Embryonated Chicken Eggs Research Articles

Molecular Detection and Isolation of Chicken Astrovirus from Commercial Chicken Flocks of Kerala, India

Reduced productivity due to enteric infections in chickens is a very common challenge in the poultry industry of India. Various studies associated Chicken astrovirus (CAstV) as a common pathogen in commercial flocks exhibiting poor weight gain, stunted growth and visceral gout. However, no published report documented the detection of the virus from Kerala so far. Hence, this study was done to investigate the presence of CAstV in 50 broiler flocks from different parts of Kerala with clinical signs such as retarded growth, diarrhoea, and a mortality of 5 to 10 %. Out of the 50, five flocks were found positive for the virus by using the Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) method with the primers targeting the partial ORF1b gene of the viral nucleic acid. The viral presence was confirmed by isolation using embryonated chicken eggs (ECE), which produced notable alteration to the infected embryos. Nucleotide sequencing of the isolates revealed a high identity of up to 100 % with the other CAstV sequences retrieved from the NCBI database. The study confirms the first report of molecular detection and isolation of CAstV from the state of Kerala.

Antibodies to Influenza A Virus in Lesser (Aythya affinis) and Greater Scaup (Aythya marila) in the USA.

Scaup, including both Lesser and Greater (Aythya affinis and Aythya marila, respectively), are a grouping of populous and widespread North American diving ducks. Few influenza type A viruses (IAV) have been reported from these species despite a high prevalence of antibodies to IAV being reported. Existing virologic and serologic data indicate that IAV infection routinely occurs in scaup, yet it is unknown which IAV subtypes are linked to these infections. In this study, we aimed to gain a more complete picture of IAV natural history in Lesser and Greater Scaup from two coastal flyways in North America in 2015-18 (302 samples from California in the Pacific Flyway and 471 samples from Maryland in the Atlantic Flyway). Low prevalence of active IAV infection was detected by real-time reverse-transcription PCR in Lesser Scaup sampled in Maryland and California (2.8% and 8.1%, respectively). A single IAV (H1N1) was isolated in embryonated chicken eggs from a bird sampled in California. Similarly low levels were observed in Greater Scaup in California (3.3%). Antibodies to the nucleoprotein as detected with a commercial blocking ELISA were observed in all species and flyway combinations. Antibody seroprevalence estimates were higher in adult Lesser Scaup than in juveniles at both the ≤0.5 (P<0.001, z=-3.582) and ≤0.7 serum-sample-to-negative-control absorbance thresholds (P=0.003, z=-2.996). Neutralizing antibodies to H1-H12, H14, and H15 were detected using a microtiter virus neutralization assay, with the highest prevalence of antibodies against H1 (38%), H6 (36%), and H11 (35%). The high prevalence of antibodies to IAV and evidence of previous exposure to numerous subtypes are consistent with a high level of population immunity and a low prevalence of infection. These results must be interpreted in the context of season (winter sampling), as results may vary with the annual influx of naïve juvenile birds.

Differential Expression of Mitogen-Activated Protein Kinase Kinase 3 (MAP2K3) and Interleukin12B (IL12B) in the Bursa of Fabricius of chicken infected with Infectious Bursal Disease Virus

Infectious Bursal Disease (IBD) caused by Infectious Bursal Disease virus (IBDV), poses a significant threat to the poultry industry, causing immunosuppression and economic losses since its discovery in 1957. To understand the host response to IBDV infection, this study examined the modulation of MAP2K3 and IL12B in chickens infected with IBDV. A local IBDV isolate propagated in embryonated chicken eggs was inoculated into 18-day-old chicken. The mRNA transcription levels of MAP2K3 and IL12B in bursal tissue were quantified by real-time quantitative polymerase chain reaction on day two post-infection and compared with those of uninfected, age-matched control birds (sham inoculated). The study revealed increased expression of the MAP2K3 and IL12B genes, indicating their role in inflammatory response during IBDV infection. Thus, the present investigation sheds light on the involvement of MAP2K3 and IL12B in the inflammatory response to IBDV infection in chickens. Keywords: Infectious bursal disease virus, immunosuppression, MAP2K3, IL12B, pro-inflammatory cytokine gene expression

Design and biological evaluation of candidate drugs against zoonotic porcine deltacoronavirus (PDCoV)

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. PDCoV spillovers were recently detected in Haitian children with acute undifferentiated febrile illness, underscoring the urgent need to develop anti-PDCoV therapeutics. Coronavirus 3C-like protease (CoV 3CLpro) is essential for viral replication, and therefore provides an attractive target for drugs directed against CoV. Here, we initially evaluated the anti-PDCoV effect of Nirmatrelvir (PF-07321332), an FDA-approved anti-SARS-CoV-2 drug targeting viral 3CLpro. Regrettably, a very limited anti-PDCoV effect was achieved. By analyzing the binding modes of Nirmatrelvir with PDCoV 3CLpro and SARS-CoV-2 3CLpro, we demonstrated that the S2 pocket of 3CLpro is the primary factor underlying the differential inhibitory potency of Nirmatrelvir against different CoV 3CLpros. Based on the specific characteristics of the S2 pocket of PDCoV 3CLpro, four derivatives of Nirmatrelvir (compounds T1–T4) with substituted P2 moieties were synthesized. Compound T1, with an isobutyl at the P2 site, displayed improved anti-PDCoV activity invitro (cell infection model) and invivo (embryonated chicken egg infection model), and therefore is a potential candidate drug to combat PDCoV. Together, our results identify the substrate-binding mode and substrate specificity of PDCoV 3CLpro, providing insight into the optimization of Nirmatrelvir as an antiviral therapeutic agent against PDCoV.

Assessment of PPMV-1 Genotype VI Virulence in Pigeons and Chickens and Protective Effectiveness of Paramyxovirus Vaccines in Pigeons.

Pigeon paramyxovirus serotype 1 (PPMV-1), an antigenic and host variant of avian paramyxovirus Newcastle disease virus (NDV), primarily originating from racing pigeons, has become a global panzootic. Egypt uses both inactivated PPMV-1 and conventional NDV vaccines to protect pigeons from disease and mortality. However, the impact of prevalent strains and the effectiveness of available vaccines in pigeons in Egypt are unclear. This study investigates the virulence of PPMV-1 (Pigeon/Egypt/Sharkia-19/2015/KX580988) and evaluates available paramyxovirus vaccines in protecting pigeons against a PPMV-1 challenge. Ten-day-old specific-pathogen-free (SPF) embryonated chicken eggs infected with this strain exhibited a mean death time (MDT) of 86.4 ± 5.88 h. The intracerebral pathogenicity index (ICPI) in day-old chickens was 0.8, while pigeons experienced an ICPI of 0.96 and an intravenous pathogenicity index (IVPI) of 2.11. These findings classify the strain as virulent and velogenic. Experimental infection of pigeons with this PPMV-1 strain at 106 EID50/0.1 mL resulted in a 62.5% mortality rate, displaying nervous and enteric distress. The virus caused extensive lesions in visceral organs, with strong immunohistochemistry signals in all examined organs, indicating the systemic spread of the virus concurrent to its neurotropic and viscerotropic tropism. Furthermore, vaccination using an inactivated PPMV-1 and live NDV LaSota vaccine regimen protected 100% of pigeons against mortality, while with a single NDV LaSota vaccine, it was 62.5%. The PPMV alone or combined with NDV LaSota induced protective levels of haemagglutination inhibition (HI) antibody titres and reduced virus shedding from buccal and cloacal cavities. Based on generalised linear gamma model analysis, both PPMV-1 and NDV LaSota are antigenically comparable by HI. These findings suggest that using both inactivated PPMV-1 (G-VI) and live attenuated NDV (LaSota) vaccines is an effective prophylactic regimen for preventing and controlling PPMV-1 and NDV in pigeons, thereby reducing the risk of interspecies transmission.

Molecular detection of lumpy skin disease virus in naturally infected cattle and buffaloes: unveiling the role of tick vectors in disease spread.

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals.

Detection of H1N1 Influenza Virus in the Bile of a Severe Influenza Mouse Model.

Influenza virus infection may lead to fatal complications including multi-organ failure and sepsis. The influenza virus was detected in various extra-pulmonary organs in autopsy studies during the 2009 pandemic. However, limited research has been conducted on the presence of viral particle or viral components in the peripheral blood. We established a mouse model for severe H1N1 influenza. The bile and blood samples were collected over time and inoculated into embryonated chicken eggs. We detected live influenza virus in bile and blood samples in early infection. Immunofluorescence showed influenza viral components in the liver tissue. No live virus was isolated in the bile in mice intragastrically administered with influenza virus, indicating that the virus was spread from the blood stream. Targeted metabolomics analysis of bile acid and liver tissues showed that a secondary bile acid (3-dehydrocholic acid) was decreased after influenza H1N1 infection. Genes related with fatty acid metabolism and bile secretion pathways were down-regulated in liver after influenza virus infection. Our study indicated that influenza virus viremia is present in severe influenza, and that the liver is a target organ for influenza viral sepsis.

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT–qPCR confirmed the presence of H9 and N2 genes in the samples. RT–PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT–qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Propagation of koi herpesvirus using embryonated chicken eggs: a potential substitute method for fish vaccine production?

Koi herpesvirus disease (KHVD) is caused by a large DNA virus that commonly infects carp, Cyprinus carpio (koi and common carp). KHVD has spread rapidly across the globe and caused high mortality in all ages of common carp and koi. Until now, no effective treatment has been applied to prevent KHV infection impacting the mass production of koi and common carp . An environmentally friendly alternative strategy for controlling fish disease is vaccination. One of the challenges facing conventional viral vaccine production is the requirement for koi or common carp cell cultures, which must be frequently maintained with expensive materials required for virus propagation. This study aims to obtain KHV that has been propagated in embryonated chicken eggs (ECE) to formulate an affordable KHV vaccine for the aquaculture industry. This research consisted of three stages; the first stage was virus inoculation into various parts of eggs (allantoic fluid, chorioallantoic membrane, amniotic fluid, and egg yolk). The second stage was observing viral growth and collecting ECE fluid and membranes. The third stage involved quantitatively determining the viral genomic copy numbers using the quantitative Polymerase Chain Reaction (qPCR) assay. KHV propagation in various parts of ECE resulted in varying viral genomic copy numbers with a high DNA copy number reported in allantoic fluid on the third day after inoculation. Further work is required to monitor virus titer in later passages and optimize methodology for using ECE as the potential alternative to cultured cells as the medium for virus propagation. In the future the system could be developed to produce promising vaccine candidates with more affordable vaccine prices for the Indonesian fish farming industry.

In-Ovo Antiviral Activity of Hibiscus sabdariffa against Newcastle Disease Virus

Newcastle disease is a highly contagious viral infection affecting poultry and wild birds. The causative agent is Avian paramyxovirus 1 (APMV-1), causing significant economic losses despite vaccination efforts due to its high mortality rate. Hibiscus sabdariffa was identified at Modibbo Adama University Yola, and laboratory assays were performed at the National Veterinary Research Institute Vom. The study explores the antiviral effects of extracts from H. sabdariffa calyx against a virulent strain of Newcastle Disease Virus (NDV) using embryonated chicken eggs (ECE). Standard methods were employed for cytotoxicity assay, embryo infective dose 50 (EID50) determination, and therapeutic antiviral assays. Methanol was used for extraction and phytochemical analysis, revealing various bioactive compounds like cardiac glycosides, alkaloids, flavonoids, terpenes, and phenols. Toxicity assay showed cytotoxicity at concentrations over 25 mg/ml, but therapeutic antiviral assays demonstrated virus replication inhibition at concentrations as low as 5 mg/ml. These findings suggest the potential of H. sabdariffa calyx extracts as safe and effective treatments for NDV, with promising therapeutic antiviral properties. Further pharmaceutical research is recommended to explore their use in developing novel Newcastle Disease treatments.

In vitro analysis of antiviral immune response against avian influenza virus in chicken tracheal epithelial cells.

Avian influenza virus (AIV) infections first affect the respiratory tract of chickens. The epithelial cells activate the host immune system, which leads to the induction of immune-related genes and the production of antiviral molecules against external environmental pathogens. In this study, we used chicken tracheal epithelial cells (TECs) in vitro model to investigate the immune response of the chicken respiratory tract against avian respiratory virus infections. Eighteen-day-old embryonic chicken eggs were used to culture the primary chicken TECs. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) analysis of epithelial cell-specific gene makers were performed to confirm the characteristics, morphology, and growth pattern of primary cultured chicken TECs. Moreover, to investigate the cellular immune response to AIV infection or polyinosinic-polycytidylic acid (poly (I:C)) treatment, the TECs were infected with the H5N1 virus or poly (I:C). Then, immune responses were validated by RT-qPCR and western blotting. The TECs exhibited polygonal morphology and formed colony-type cell clusters. The RT-qPCR results showed that H5N1 infection induced a significant expression of antiviral genes in TECs. We found that TECs treated with poly (I:C) and exposed to AIV infection-mediated activation of signaling pathways, leading to the production of antiviral molecules (e.g., pro-inflammatory cytokines and chemokines), were damaged due to the loss of junction proteins. We observed the activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which are involved in inflammatory response by modulating the release of pro-inflammatory cytokines and chemokines in TECs treated with poly (I:C) and pathway inhibitors. Furthermore, our findings indicated that poly (I:C) treatment compromises the epithelial cell barrier by affecting junction proteins in the cell membrane. Our study highlights the utility of in vitro TEC models for unraveling the mechanisms of viral infection and understanding host immune responses in the chicken respiratory tract.

Inactivation of Highly Pathogenic Avian Influenza Virus with High-temperature Short Time Continuous Flow Pasteurization and Virus Detection in Bulk Milk Tanks

Infections of dairy cattle with clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) were reported in March 2024 in the U.S. and viable virus was detected at high levels in raw milk from infected cows. This study aimed to determine the potential quantities of infectious HPAIV in raw milk in affected states where herds were confirmed positive by USDA for HPAIV (and therefore were not representative of the entire population), and to confirm that the commonly used continuous flow pasteurization using the FDA approved 72 °C (161°F) for 15 s conditions for high−temperature short time (HTST) processing, will inactivate the virus. Double-blinded raw milk samples from bulk storage tanks from farms (n = 275) were collected in four affected states. Samples were screened for influenza A using quantitative real-time RT-PCR (qrRT-PCR) of which 158 (57.5%) were positive and were subsequently quantified in embryonating chicken eggs. Thirty-nine qrRT-PCR positive samples (24.8%) were positive for infectious virus with a median titer of 3.5 log10 50% egg infectious doses (EID50) per mL. To closely simulate commercial milk pasteurization processing systems, a pilot-scale continuous flow pasteurizer was used to evaluate HPAIV inactivation in artificially contaminated raw milk using the most common legal conditions in the US: 72 °C (161°F) for 15 s. Among all replicates at two flow rates (n = 5 at 0.5 L/min; n = 4 at 1 L/min), no viable virus was detected. A mean reduction of ≥5.8 ± 0.2 log10 EID50/mL occurred during the heating phase where the milk is brought to 72.5 °C before the holding tube. Estimates from heat-transfer analysis support that standard U.S. continuous flow HTST pasteurization parameters will inactivate >12 log10 EID50/mL of HPAIV, which is ∼9 log10 EID50/mL greater than the median quantity of infectious virus detected in raw milk from bulk storage tank samples. These findings demonstrate that the US milk supply is safe when pasteurized.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

RT-PCR based detection and pathotypical characterization of Newcastle disease virus isolated from layer chickens in Mymensingh, Bangladesh

The Newcastle disease virus (NDV) poses a significant threat to commercial chicken operations and is prevalent in Bangladesh. This rapidly spreading viral illness has severe economic implications for the poultry industry globally. In Bangladesh, the velogenic strain of NDV is particularly widespread. This study utilized primers from the Fusion (F) and Matrix (M) genes along with haemagglutination (HA) testing and reverse transcription-polymerase chain reaction (RT-PCR) to isolate and identify NDV. Post-mortem examinations provided 12 samples (from brain, trachea, and proventriculus) taken from four birds suspected of having NDV from Trishal and Mymensingh Sadar. The samples were injected into 10-day-old embryonated chicken eggs using the chorioallantoic membrane technique. Embryos and allantoic fluid (AF) were collected at 48 and 96 hours post-infection. NDV-infected embryos showed edema and hemorrhage. Significant hemagglutination was observed in the allantoic fluid tested with HA. Through the HA test and lesion observation, four NDV isolates were identified. Pathotypic characterization of the field isolates using the Intracerebral Pathogenicity Index (ICPI) and Mean Death Time (MDT) indicated velogenic strains (ICPI: 1.5–2.0, MDT: &lt;60). RT-PCR confirmed the NDV status of all four isolates using primers for partial amplification of the F gene (839 bp) and the common sequence of the ‘F-M’ genes (970 bp). This study demonstrates that RT-PCR is an effective molecular technique for the rapid and confirmatory identification of velogenic NDV strains, particularly prevalent in Mymensingh at the field level.

Isolation and Genetic Characterization of Genotype VII Velogenic Pathotype Newcastle Disease Virus from Commercial Chicken Farms in Central Ethiopia, Distinct from the Local Vaccine Strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

Synergistic activity of an RNA polymerase PA-PB1 interaction inhibitor with oseltamivir against human and avian influenza viruses in cell culture and in ovo

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections.

Detection and genomic characterization of an avian influenza virus A/mute swan/Mangystau/1-S24R-2/2024 (H5N1; clade 2.3.4.4b) strain isolated from the lung of a dead swan in Kazakhstan.

The influenza virus strain A/mute swan/Mangystau/1-S24R-2/2024 (H5N1; clade 2.3.4.4b) was isolated in embryonated chicken eggs from the lung of a dead swan found around Lake Karakol (Kazakhstan) during a highly pathogenic avian influenza outbreak in 2024. The aim of this study was to characterize the genetic profile of the isolated strain.

Antiviral Effect of Different Essential Oils on Avian Coronavirus.

Plant essential oils (EOs) possess established antimicrobial properties; however, research on their antiviral activity, particularly against avian coronaviruses, remains limited. EOs offer a promising plant-based alternative for viral control, especially in scenarios where conventional chemical use is restricted. This study aimed to evaluate the antiviral effects of six different essential oils derived from Syzygium aromaticum, Origanum vulgare, Cymbopogon martinii, Cymbopogon citratus, Mentha piperita, and Mentha spicata against avian coronavirus (AvCov) at 0.1% and 1% dilutions. The antiviral effects of the EOs were assessed via virus isolation from embryonated chicken eggs, and the ability of the EOs to inhibit AvCoV replication was evaluated. The EOs from Syzygium aromaticum, Origanum vulgare, Cymbopogon martinii, and Cymbopogon citratus completely inhibited AvCov replication at a 1% dilution. Conversely, absent to partial inhibitory effect was observed at the 0.1% dilution for all tested EOs, with O. vulgare derived EO exhibiting the greatest inhibitory effect (over 70%). Notably, EOs from Mentha piperita and Mentha spicata were unable to completely inhibit AvCov at either concentration. Our findings highlight the potent antiviral activity of the EOs from Syzygium aromaticum, Origanum vulgare, Cymbopogon martinii, and Cymbopogon citratus against AvCov at a 1% dilution. The complete inhibition observed for these EOs suggests their potential for AvCov control. However, further research is necessary to elucidate the mechanisms of action, optimize formulations, and evaluate the efficacy against other coronaviruses, including those relevant to human health.

First Isolation and Genetic Characterization of Avian Nephritis Virus 4 from Commercial Poultry in India.

Avian nephritis virus (ANV), which belongs to the family Astroviridae, is associated with different clinical manifestations (enteric and kidney disorders) in poultry. Despite being a significant pathogen of the avian industry worldwide, information regarding genetic features of these viruses in India is scarce. In this study, 386 intestinal samples collected from 37 slaughterhouses in two north Indian states (Rajasthan and Haryana) were screened for ANV with reverse transcription PCR (RT-PCR) targeting the conserved ORF1b gene, followed by nucleotide sequencing of the amplified product. RT-PCR and sequencing confirmed the presence of ANV in 32 clinical samples (8.29%), with concurrent infections of infectious bronchitis virus, chicken astrovirus, and fowl adenoviruses observed in some clinical samples (n = 4). Virus isolations were successful from four out of 12 ANV-positive clinical samples passaged via the yolk-sac route in specific-pathogen-free embryonated chicken eggs. Additionally, the near-complete genomes of two viruses were determined through sequencing. Phylogenetic analysis based on full-length capsid protein sequences classified the viruses into ANV genotype 4 (ANV4), and to the best of our knowledge this is the first report of ANV4 from India. This study revealed the presence and circulation of new strains of ANV in Indian poultry. Genetic profiling and isolation of the viruses in this study will not only aid in the development of diagnostic tools and vaccines for ANV but also offer valuable insights into its epidemiology.

Parallel Multifactorial Process Optimization and Intensification for High-Yield Production of Live YF17D-Vectored Zika Vaccine.

The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEKDyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.