Kinetic stability modulation of polymeric nanoparticles for enhanced detection of influenza virus via penetration of viral fusion peptides.

Specific interactions between viruses and host cells provide essential insights into material science-based strategies to combat emerging viral diseases. pH-triggered viral fusion is ubiquitous to multiple viral families and is important for understanding the viral infection cycle. Inspired by this process, virus detection has been achieved using nanomaterials with host-mimetic membranes, enabling interactions with amphiphilic hemagglutinin fusion peptides of viruses. Most research has been on designing functional nanoparticles with fusogenic capability for virus detection, and there has been little exploitation of the kinetic stability to alter the ability of nanoparticles to interact with viral membranes and improve their sensing performance. In this study, a homogeneous fluorescent assay using self-assembled polymeric nanoparticles (PNPs) with tunable responsiveness to external stimuli is developed for rapid and straightforward detection of an activated influenza A virus. Dissociation of PNPs induced by virus insertion can be readily controlled by varying the fraction of hydrophilic segments in copolymers constituting PNPs, giving rise to fluorescence signals within 30 min and detection of various influenza viruses, including H9N2, CA04(H1N1), H4N6, and H6N8. Therefore, the designs demonstrated in this study propose underlying approaches for utilizing engineered PNPs through modulation of their kinetic stability for direct and sensitive identification of infectious viruses.

A Pigeon-Derived Sub-Genotype XXI.1.2 Newcastle Disease Virus from Bangladesh Induces High Mortality in Chickens.

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014-2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.

Analysis of Single Nucleotide Variants (SNVs) Induced by Passages of Equine Influenza Virus H3N8 in Embryonated Chicken Eggs.

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.

Innate immune responses of domestic pigeons to the infection of pigeon paramyxovirus type 1 virus.

Pigeon paramyxovirus type 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus type-1. The PPMV-1-associated disease poses a great threat to the pigeon industry. The innate immune response is crucial for antiviral infections and revealing the pathogenic mechanisms of PPMV-1. In this study, we evaluated the pathogenicity of a PPMV-1 strain LHLJ/110822 in one-month-old domestic pigeons, as well as the host immune responses in PPMV-1-infected pigeons. We observed typically clinical sign in infected pigeons by 3 dpi. The morbidity rate and the mortality in pigeons inoculated with the PPMV-1 strain were up to 100% and 30%, respectively. The virus could replicate in all of the examined tissues, namely trachea, lung, liver, spleen, and bursa of Fabricius. In addition, the infected pigeons had developed anti-PPMV-1 antibodies as early as 8 dpi; and the antibody level increased over the time in this study. The expression level of toll-like receptor (TLR) 2, TLR3 TLR15, IFN-γ, and IL-6 were significantly upregulated by the PPMV-1 infection in some tissues of pigeons. By contrast, PPMV-1 infection results in downregulation of IL-18 expression in most of investigated tissues except for bursa of Fabricius in this study. The current results confirmed that this virus could replicate in pigeons and induce host immune responses, then leading to produce serum antibody titers. Meanwhile, the PPMV-1 infection induces strong innate immune responses and intense inflammatory responses at early stage in pigeon which may associate with the viral pathogenesis.

Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets.

Poultry production plays an important role in the economy and livelihoods of rural households in Kenya. As part of a surveillance program, avian influenza virus (AIV)-specific real-time RT-PCR (RRT-PCR) was used to screen 282 oropharyngeal swabs collected from chickens at six live bird markets (LBMs) and 33 backyard poultry farms in Kenya and 8 positive samples were detected. Virus was isolated in eggs from five samples, sequenced, and identified as H9N2 low pathogenic AIV (LPAIV) G1 lineage, with highest nucleotide sequence identity (98.6-99.9%) to a 2017 Ugandan H9N2 isolate. The H9N2 contained molecular markers for mammalian receptor specificity, implying their zoonotic potential. Virus pathogenesis and transmissibility was assessed by inoculating low and medium virus doses of a representative Kenyan H9N2 LPAIV isolate into experimental chickens and exposing them to naïve uninfected chickens at 2 -days post inoculation (dpi). Virus shedding was determined at 2/4/7 dpi and 2/5 days post placement (dpp), and seroconversion determined at 14 dpi/12 dpp. None of the directly-inoculated or contact birds exhibited any mortality or clinical disease signs. All directly-inoculated birds in the low dose group shed virus during the experiment, while only one contact bird shed virus at 2 dpp. Only two directly-inoculated birds that shed high virus titers seroconverted in that group. All birds in the medium dose group shed virus at 4/7 dpi and at 5 dpp, and they all seroconverted at 12/14 dpp. This is the first reported detection of H9N2 LPAIV from Kenya and it was shown to be infectious and transmissible in chickens by direct contact and represents a new disease threat to poultry and potentially to people.

Challenges in the development of egg-independent vaccines for influenza.

Introduction: Current influenza vaccines are mainly produced from viruses propagated in eggs, an established process developed over 70 years. However, this technology presents some drawbacks and other platforms are under development or have been already developed in order to replace or to be used alongside the old one. Area covered: The present review provides an overview of influenza vaccine production, starting from egg-based technology, to cell-derived vaccines, until the novel platforms/technologies for the production of influenza vaccines such as DNA-based vaccines, virus-like particles and plant-based technology. Expert opinion: The ideal method of production should have certain characteristics such as great flexibility and scalability, the viruses should be representative of the circulating strains, process should be standardized and controlled, and it should be possible to start production as soon as the sequence of the new influenza strain is available. However, it is important not to underestimate the fact that some parts of the vaccine production process have been established for egg-based vaccines. Thus, changes in vaccine production methods are not merely 'technical changes'; rather, they involve various aspects that slow down the authorization of new influenza vaccines and make the complete replacement of egg-based production unlikely in the near future.

Attenuation of Infectious Bronchitis Virus in Eggs Results in Different Patterns of Genomic Variation across Multiple Replicates.

The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs; however, the molecular mechanism of attenuation is unknown. M41-CK, a virulent lab-adapted strain of IBV, was egg passaged over 100 times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, and the results showed that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hot spots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation; however, little is known about the evolution of the viral population during the process of attenuation. High-throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hot spots of variation in the attenuated viruses have the potential to be used in the rational attenuation of virulent IBV for next-generation vaccine design.

Managing Marek's disease in the egg industry.

The industrialization of farming has had an enormous impact. To most, this impact is viewed solely in the context of productivity, but the denser living conditions and shorter rearing periods of industrial livestock farms provide pathogens with an ideal opportunity to spread and evolve. For example, the industrialization of poultry farms drove the Marek's disease virus (MDV) to evolve from a mild paralytic syndrome to a highly contagious, globally prevalent, deadly disease. Fortunately, the economic catastrophe that would occur from MDV evolution is prevented through the widespread use of live imperfect vaccines that limit disease symptoms, but fail to prevent transmission. Unfortunately, the continued rollout of such imperfect vaccines is steering MDV evolution towards even greater virulence, and the ability to evade vaccine protection. Thus, there is a need to investigate alternative economically viable control measures for their ability to inhibit MDV spread and evolution. In what follows we examine the economic viability of standard husbandry practices for their ability to inhibit the spread of both virulent MDV and very virulent MDV throughout an industrialized egg farm. To do this, we parameterize a MDV transmission model and calculate the loss in egg production due to MDV. We find that MDV strain and the cohort duration have the greatest influence on both disease burden and egg production. Additionally, our findings show that for long cohort durations, conventional cages result in the least per capita loss in egg production due to MDV infection, while Aviary systems perform best over shorter cohort durations. Finally, we find that the least per capita loss in egg production for flocks infected with the more virulent MDV strains occurs when cohort durations are sufficiently short. These results highlight the important decisions that managers will face when implementing new hen husbandry practices.

Assessing the susceptibility of highly pathogenic avian influenza H5N1 viruses to oseltamivir using embryonated chicken eggs.

Background & objectives: The susceptibility of influenza viruses to neuraminidase inhibitors (NAIs) is studied using enzyme-based assays, sequence analysis and in vitro and in vivo studies. Oseltamivir carboxylate (OC) is the active prodrug of the NAI oseltamivir. There is lack of information on the use of embryonated chicken eggs for studying susceptibility of highly pathogenic avian influenza (HPAI) H5N1 viruses to antiviral drugs. The aim of the present study was to assess the use of 10 day old embryonated chicken eggs for studying antiviral susceptibility of HPAI H5N1 viruses. Methods: Two HPAI H5N1 viruses isolated from India were used in the study. Fluorescence-based NAI assay was performed to determine antiviral susceptibility of these viruses. In ovo antiviral assays were carried out using 10 day old embryonated chicken eggs. The virus dilutions were incubated with 14 µg/ml of OC and inoculated in the allantoic cavity. In the eggs, 50 per cent egg infectious dose (EID50) titres as well as mortality were quantitated. Results: The two viruses used were susceptible to OC in the NAI assay. It was found that there was a significant drop in EID50titres; however, no significant protection from mortality after OC treatment was observed. Interpretation & conclusions: By measuring viral titres, the egg model was suitable to study the susceptibility of HPAI viruses to antiviral drugs along with NAI assay. The present study highlights the use of eggs as a model to study susceptibility of HPAI viruses to OC.

Quantitative patterns of vertical transmission of deformed wing virus in honey bees.

Deformed wing virus (DWV) is an important pathogen in a broad range of insects, including honey bees. Concordant with the spread of Varroa, DWV is present in the majority of honey bee colonies and can result in either low-level infections with asymptomatic bees that nonetheless exhibit increased colony loss under stress, or high-level infections with acute effects on bee health and viability. DWV can be transmitted vertically or horizontally and evidence suggests that horizontal transmission via Varroa is associated with acute symptomatic infections. Vertical transmission also occurs and is presumably important for the maintenance of DWV in honey bee populations. To further our understanding the vertical transmission of DWV through queens, we performed three experiments: we studied the quantitative effectiveness of vertical transmission, surveyed the prevalence of successful egg infection under commercial conditions, and distinguished among three possible mechanisms of transmission. We find that queen-infection level predicts the DWV titers in their eggs, although the transmission is not very efficient. Our quantitative assessment of DWV demonstrates that eggs in 1/3 of the colonies are infected with DWV and highly infected eggs are rare in newly-installed spring colonies. Additionally, our results indicate that DWV transmission occurs predominantly by virus adhering to the surface of eggs (transovum) rather than intracellularly. Our combined results suggest that the queens' DWV vectoring capacity in practice is not as high as its theoretical potential. Thus, DWV transmission by honey bee queens is part of the DWV epidemic with relevant practical implications, which should be further studied.

Electrochemical detection of influenza virus H9N2 based on both immunomagnetic extraction and gold catalysis using an immobilization-free screen printed carbon microelectrode.

Influenza is a viral infectious disease considered as a source of many health problems and enormous socioeconomic disruptions. Conventional methods are inadequate for in-field detection of the virus and generally suffer from being laborious and time-consuming. Thus, studies aiming to develop effective alternatives to conventional methods are urgently needed. In this work, we developed an approach for the isolation and detection of influenza A virus subtype H9N2. For this aim, two specific influenza receptors were used. The first, anti-matrix protein 2 (M2) antibody, was attached to iron magnetic nanoparticles (MNPs) and used for the isolation of the virus from allantoic fluid. The second biomolecule, Fetuin A, was attached to an electrochemical detectable label, gold nanoparticles (AuNPs), and used to detect the virus tacking advantage from fetuin-hemagglutinin interaction. The MNP-Influenza virus-AuNP formed complex was isolated and treated by an acid solution then the collected gold nanoparticles were deposited onto a screen printed carbon electrode. AuNPs catalyzes the hydrogen ions reduction in acidic medium while applying an appropriate potential, and the generated current signal was proportional to the virus titer. This approach allows the rapid detection of influenza virus A/H9N2 at a less than 16 HAU titer.

Poor Immunogenicity, Not Vaccine Strain Egg Adaptation, May Explain the Low H3N2 Influenza Vaccine Effectiveness in 2012-2013.

Background: Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012-2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3). Methods: We investigated the basis of low VE in 2012-2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination. Results: We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer. Conclusions: In contrast to analyses based on ferret studies, low H3N2 VE in 2012-2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population.

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice.

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients' respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.

Detección e identificación de Salmonella spp. en huevos para consumo humano, provenientes de diferentes localidades de Bogotá, Colombia, 2015 / Salmonella spp. isolation and identification in eggs for human consumption from different urban areas in Bogotá, Colombia, 2015

Objetivo: Determinar la prevalencia de Salmonella spp. en muestras de huevos para consumo humano en localidades de Bogotá. Materiales y métodos: Se obtuvieron 96 muestras en tiendas y plazas de mercado de 4 localidades de la ciudad. Se procesó de forma separada la cáscara y el contenido interno mediante métodos microbiológicos y moleculares para aislamiento e identificación Salmonella spp. Resultados: Se determinó una prevalencia total de Salmonella spp. de 9,4% (n=9), de ésta el 55% (n = 5) provenían del contenido interno y 44% (n = 4) de cáscara, sin embargo no se logró tipificar el serovar de Salmonella enterica presente. Las localidades con mayor presencia del patógeno fueron Usaquén y Fontibón. Discusión: Estudios realizados en Colombia evidencian bajas prevalencias de Salmonella spp. (0 -1,74%) en muestras de huevos, sin embargo los hallazgos de este estudio evidencian una mayor recuperación, lo que podría asociarse con inadecuadas condiciones de manejo y/o almacenamiento del producto. Conclusión: Se estableció la presencia de Salmonella spp. en las muestras de huevo evaluadas, lo que implica un riesgo potencial para la salud pública, por lo que es necesario ampliar este tipo de estudios para conocer la situación real a nivel nacional frente a este patógeno.

Objective: To determine the prevalence of Salmonella spp presence in eggs for human consumption in urban areas in Bogota. Materials and methods: 96 samples were collected from convenience stores and markets in 4 urban areas in the city. The eggshells were separated from the egg's internal content and both were processed separately using microbiological and molecular techniques to isolate and identify Salmonella spp. strains. Results: A Salmonella spp. prevalence of 9.4% (n=9) was found. Salmonella spp was isolated from the egg's internal content in 55% (n=5) of samples and 44% (n=4) from the eggshells. The Salmonella enterica serovar could not be identified. The pathogen was more frequently isolate in samples from Usaquén and Fontibón urban areas. Discussion: Studies of Salmonella spp. prevalence in eggs done in Colombia have shown it to be low (0-1.74%); However, this study determined a higher prevalence. These results suggest that inadequate handling/storage conditions could have been associated with them. Conclusion: Salmonella spp. was isolated from the egg samples from 4 different urban areas in Bogotá. These findings suggest the existence of a public health risk; therefore, there is a need to perform wider and more complete studies to determine the actual situation of Salmonella ssp. egg contamination in the country.

Rationally Designed Influenza Virus Vaccines That Are Antigenically Stable during Growth in Eggs.

Influenza virus vaccine production is currently limited by the ability to grow circulating human strains in chicken eggs or in cell culture. To facilitate cost-effective growth, vaccine strains are serially passaged under production conditions, which frequently results in mutations of the major antigenic protein, the viral hemagglutinin (HA). Human vaccination with an antigenically drifted strain is known to contribute to poor vaccine efficacy. To address this problem, we developed a replication-competent influenza A virus (IAV) with an artificial genomic organization that allowed the incorporation of two independent and functional HA proteins with different growth requirements onto the same virion. Vaccination with these viruses induced protective immunity against both strains from which the HA proteins were derived, and the magnitude of the response was as high as or higher than vaccination with either of the monovalent parental strains alone. Dual-HA viruses also displayed remarkable antigenic stability; even when using an HA protein known to be highly unstable during growth in eggs, we observed high-titer virus amplification without a single adaptive mutation. Thus, the viral genomic design described in this work can be used to grow influenza virus vaccines to high titers without introducing antigenic mutations.IMPORTANCE Influenza A virus (IAV) is a major public health threat, and vaccination is currently the best available strategy to prevent infection. While there have been many advances in influenza vaccine production, the fact that we cannot predict the growth characteristics of a given strain under vaccine production conditions a priori introduces fundamental uncertainty into the process. Clinically relevant IAV strains frequently grow poorly under vaccine conditions, and this poor growth can result in the delay of vaccine production or the exchange of the recommended strain for one with favorable growth properties. Even in strains that grow to high titers, adaptive mutations in the antigenic protein hemagglutinin (HA) that make it antigenically dissimilar to the circulating strain are common. The genomic restructuring of the influenza virus described in this work offers a solution to the problem of uncertain or unstable growth of IAV during vaccine production.

Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus.

BACKGROUND: Influenza virus isolation in embryonated chicken eggs (ECEs) is not applicable for rapid diagnosis, however it allows the recovery and propagation of the viable virus. A low number of infectious virus particles in the swabs, poor quality of samples or individual strain properties can lead to difficulties during the virus isolation process. We propose to utilize chorioallantoic membranes (CAM) of ECEs with the assistance of real-time RT PCR to facilitate equine influenza virus isolation. METHODS: Real-time RT PCR was used to detect influenza virus genetic material in amniotic/allantoic fluids (AF) and CAM of ECEs. Haemagglutination assay was used for AF. We used highly diluted virus as a substitute of clinical specimen for ECEs inoculation. RESULTS: Our study demonstrated that real-time RT PCR testing of CAM homogenates was more useful than testing of AF for EIV detection in ECEs. Positive results from CAM allowed to select the embryos from those with haemagglutination assay (HA) - and real-time RT PCR-negative AF for further passages. Using homogenates of CAM for subsequent passages, we finally obtained HA-positive AF, which confirmed virus replication. CONCLUSION: We postulate that real-time RT PCR testing of CAM homogenates and their subsequent passages may facilitate the isolation of equine influenza viruses.

In vitro characterization of the novel H3N1 reassortant influenza viruses from quail.

Quail is considered as an intermediate host for generation of the novel reassortant influenza A viruses (IAVs). In this study, we evaluated the replication ability of the three novel H3N1 reassortant viruses recovered from pandemic H1N1 2009 (pH1N1) and duck H3N2 (dkH3N2) co-infected quail generated from our previous study in embryonated chicken eggs, mammalian (MDCK) and human lung derived (A549) cells. Our study demonstrated that all of the reassortant viruses replicated efficiently in avian and mammalian cells, albeit with slightly lower titers than the parental viruses. Of note, all of the reassortant viruses showed enhanced replication in human lung derived A549 cells compared to their parental viruses. Interestingly, among the reassortant viruses tested, a reassortant virus (P(NA,NS)-DK) containing NA and NS genes derived from pH1N1 and the other genes from dkH3N2 exhibited the highest replication ability in all in vitro models, indicating a high level of gene compatibility of this reassortant virus. Our results highlight the potential role of quail as intermediate hosts for the generation of the viable reassortant viruses with ability to replicate efficiently in avian, mammalian, and particularly human lung derived cells. These findings emphasize the need for the continuous IAV surveillance in quail to prevent the risk of the emergence of the novel viable reassortant viruses.

The Ability of a Non-Egg Adapted (Cell-Like) A(H1N1)pdm09 Virus to Egg-Adapt at HA Loci Other than 222 and 223 and Its Effect on the Yield of Viral Protein.

Previous studies on influenza A(H1N1)pdm09 candidate vaccine viruses (CVVs) that had adapted to growth in embryonated chicken eggs by the acquisition of amino acid substitutions at HA positions 222 or 223 showed that improved protein yield could be conferred by additional amino acid substitutions in the haemagglutinin (HA) that arose naturally during passaging of the virus in eggs. In this study we investigated, by means of reverse genetics, the ability of a non-egg adapted (cell-like) A(H1N1)pdm09 virus to egg-adapt at HA loci other than 222/223, introducing amino acid substitutions previously identified as egg adaptations in pre-H1N1pdm09 H1N1 viruses and assessing their effect on protein yield and antigenicity. We also investigated the effect on the protein yield of these substitutions in viruses that had A(H1N1)pdm09 internal genes rather than the traditional PR8 internal genes of a CVV. The data show that a cell-like A/Christchurch/16/2010 can be egg-adapted via amino acid substitutions in at least three alternative HA loci (187, 190 and 216), in viruses with either PR8 or A/California/7/2009 internal genes, but that the effects on protein yield vary depending on the amino acid substitution and the internal genes of the virus. Since CVVs need to produce high protein yields to be suitable for vaccine manufacture, the findings of this study will assist in the future characterisation of both wild type viruses and lab-derived CVVs for vaccine use.

Treatment of Salmonella-Contaminated Eggs and Pork with a Broad-Spectrum, Single Bacteriophage: Assessment of Efficacy and Resistance Development.

Numerous studies have assessed the efficacy of phage-based methods to inhibit Salmonella contamination in food products. As with most antibacterials, bacteria can develop resistance to phage in vitro. Here, we applied a single broad-spectrum Salmonella phage, vB_SalS_SJ_2 (SJ2; 108 PFU; MOI = 10), to Salmonella-contaminated meat and eggs to quantify the development of resistance in actual food matrices. Treatment with a single phage significantly reduced Salmonella Typhimurium contamination in both ground pork and liquid egg at various time points. Similarly, the same phage significantly reduced Salmonella Enteritidis in both food matrices. Efficacy was temperature dependent as larger reductions were seen at higher temperatures (21°C) versus lower temperatures (4°C) at 24 h. Following phage treatment, over 10,000 Salmonella isolates were examined for resistance to the treatment phage. The percentages of phage-resistant Salmonella (either serovar) recovered from phage-treated versus untreated pork did not differ. Conversely, significantly (p < 0.05) higher percentages of phage-resistant Salmonella Typhimurium (92.50% vs. 0.56% of control) and Salmonella Enteritidis (50.83% vs. 0.56% of control) isolates were observed in phage-treated versus untreated egg samples after incubation at room temperature for 48 h. Taken together, these data indicate that the food matrix may influence the emergence of phage resistance with resistance developing more rapidly in foods with less complex microbial communities. Future studies will focus on the impact the development of resistance in production and processing settings may have on the efficacy of phage treatments for longer term biocontrol of pathogens.

Surveillance of maternal antibodies against West Nile virus in chicken eggs in South-West Germany.

OBJECTIVE: The emergence of West Nile virus (WNV) in several European countries increases the risk of its introduction to Germany. This study evaluated a new method for WNV surveillance by testing for maternal antibodies in chicken eggs. METHODS: A total of 1,990 eggs were collected in 35 sampling sites in the south-west of Germany and tested for WNV-specific antibodies. RESULTS: The results did not indicate evidence for WNV circulation in the study area. CONCLUSION: This work serves as a proof-of-concept that such a method is useful and a potential alternative to use of sentinel chicken for regular WNV surveillance.