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Salmonella enterica serovars are zoonotic bacterial that cause foodborne enteritis. Due to bacteria's antibiotic resistance, using bacteriophages for biocontrol and treatment is a new therapeutic approach. In this study, we isolated, characterized, and analyzed the genome of vB_SenS_TUMS_E19 (E19), a broad host range Salmonella bacteriophage, and evaluated the influence of E19 on liquid eggs infected with Salmonella enterica serovar Enteritidis. Transmission electron microscopy showed that the isolated bacteriophage had a siphovirus morphotype. E19 showed rapid adsorption (92% in 5 min), a short latent period (18 min), a large burst size (156 PFU per cell), and a broad host range against different Salmonella enterica serovars. Whole-genome sequencing analysis indicated that the isolated phage had a 42 813 bp long genome with 49.8% G + C content. Neither tRNA genes nor those associated with antibiotic resistance, virulence factors, or lysogenic formation were detected in the genome. The efficacy of E19 was evaluated in liquid eggs inoculated with S . Enteritidis at 4 and 25 °C, and results showed that it could effectively eradicate S . Enteritidis in just 30 min and prevented its growth up to 72 h. Our findings indicate that E19 can be an alternative to a preservative to control Salmonella in food samples and help prevent and treat salmonellosis., Competing Interests: The authors declare no conflict of interest.

Salmonella Typhimurium ( S. Typhimurium) contamination poses a significant challenge to breeder egg hatchability and chick health, necessitating the exploration of alternative disinfection methods. This study investigates the potential of phage vB_SPuM_SP02 (SP02) as a novel disinfectant for breeder eggs contaminated with S. Typhimurium SM022. Phage SP02 was isolated from poultry farm effluent and characterized for morphology, biological properties, and genome properties. Experimental groups of specific pathogen-free (SPF) eggs were treated with Salmonella and phage SP02, and efficacy was assessed through hatching rates, chick survival, weight, Salmonella load, immune organ indices, and intestinal flora. Phage treatment effectively eradicated Salmonella contamination on eggshells within 12 h, resulting in increased hatching and survival rates compared to controls. Furthermore, phage treatment mitigated weight loss and tissue Salmonella load in chicks without causing immune organ damage while reducing Salmonella spp. abundance in the intestinal tract. This study demonstrates the potential of phage SP02 as an eco-friendly and efficient disinfectant for S . Typhimurium-contaminated breeder eggs, offering promising prospects for practical application in poultry production.

Salmonella, a prominent foodborne pathogen, has posed enduring challenges to the advancement of food safety and global public health. The escalating concern over antibiotic misuse, resulting in the excessive presence of drug residues in animal-derived food products, necessitates urgent exploration of alternative strategies for Salmonella control. Bacteriophages emerge as promising green biocontrol agents against pathogenic bacteria. This study delineates the identification of two novel virulent Salmonella phages, namely phage vB_SalS_ABTNLsp11241 (referred to as sp11241) and phage 8-19 (referred to as 8-19). Both phages exhibited efficient infectivity against Salmonella enterica serotype Enteritidis (SE). Furthermore, this study evaluated the effectiveness of two phages to control SE in three different foods (whole chicken eggs, raw chicken meat, and lettuce) at different MOIs (1, 100, and 10000) at 4°C. It's worth noting that sp11241 and 8-19 achieved complete elimination of SE on eggs after 3 h and 6 h at MOI = 100, and after 2 h and 5 h at MOI = 10000, respectively. After 12 h of treatment with sp11241, a maximum reduction of 3.17 log 10 CFU/mL in SE was achieved on raw chicken meat, and a maximum reduction of 3.00 log 10 CFU/mL was achieved on lettuce. Phage 8-19 has the same effect on lettuce as sp11241, but is slightly less effective than sp11241 on chicken meat (a maximum 2.69 log 10 CFU/mL reduction). In conclusion, sp11241 and 8-19 exhibit considerable potential for controlling Salmonella contamination in food at a low temperature and represent viable candidates as green antibacterial agents for food applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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., (Copyright © 2020. Published by Elsevier Inc.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All authors read and approved the final version of the manuscript., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 (EID 50 ) 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 EID 50 titres; 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., Competing Interests: None

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.

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., (Copyright © 2019 Oade et al.)

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2017 Harding et al.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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&#95;SalS&#95;SJ&#95;2 (SJ2; 10 8 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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2016 John Wiley & Sons Ltd.)

Unlabelled: Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines., Importance: Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The emergence of avian influenza A(H7N9) virus in poultry causing zoonotic human infections was reported on March 31, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was initiated without delay. Candidate vaccine viruses were derived by reverse genetics using the internal genes of A/Puerto/Rico/8/34 (PR8). The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation., Methods: Two CVVs derived by reverse genetics were serially passaged in embryonated eggs to improve the hemagglutinin (HA) antigen yield. The total viral protein and HA antigen yields of six egg-passaged CVVs were determined by the BCA assay and isotope dilution mass spectrometry (IDMS) analysis, respectively. CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera., Results: Improvement of total viral protein yield was observed for the six egg-passaged CVVs; HA quantification by IDMS indicated approximately a twofold increase in yield of several egg-passaged viruses as compared to that of the parental CVV. Several different amino acid substitutions were identified in the HA of all viruses after serial passage. However, HI tests indicated that the antigenic properties of two CVVs remained unchanged., Conclusions: If influenza A(H7N9) viruses were to acquire sustained human-to-human transmissibility, the improved HA yield of the egg-passaged CVVs generated in this study could expedite vaccine manufacturing for pandemic mitigation., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Influenza is an infectious respiratory disease with frequent seasonal epidemics that causes a high rate of mortality and morbidity in humans, poultry, and animals. Influenza is a serious economic concern due to the costly countermeasures it necessitates. In this study, we compared the antiviral activities of several flavonols and other flavonoids with similar, but distinct, hydroxyl or methyl substitution patterns at the 3, 3', and 4' positions of the 15-carbon flavonoid skeleton, and found that the strongest antiviral effect was induced by isorhamnetin. Similar to quercetin and kaempferol, isorhamnetin possesses a hydroxyl group on the C ring, but it has a 3'-methyl group on the B ring that is absent in quercetin and kaempferol. Co-treatment and pre-treatment with isorhamnetin produced a strong antiviral effect against the influenza virus A/PR/08/34(H1N1). However, isorhamnetin showed the most potent antiviral potency when administered after viral exposure (post-treatment method) in vitro. Isorhamnetin treatment reduced virus-induced ROS generation and blocked cytoplasmic lysosome acidification and the lipidation of microtubule associated protein1 light chain 3-B (LC3B). Oral administration of isorhamnetin in mice infected with the influenza A virus significantly decreased lung virus titer by 2 folds, increased the survival rate which ranged from 70-80%, and decreased body weight loss by 25%. In addition, isorhamnetin decreased the virus titer in ovo using embryonated chicken eggs. The structure-activity relationship (SAR) of isorhamnetin could explain its strong anti-influenza virus potency; the methyl group located on the B ring of isorhamnetin may contribute to its strong antiviral potency against influenza virus in comparison with other flavonoids.

High pathogenicity avian influenza virus (HPAIV) infections in chickens negatively impact egg production and cause egg contamination. Previously, vaccination maintained egg production and reduced egg contamination when challenged with a North American H5N2 HPAIV. However, Asian H5N1 HPAIV infection has some characteristics of increased pathogenicity compared to other H5 HPAIV such as more rapid drop and complete cessation in egg production. Sham (vaccinated at 25 and 28 weeks of age), inactivated H5N1 Once (1X-H5-Vax; vaccinated at 28 weeks of age only) and inactivated H5N1 Twice (2X-H5-Vax; vaccinated at 25 and 28 weeks of age) vaccinated adult White Leghorn hens were challenged intranasally at 31 weeks of age with 6.1 log10 mean embryo infectious doses (EID50) of clade 2.3.2.1a H5N1 HPAIV (A/chicken/Vietnam/NCVD-675/2011) which was homologous to the inactivated vaccine. Sham-vaccinated layers experienced 100% mortality within 3 days post-challenge; laid soft and thin-shelled eggs; had recovery of virus from oral swabs and in 53% of the eggs from eggshell surface (35%), yolk (24%), and albumin (41%); and had very high titers of virus (average 7.91 log10 EID50/g) in all segments of the oviduct and ovary. By comparison, 1X- and 2X-H5-Vax challenged hens survived infection, laid similar number of eggs pre- and post-challenge, all eggs had normal egg shell quality, and had significantly fewer contaminated eggs with reduced virus quantity. The 2X-H5-Vax hens had significantly higher HI titers by the day of challenge (304 GMT) and at termination (512 GMT) than 1X-H5-Vax hens (45 GMT and 128 GMT). The current study demonstrated that AIV infections caused by clade 2.3.2.1a H5N1 variants can be effectively controlled by either double or single homologous vaccination., (Published by Elsevier Ltd.)

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs.

Specific Pathogen Free (SPF) embryonated eggs are used for the production of many veterinary and human vaccines. We have used High Throughput Sequencing to screen allantoic fluids and embryos for the presence of encapsidated viral genomes and viral transcripts, respectively. SPF eggs from two different producers were tested. We evidenced sequences corresponding to known endogenous retroviruses and sequences of Avian Leukosis Virus, but no sequence that might suggest a productive infection of eggs with a virus even distant from known viruses. Our results strongly suggest that SPF eggs such as those used for this study represent a safe substrate for the production of vaccines., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Chicks possess maternally derived antibody (MDA) against pathogens and vaccines previously encountered by the dams. This passive immunity is important in early life, when the immune system is immature and unable to fight off infection. On the other hand, MDA can also affect the development of the immune system and interfere with vaccination against avian diseases such as Newcastle disease (ND) and avian influenza (AI). The effect of MDA is generally investigated by studying the progeny of vaccinated dams, which is time-consuming, poorly flexible and expensive. Moreover, the antibody titres obtained are not homogeneous. In this study, a model was developed to offer a faster, more reproducible and cheaper way to study passive immunity in specific pathogen free chickens by injection of a polyclonal serum into the egg yolk at embryonic day 14, combined with an intraperitoneal injection at day 1. A satisfactory model, with consistent, homogeneous antibody titres, as well as persistence close to natural passive immunity, could be obtained for ND virus. On the other hand, the application of this optimized protocol in an H5 AI context induced only a low artificial passive immunity compared with that described in the literature for the progeny of AI vaccinated dams. This artificial model should facilitate future studies regarding the effect of passive immunity on vaccine efficacy at a young age and its effect on immune system development.

Influenza viruses can take on two distinct morphologies: filamentous or spherical. While the functional significance of each virion type is unclear, filaments are generally observed in low-passage-number isolates, while an exclusively spherical morphology is seen in strains grown extensively in laboratory substrates. Previous studies have shown that filamentous morphology is lost upon passage in eggs. The fact that the filamentous morphology is maintained in nature but not in the laboratory suggests that filaments provide an advantage in the host that is not necessary for growth in laboratory substrates. To test this hypothesis and identify naturally occurring mutations that alter morphology, we examined the effect of serial adaptation in eggs, MDCK cells, and guinea pigs. Two filamentous strains, A/Netherlands/602/2009 (H1N1) and A/Georgia/M5081/2012 (H1N1), were passaged in eggs and MDCK cells. Conversely, the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) was passaged in guinea pigs. We found that although passage in eggs and MDCK cells can lead to a loss of filaments, an exclusively spherical morphology is not required for highly efficient growth in either substrate. We did, however, identify two point mutations in the matrix of egg passage 10 isolates that confer spherical morphology and increased growth in eggs. In contrast, serial passage in guinea pigs resulted in the selection of filament-forming variants. Sequencing revealed point mutations to the PR8 matrix that, when introduced individually, yielded filaments. These findings suggest a functional role for filaments in the infected host and expand the breadth of mutations known to affect influenza virus shape.

Infection with high-pathogenicity avian influenza virus (HPAIV) has been associated with a wide range of clinical manifestations in poultry, including severe depression in egg production and isolation of HPAIV from eggs laid by infected hens. To evaluate the pathobiology in the reproductive tract of chickens, adult hens were inoculated intranasally with 3 HPAIV strains. All 3 strains induced lesions in the reproductive tract 36 to 72 hours after inoculation. Positive immunostaining was observed in all segments of the reproductive tract, occurring predominantly in stromal cells and superficial germinal epithelium of the ovary, in mucosal epithelial cells and less often glandular epithelium throughout the oviduct, and in vascular endothelium. This study generates important data and explains previously reported virus isolation from yolk, due to ovarian virus replication, and virus recovery from albumin, due to virus replication in epithelial cells in several segments of the oviduct.

The influenza surface glycoprotein hemagglutinin (HA) binds to sialylglycoproteins and sialylglycolipids on the surface of host cells. These sialyl-glycans, usually linked to galactose in either α2,6 or α2,3 configurations, are the receptors for the viral HA, the binding to which promotes viral attachment, membrane fusion, and internalization of the virus. This review examines all of the available receptor binding data on the influenza B HA and provides a structure recognition perspective for the receptor binding preferences of influenza B virus HA regional and egg-adapted variants. Overall, the review serves as an up-to-date compendium of the literature binding data, and the presented discussions assist the reader in reaching a consensus understanding of the receptor specificity determinants for the influenza B HA., (Copyright © 2013 John Wiley & Sons, Ltd.)

H5N1 infection and the potential for spread from human to human continue to pose a severe public health concern. Since vaccination remains the most effective way to prevent a potential H5N1 pandemic, the World Health Organization (WHO) Collaborating Centers (CCs) and Essential Regulatory Laboratories (ERLs) engineered and developed a panel of H5N1 pre-pandemic vaccine viruses for pandemic vaccine preparedness as well as production of antigen potency testing reagents (reference antigen and reference anti-serum) for vaccine standardization. To develop a strategy utilizing a number of biochemical methods for the characterization of the viral growth properties and protein yield in eggs, we have selected eight H5N1 pre-pandemic viruses and determined the viral Egg Infectious Dose 50 (EID50), total protein yield, hemagglutinin (HA) to nucleoprotein (NP) ratios (HA:NP), and HA1 content of each virus. Our results showed that all the tested H5N1 vaccine viruses grew to high titers in eggs. The total viral protein yield varies within a narrow range, whereas there were greater differences in the HA:NP protein ratios among the eight viruses. The RP-HPLC based HA1 content analysis demonstrated that the viruses A/Anhui/1/2010, A/Hubei/1/2005, and A/goose/Guiyang/337/2006 contained higher HA contents than other five viruses including A/Vietnam/1203/2003. Our approach for analyzing virus growth and protein yield will allow us identify optimal vaccine virus in a timely manner. In addition, we successfully purified the HA proteins of H5N1 vaccine viruses by optimizing bromelain cleavage conditions. Our studies on the HA protein purification may improve the quality control of the production of influenza vaccine test reagent., (Published by Elsevier Ltd.)

Despite global efforts to control influenza viruses, they have taken a heavy toll on human public health worldwide. Among particular threats is highly pathogenic avian H5N1 influenza virus (HPAI) due to not only its high mortality in humans but also possible human-to-human transmission either through reassortment with other human influenza viruses such as 2009 pandemic H1N1 influenza virus, or by genetic mutations. With the aim of developing effective vaccines against the H5N1 viruses, we generated two live attenuated H5N1 vaccine candidates against A/Indonesia/05/2005 (clade 2.1) and A/chicken/Korea/ES/2003 (clade 2.5) strains, in the genetic background of the cold-adapted donor strain of X-31. In mice, a single dose of immunization with each of the two vaccines was highly immunogenic inducing high titers of serum viral-neutralizing and hemagglutinin-inhibiting antibodies against the homologous H5N1 strain. Furthermore, significant levels of cross-clade antibody responses were induced by the vaccines, suggesting a broad-spectrum cross-reactivity against the heterologous H5N1 strains. The immunizations provided solid protections against heterologous lethal challenges with H5N2 virus, significantly reducing the morbidity and challenge virus replications in the respiratory tracts. The robustness of the antibody responses against both the homologous and heterologous strains, together with efficient protection against the lethal H5N2 challenge, strongly support the protection against wild type H5N1 infections. These results could serve as an experimental basis for the development of safe and effective H5N1 pre-pandemic vaccines while further addressing the biosecurity concerns associated with H5N1 HPAI., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The use of lytic bacteriophages for the biocontrol of food-borne pathogens in food and in the food industry is gaining increasing acceptance. In this study, the effectiveness of a bacteriophage cocktail composed of three different lytic bacteriophages (UAB&#95;Phi 20, UAB&#95;Phi78, and UAB&#95;Phi87) was determined in four different food matrices (pig skin, chicken breasts, fresh eggs, and packaged lettuce) experimentally contaminated with Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis. A significant bacterial reduction (>4 and 2 log/cm(2) for S. Typhimurium and S. Enteritidis, respectively; p≤0.005) was obtained in pig skin sprayed with the bacteriophage cocktail and then incubated at 33 °C for 6h. Significant decreases in the concentration of S. Typhimurium and S. Enteritidis were also measured in chicken breasts dipped for 5 min in a solution containing the bacteriophage cocktail and then refrigerated at 4 °C for 7 days (2.2 and 0.9 log10 cfu/g, respectively; p≤0.0001) as well as in lettuce similarly treated for 60 min at room temperature (3.9 and 2.2 log10 cfu/g, respectively; p≤0.005). However, only a minor reduction of the bacterial concentration (0.9 log10 cfu/cm(2) of S. Enteritidis and S. Typhimurium; p≤0.005) was achieved in fresh eggs sprayed with the bacteriophage cocktail and then incubated at 25 °C for 2 h. These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent of Salmonella in several food matrices under conditions similar to those used in their production., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the frequency of the Newcastle disease virus (NDV) infection and its virulence in exotic cage birds over a limited area and time period. A set of 335 samples was collected from 24 different species of exotic unvaccinated cage birds kept in the zoological gardens and bird markets of the Tehran province of Iran during 1.5 years. Except for three pigeons, all of the sampled birds were healthy with no clinical signs of Newcastle disease. NDV was detected in three sick pigeons by haemagglutination assay (HA), haemagglutination inhibition (HI) and reverse transcription-polymerase chain reaction (RT-PCR) tests while two of them were identified as virulent types by RT-PCR. Although the remaining samples were negative by Newcastle-disease-specific HA and HI tests, 35 of them (10%) were identified as positive and 25 (72%) were determined as the velogenic type by RT-PCR test. Five PCR products were sequenced and all were confirmed as NDV but sequences were different from each other and from other sequences from Iran. In total, 14 species (58%) were infected and 10 species were uninfected with the velogenic type without showing any signs. Pigeons are very sensitive to NDV infection and play an important role in its epidemiology. In this study, the PCR test was found to be a more sensitive and powerful method than the HA and HI tests for detection of NDV reservoirs and carrier status in exotic birds. Also, the frequency of infection with the virulent type showed that the exotic birds should probably be considered one of the main causes of recurrent annual epidemics of Newcastle disease in endemic regions.

The 2009 swine-origin influenza A virus (H1N1) and its initial reassortant vaccine strains did not grow well in embryonated eggs. The glutamine to arginine mutation at the amino acid position 223 (Q223R) of the hemagglutinin (HA) gene is the major mutation previously found in egg-adapted 2009 H1N1 strains and shown to enhance viral growth in embryonated eggs. However, the effect of this mutation on the receptor-binding preference had not been directly demonstrated. In this study, the Q223R mutation was shown to change the viral HA binding preference from the human-type receptor, α2,6-linked sialic acid, to the avian-type receptor, α2,3-linked sialic acid; and to enhance the viral growth in embryonated eggs but not in cell culture.

Even though low pathogenic avian influenza viruses (LPAIv) affect the poultry industry of several countries in the world, information about their transmission characteristics in poultry is sparse. Outbreak reports of LPAIv in layer chickens have described drops in egg production that appear to be correlated with the virus transmission dynamics. The objective of this study was to use egg production data from LPAIv infected layer flocks to quantify the within-flock transmission parameters of the virus. Egg production data from two commercial layer chicken flocks which were infected with an H7N3 LPAIv were used for this study. In addition, an isolate of the H7N3 LPAIv causing these outbreaks was used in a transmission experiment. The field and experimental estimates showed that this is a virus with high transmission characteristics. Furthermore, with the field method, the day of introduction of the virus into the flock was estimated. The method here presented uses compartmental models that assume homogeneous mixing. This method is, therefore, best suited to study transmission in commercial flocks with a litter (floor-reared) housing system. It would also perform better, when used to study transmission retrospectively, after the outbreak has finished and there is egg production data from recovered chickens. This method cannot be used when a flock was affected with a LPAIv with low transmission characteristics (R(0)<2), since the drop in egg production would be low and likely to be confounded with the expected decrease in production due to aging of the flock. Because only two flocks were used for this analysis, this study is a preliminary basis for a proof of principle that transmission parameters of LPAIv infections in layer chicken flocks could be quantified using the egg production data from affected flocks., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The negative impact of high pathogenicity avian influenza virus (HPAIV) infection on egg production and deposition of virus in eggs, as well as any protective effect of vaccination, is unknown. Individually housed non-vaccinated, sham-vaccinated and inactivated H5N9 vaccinated Once or Twice adult White leghorn hens were challenged intranasally/intratracheally 3-weeks post-vaccination with H5N2 HPAIV. The non-/sham-vaccinated layers experienced 100% mortality (0% survivability) within 3-4 days post-challenge (DPC), and major changes to reproductive parameters including precipitous drops in egg production (79-0% in <5 days), production of soft and thin-shelled eggs, and deposition of virus in albumin and yolk, and on the egg shell surface of 53% of eggs. By comparison, the three H5-vaccinated groups had 83%, 100% and 100% survivability after challenge; the two H5-vaccinated Once hens that died had low pre-challenge HI titers (GMT=16). H5-vaccinated Once or Twice groups maintained egg production after challenge (63%), but there was a mild and significant reduction in egg production as compared to pre-challenge egg production (79%). H5-vaccinated groups had reduced number of virus contaminated eggs (28%), and in most groups, reduced quantity of virus in contaminated eggs compared to non-/sham-vaccinated groups. No HPAIV-positive eggs were laid on or after 5 DPC. In conclusion, HPAIV infection had major negative impact on egg production and other reproductive parameters. H5-vaccination Once or Twice prevented declines in egg production after HPAIV challenge, reduced number of virus-infected eggs, and typically reduced the titer of virus in internal contents and on eggshell surface., (Published by Elsevier Ltd.)

Aim: Detection of antibodies against neuraminidase (NA) of A/California/07/2009 (H1N1) influenza virus in blood sera of volunteers after the immunization with live monovalent influenza vaccine (LIV)., Materials and Methods: Neuraminidase enzyme activity inhibition by antibodies test with reassortant strain A(H7N1) containing NA of pandemic strain was used. Anti-neuraminidase IgG antibodies against whole reassortant virus A(H7N1) and purified NA of A/California/07/2009 (H1N1) strains were determined by enzyme immunoassay (EIA)., Results: After two immunizations with LIV of seronegative individuals a 1.5 times mean increase of antibodies against homologous neuraminidase was detected (by hemagglutinin inhibition reaction). The high level of anti-neuraminidase antibodies were detected in individuals that had been naturally infected. A correlation between anti-neuraminidase IgG antibody titers obtained in EIA with whole reassortant virus A(H7N1) and purified protein was demonstrated., Conclusion: Modified sialidase activity inhibition method and EIA with reassortant diagnostic strain can be applied to evaluate anti-neuraminidase antibodies.

To establish an accurate, rapid, and a quantifiable method for the detection of the newly emerged duck Tembusu virus (DTMUV) that recently caused a widespread infectious disease in ducks in China, we developed a TaqMan-based real-time PCR assay by using E gene-specific primers and a TaqMan probe. This real-time PCR assay was 100 times more sensitive than the conventional PCR. The reproducibility and specificity of the real-time PCR assay were confirmed using plasmids containing E genes or RNAs and DNAs extracted from well-known viruses causing duck diseases. The reliability of this real-time PCR assay was confirmed in 19 of the 24 swab samples, 22 of the 24 tissue samples collected from experimentally infected ducks, as well as 15 of the 21 clinical samples collected from sick ducks since they were verified as DTMUV-positive. The results reveal that the newly established real-time PCR assay might be a useful diagnostic method for epidemiologically investigating and closely observing the newly emerged DTMUV.

Circulation of an antigenically variant lineage of highly pathogenic avian influenza (HPAI) H5N1 virus in chicken breeder flocks in Egypt is a continuing problem. The protective efficacy of multiple repeated vaccinations using the currently available H5N2 vaccines is unclear. Here, broiler breeder chickens were vaccinated at weeks 6, 12 and 18 with an inactivated H5N2 commercial vaccine. HI-titer against an Egyptian H5N1 field isolate of classic clade 2.2.1 (EGYcls/H5N1) were significantly lower after the first immunization but increased after booster vaccinations. In contrast, no HI titers were induced against an antigenically distinct field virus of the variant lineage of clade 2.2.1 (EGYvar/H5N1). Upon challenge at week 50 mild, if any, clinical signs were observed in the group infected with EGYcls/H5N1 although one of eight (12.5%) birds died. Mortality reached 6/8 (75%) in the EGYvar/H5N1 challenge group. Virus excretion in all vaccinated groups was reduced in amplitude, but in vaccinated surviving birds, time of virus excretion was extended to up to ten days. Strikingly, challenged vaccinated birds kept laying eggs almost throughout the observation period. Virus was detected on the outer egg-shell of 17 of 40 eggs. The majority of the infected eggs were derived from the EGYcls/H5N1 challenged animals; here the virus was detected also in the yolk and albumin. Repeated vaccination using a commercial H5N2-based vaccine broadened the antigen profile of induced antibodies but did not provide adequate protection against heterologous virus variant. In addition, the observation of AIV contaminated eggs from infected flocks highlights the risk of silent virus spread by vaccinated animals and point to eggs as a possible vector., (Copyright © 2011. Published by Elsevier Ltd.)

High-pathogenicity avian influenza (HPAI) virus, low-pathogenicity avian influenza (LPAI) virus, virulent Newcastle disease virus (vNDV) and low-virulent Newcastle disease virus (lNDV) can be present on the eggshell surface, and HPAI viruses and vNDV can be present in the internal contents of chicken eggs laid by infected hens. With the increase in global trade, egg products could present potential biosecurity problems and affect international trade in liquid and dried egg products. Therefore, the generation of survival curves to determine decimal reduction times (D(T)-values) and change in heat resistance of the viruses (z(D)-value) within fat-free egg product could provide valuable information for development of risk reduction strategies. Thermal inactivation studies using A/chicken/Pennsylvania/1370/83 (H5N2) HPAI virus resulted in D(55)-, D(56)-, D(56.7)-, D(57)-, D(58)-, and D(59)-values of 18.6, 8.5, 3.6, 2.5, 0.4, and 0.4 min, respectively. The z(D)-value was 4.4 °C. LPAI virus A/chicken/New York/13142/94 (H7N2) had D(55)-, D(56.7)-, D(57)-, D(58)-, D(59)-, and D(60)-values of 2.9, 1.4, 0.8, 0.7, 0.7, and 0.5 min, respectively, and a z-value of 0.4 °C. vNDV avian paramyxoviruses of serotype 1 (AMPV-1)/chicken/California/212676/2002 had D(55)-, D(56)-, D(56.7)-, D(57)-, D(58)-, and D(59)-values of 12.4, 9.3, 6.2, 5, 3.7, and 1.7 min, respectively. The z(D)-value was 4.7 °C. lNDV AMPV-1/chicken/United States/B1/1948 had D(55)-, D(57)-, D(58)-, D(59)-, D(61)-, and D(63)-values of 5.3, 2.2, 1.1, 0.55, 0.19, and 0.17 min, respectively, and a z(D)-value of 1.0 °C. Use of these data in developing egg pasteurization standards for AI and NDV-infected countries should allow safer trade in liquid egg products., (Copyright ©, International Association for Food Protection)

The extensive circulation of the H5N1 highly pathogenic avian influenza (HPAI) virus in animals and the human health implications which it poses have led to extensive research in unexplored fields and thus a re-assessment of our understanding of this infection. Moreover, widespread infection of poultry has raised concerns about the food safety and trade implications of this infection, necessitating revised international trade regulations. The role of wild birds has been much debated and resources have been invested to clarify the role that they may play in the spread of infection. It is now clear that wild birds may be responsible for primary introduction in a previously free area. To date it is still unclear whether HPAI infection may be maintained in wild bird populations for extended periods of time. This paper reviews existing knowledge on the transboundary spread of HPAI through poultry and poultry commodities and summarises evidence of spread through wild birds.

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The international trade in poultry hatching eggs could potentially facilitate the global dissemination of poultry disease. Provided the guidelines of the World Organisation for Animal Health (OIE) on breeding flock hygiene are followed, of those avian diseases currently listed by the OlE, only highly pathogenic avian influenza (HPAI), Newcastle disease (ND), and avian mycoplasmosis (caused by Mycoplasma gallisepticum or M. synoviae) should be considered likely to be spread though trade in this commodity. Furthermore, the impact of HPAI and ND on egg production and hatchability will constrain the potential for these agents to be spread by poultry hatching eggs.