV. Knockdown of miR-155 also supported these above conclusions. Our findings uncover a new mechanism of REV pathogenesis in CEFs, and also provide a theoretical basis for uncovering new effective treatment and prevention methods for RE based on miR-155.

Antiviral activity of Isatidis Radix derived glucosinolate isomers and their breakdown products against influenza A in vitro/ovo and mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Isatidis Radix, the sun-dried roots of Isatis indigotica Fortune ex Lindl., is one of the most usually used traditional Chinese medicines. For centuries, the herb has been employed in clinical practice for treatment of virus infection and inflammation. However, its active ingredients remain unclear. AIM OF THE STUDY: In the present study, the anti-influenza virus activity of epiprogoitrin, progoitrin, epigoitrin and goitrin, the Isatidis Radix derived glucosinolate isomers and their breakdown products, was firstly evaluated in vitro and in ovo and their mechanism of action was investigated. MATERIALS AND METHODS: Epiprogoitrin, progoitrin, epigoitrin and goitrin were isolated from Isatidis Radix by chiral separation. In vitro and in ovo evaluations were performed on Madin-Darby canine kidney (MDCK) cells and embryonated eggs respectively, both using protocols including prevention, treatment and virus neutralization. Hemagglutination (HA) and neuraminidase (NA) inhibition assays were performed for further understanding of the antiviral mechanism. RESULTS: Isatidis Radix derived glucosinolate isomers and their breakdown products all exhibited dose-dependent inhibition effect against influenza A virus (H1N1) without toxicity. The antiviral potency of the components was in the order of progoitrin > goitrin > epigoitrin > epiprogoitrin. The attachment of the constituents to the viral envelope conduced to the mechanism of their antiviral action without disturbing viral adsorption or budding. CONCLUSION: Taken together, these results are promising for further development of Isatidis Radix and may contribute an adjunct to pharmacotherapy for influenza virus infection.

GGERV20, a recently integrated, segregating endogenous retrovirus in Gallus gallus.

Endogenous retroviruses (ERVs) are widespread in vertebrate genomes. The recent availability of whole eukaryotic genomes has enabled their characterization in many organisms, including Gallus gallus (red jungle fowl), the progenitor of the domesticated chicken. Our bioinformatics analysis of a G. gallus ERV previously designated GGERV20 identified 35 proviruses with complete long terminal repeats (LTRs) and gag-pol open reading frames (ORFs) in the Genome Reference Consortium Chicken Build 6a, of which 8 showed potential for translation of functional retroviral polyproteins, including the integrase and reverse transcriptase enzymes. No elements were discovered with an env gene. Fifteen loci had LTR sequences with 100 % identity, indicative of recent integration. Chicken embryo fibroblast RNA-seq datasets showed reads representing the entire length of the GGERV20 provirus, supporting their potential for expressing viral proteins. To investigate the possibility that GGERV20 elements may not be fixed in the genome, we assessed the integration status of five loci in a meat-type chicken. PCRs targeting a GGERV20 locus on G. gallus chromosome one (GGERV201-1) reproducibly amplified both LTRs and the preintegration state, indicating that the bird from which the DNA was sampled was hemizygous at this locus. The four other loci examined only produced the preintegration state amplicons. These results reveal that GGERV20 is not fixed in the G. gallus population, and taken together with the lack of mutations seen in several provirus LTRs and their transcriptional activity, suggest that GGERV20 retroviruses have recently been and continue to be active in the chicken genome.

Polymorphisms of the Chicken Mx Gene Promoter and Association with Chicken Embryos' Susceptibility to Virulent Newcastle Disease Virus Challenge.

Newcastle disease is a devastating viral disease of chicken in low- and middle-income countries where the backyard production system is predominant. Marker-assisted selection of chickens that are resistant to Newcastle disease virus (NDV) is the promising strategy that needs to be explored. The aim of the present study was to investigate polymorphisms of the promoter region of the chicken Mx gene and association with Kuroiler, Sasso, and local Tanzanian chicken embryos' survival variability to virulent NDV infection. Chicken embryos were initially challenged with a minimum lethal dose of virulent NDV suspension and then were followed over time to gather information on their survival variability. Using the survival data, high and less susceptible cohorts were established, and a total of 88 DNA samples from high and less susceptible groups were genotypes by sequencing. Five single-nucleotide polymorphisms (SNPs), which were previously reported, were detected. Interestingly, for the first time, the findings demonstrated the association of the promoter region of chicken myxovirus-resistance (Mx) gene polymorphisms with chicken embryos' susceptibility to the virulent NDV challenge. At the genotypic level, the SNP4 G > A mutation that was located within the IFN-stimulating response element was associated (LR: 6.97, P=0.03) with chicken embryos' susceptibility to the virulent NDV challenge. An allele G frequency was higher in the less susceptible cohort, whereas an allele A frequency was higher in the high susceptible cohort. At the haplotype level, the haplotype group ACGC was associated (OR: 9.8, 95% CI: 1.06-79.43, P=0.042) with the same trait and had a resistant effect. In conclusion, the results have demonstrated the association of chicken Mx gene promoter polymorphisms and chicken embryos' survival variability to the virulent NDV challenge, and the information is useful for breeding programs designed to develop chicken genotypes that are resistant to Newcastle disease virus.

Pathogenicity of an FAdV-4 isolate to chickens and its genomic analysis.

Fowl adenovirus serotype 4 (FAdV-4) strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province, China. The isolate was cultured in primary chicken embryo kidney cells. A study of pathogenicity indicated that SD1511 readily infected 7-35-d-old chickens by intramuscular injection and intranasal and oral routes, causing 50%-100% mortality. The 35-d-old chickens suffered more severe infection than 7- and 21-d-old chickens with mortality highest in the intramuscular injection group. The serum from surviving chickens showed potent viral neutralizing capability. The complete genome of SD1511 was sequenced and analyzed. The strain was found to belong to the FAdV-4 cluster with more than 99% identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations, including deletions of open reading frame 27 (ORF27), ORF48, and part of ORF19. Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.

Assessment of replicate numbers for titrating avian influenza virus using dose-response models.

Embryonating chicken eggs (ECEs) are among the most sensitive laboratory host systems for avian influenza virus (AIV) titration, but ECEs are expensive and require space for storage and incubation. Therefore, reducing ECE use would conserve resources. We utilized statistical modeling to evaluate the accuracy and precision of AIV titration with 3 instead of 5 ECEs for each dilution by the Reed-Muench method for 50% endpoint calculation. Beta-Poisson and exponential dose-response models were used in a simulation study to evaluate observations from actual titration data from 18 AIV isolates. The reproducibility among replicates of a titration was evaluated with one AIV isolate titrated in 3 replicates with the beta-Poisson, exponential, and Weibull dose-response models. The standard deviation (SD) of the error between input and estimated virus titers was estimated with Monte Carlo simulations using the fitted dose-response models. Good fit was observed with all models that were utilized. Reducing the number of ECEs per dilution from 5 to 3 resulted in the width of the 95% confidence interval increasing from ±0.64 to ±0.75 log10 50% ECE infectious doses (EID50) and the SD of the error increased by 0.03 log10 EID50. Our study suggests that using fewer ECEs per dilution is a viable approach that will allow laboratories to reduce costs and improve efficiency.

A viral race for primacy: co-infection of a natural pair of low and highly pathogenic H7N7 avian influenza viruses in chickens and embryonated chicken eggs.

Highly pathogenic avian influenza virus (HPAIV) infection in poultry caused devastating mortality and economic losses. HPAIV of subtypes H5 and H7 emerge from precursor viruses of low pathogenicity (LP) by spontaneous mutation associated with a shift in the susceptibility of the endoproteolytic cleavage site of the viral hemagglutinin protein from trypsin- to furin-like proteases. A recently described natural pair of LP/HP H7N7 viruses derived from two spatio-temporally linked outbreaks in layer chickens was used to study how a minority of mutated HP virions after de novo generation in a single host might gain primacy. Co-infection experiments in embryonated eggs and in chickens were conducted to investigate amplification, spread and transmissionof HPAIV within a poultry population that experiences concurrent infection by an antigenically identical LP precursor virus. Simultaneous LPAIV co-infection (inoculum dose of 106 egg-infectious dose 50% endpoint (EID50)/0.5 mL) withincreasing titers of HPAIV from 101 to 105.7 EID50/0.5 mL) had a significant impeding impact on HP H7 replication, viral excretion kinetics, clinical signs and histopathological lesions (in vivo) and on embryo mortality (in ovo). LP/HP co-infected chickens required a hundredfold higher virus dose (HPAIV inoculum of 105 EID50) compared to HPAIV mono-infection (HPAIV inoculum of 103 EID50) to develop overt clinical signs, mortality and virus spread to uninfected sentinels. Escape and spread of HP phenotypes after de novo generation in an index host may therefore be highly precarious due to significant competition with co-circulating LP precursor virus.

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens.

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.

Evolution of H9N2 avian influenza virus in embryonated chicken eggs with or without homologous vaccine antibodies.

BACKGROUND: Vaccines constitute a unique selective pressure, different from natural selection, drives the evolution of influenza virus. In this study, A/Chicken/Shanghai/F/1998 (H9N2) was continually passaged in specific pathogen-free embryonated chicken eggs with or without selective pressures from antibodies induced by homologous maternal antibodies. Genetic mutations, antigenic drift, replication, and pathogenicity of the passaged virus were evaluated. RESULTS: Antigenic drift of the passaged viruses occurred in the 47th generation (vF47) under selective pressure on antibodies and in the 52nd generation (nF52) without selective pressure from antibodies. Seven mutations were observed in the vF47 virus, with three in PB2 and four in HA, whereas 12 mutations occurred in the nF52 virus, with three in PB2, two in PB1, four in HA, one in NP, one in NA, and one in NS. Remarkably, the sequences of the HA segment from vF47 were 100% homologous with those of the nF52 virus. Both the vF47 and nF52 viruses showed enhanced replication compared to the parental virus F/98, but higher levels of replication and pathogenicity were displayed by nF52 than by vF47. An inactive vaccine derived from the parental virus F/98 did not confer protection against challenges by either the vF47 or nF52 virus, but inactive vaccines derived from the vF47 or nF52 virus were able to provide protection against a challenge using F/98. CONCLUSION: Taken together, the passage of H9N2 viruses with or without selective pressure of the antibodies induced by homologous maternal antibodies showed genetic variation, enhanced replication, and variant antigenicity. Selective pressure of the antibody does not seem to play a key role in antigenic drift in the egg model but may impact the genetic variation and replication ability of H9N2 viruses. These results improve understanding of the evolution of the H9N2 influenza virus and may aid in selecting appropriate vaccine seeds.

Discovery of C-1 modified oseltamivir derivatives as potent influenza neuraminidase inhibitors.

Inspired by our initial discovery about a series of neuraminidase (NA) inhibitors targeting the 150-cavity, in present study, we designed, synthesized, and biologically tested a panel of novel oseltamivir derivatives with C-1 modification, targeting the 430-cavity, an additional binding site which widely and stably existed in both group-1 and group-2 NAs. Some of the synthesized compounds displayed robust anti-influenza potencies against H5N1 and H5N6 viruses. Among them, compound 8b exerted the greatest inhibition, with IC50 values of 0.088 and 0.097 µM and EC50 values of 4.26 and 1.31 µM against H5N1 and H5N6 strains, respectively, which are similar to those of oseltamivir carboxylate (OSC). And its potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC. Molecular modeling revealed the elongated group at C-1 position being projected toward the 430-cavity. Notably, although compound 8b was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model, it displayed greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L. Overall, this work provided unique insights in the discovery of potent inhibitors against both group-1 and group-2 NAs.