Embryonated Chicken Eggs Research Articles

Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Isolation and Genotypic Characterization of New Emerging Avian Reovirus Genetic Variants in Egypt

Avian reovirus (ARV) strains cause a variety of symptoms in chickens, including viral arthritis/tenosynovitis, a disease that has emerged as a significant cause of economic losses in commercial chicken flocks in recent years in various countries, including Egypt. Furthermore, ARV strains are frequently isolated from birds suffering from malabsorption. In the actual study, seventy-five samples were collected in 2021 and 2022 from broiler and vaccinated broiler breeder flocks at different farms in Giza Province, Egypt, with reovirus-like symptoms such as significant weight fluctuation and arthritis/malabsorption. ARV was screened using real-time PCR, and fifteen positive samples were detected (20%), which were then subjected to embryonated chicken egg (ECE) isolation and molecular characterization (11/15 sample) of a partial segment of the sigma (σ)C gene (S1-gene). Phylogenetically, nine strains were found to belong to genotypic cluster IV, with 82–89% identity with Israeli ARV 2018, and two strains belong to genotypic cluster V with a 78% nucleotide identity with Japan ARV 2021. No correlation between lesions and genotype was found. The strains under study had a low sequence identity (43–55%) when compared with various commercial vaccines belonging to genotypic cluster I (e.g., strain S1133). These findings imply that novel ARV genotypes representing clusters IV and V have recently been introduced to Egyptian poultry farms. A homologous vaccine is suggested; because this variation raises the possibility that commercial vaccines may not offer protection against circulating ARVs.

Synthesis, anti-angiogenic activity and prediction toxicity of (E)-3-(3-methoxyphenyl) propenoic acid.

Anti-angiogenic medications, one of cancer chemo preventive mechanism were permitted for different cancers. Nevertheless, major primary and secondary resistance obstruct efficacy in several tumor types. Moreover, the improvement of safe and effective NSAIDs for angiogenesis inhibition is complicated, because of their serious toxicity. So, we require improving clinically appropriate strategies to boost efficacy of anti-angiogenic drugs with low risk of toxicity. The present study aimed to synthesize the (E)-3- (3-methoxyphenyl)propenoic acid (3MPCA), to determine the anti-angiogenic activity and predict its toxicity. 3MPCA was obtained by Knoevenagel reaction using microwave irradiation at 400 Watt. The anti-angiogenesis experimental was performed using chorioallantois membrane of embryonated chicken eggs induced by b-FGF. The potency of 3MPCA was verified at dosage 30 and 60 ng and compared with celecoxib 60 ng. Toxicity prediction of 3MPCA was performed by ProTox II online program. The results showed that 3MPCA was achieved in good yield (89%). Anti angogenic activity was showed by endothelial cells growth in neovascular capillaries of new blood vessel of chorioallantois membrane of embryonated chicken eggs. The endothelial cells growth decreased until 41.7-83%. The prediction LD50 was 1772mg/kg. (E)-3-(3-methoxyphenyl)propenoic acid can be obtained through Knovenagel reaction using microwave irradiation and it has potential as anti-angiogenesis inhibitor with low toxicity.

A Phase 1, Randomized, Double-Blinded, Placebo-Controlled and Dose-Escalation Study to Evaluate the Safety and Immunogenicity of the Intranasal DelNS1-nCoV-RBD LAIV for COVID-19 in Healthy Adults.

An intranasal COVID-19 vaccine, DelNS1-based RBD vaccines composed of H1N1 subtype (DelNS1-nCoV-RBD LAIV) was developed to evaluate the safety and immunogenicity in healthy adults. We conducted a phase 1 randomized, double-blinded, placebo-controlled study on healthy participants, age 18-55 and COVID-19 vaccines naïve, between March and September 2021. Participants were enrolled and randomly assigned (2:2:1) into the low and high dose DelNS1-nCoV-RBD LAIV manufactured in chicken embryonated eggs or placebo groups. The low and high-dose vaccine were composed of 1 × 107 EID50/ dose and 1 × 107.7 EID50/ dose in 0.2 mL respectively. The placebo vaccine was composed of inert excipients/dose in 0.2 mL. Recruited participants were administered the vaccine intranasally on day 0 and day 28. The primary end-point was the safety of the vaccine. The secondary endpoints included cellular, humoral, and mucosal immune responses post-vaccination at pre-specified time-points. The cellular response was measured by the T-cell ELISpot assay. The humoral response was measured by the serum anti-RBD IgG and live-virus neutralizing antibody against SARS-CoV-2. The saliva total Ig antibody responses in mucosal secretion against SARS-CoV-2 RBD was also assessed. Twenty-nine healthy Chinese participants were vaccinated (low-dose: 11; high-dose: 12 and placebo: 6). The median age was 26 years. Twenty participants (69%) were male. No participant was discontinued due to an adverse event or COVID-19 infection during the clinical trial. There was no significant difference in the incidence of adverse events (p = 0.620). For the T-cell response elicited after full vaccination, the positive PBMC in the high-dose group increased to 12.5 SFU/106 PMBC (day 42) from 0 (baseline), while it increased to 5 SFU/106 PBMC (day 42) from 2.5 SFU/106 PBMC (baseline) in the placebo group. The high-dose group showed a slightly higher level of mucosal Ig than the control group after receiving two doses of the vaccine (day 31, 0.24 vs. 0.21, p = 0.046; day 56 0.31 vs. 0.15, p = 0.45). There was no difference in the T-cell and saliva Ig response between the low-dose and placebo groups. The serum anti-RBD IgG and live virus neutralizing antibody against SARS-CoV-2 were undetectable in all samples. The high-dose intranasal DelNS1-nCoV-RBD LAIV is safe with moderate mucosal immunogenicity. A phase-2 booster trial with a two-dose regimen of the high-dose intranasal DelNS1-nCoV-RBD LAIV is warranted.

Synthesis, anti-angiogenic activity and prediction toxicity of (E)-3-(3-methoxyphenyl) propenoic acid

Background: Anti-angiogenic medications, one of cancer chemo preventive mechanism were permitted for different cancers. Nevertheless, major primary and secondary resistance obstruct efficacy in several tumor types. Moreover, the improvement of safe and effective NSAIDs for angiogenesis inhibition is complicated, because of their serious toxicity. So, we require improving clinically appropriate strategies to boost efficacy of anti-angiogenic drugs with low risk of toxicity. Objectives: The present study aimed to synthesize the (E)-3- (3-methoxyphenyl)propenoic acid (3MPCA), to determine the anti-angiogenic activity and predict its toxicity. Methods: 3MPCA was obtained by Knoevenagel reaction using microwave irradiation at 400 Watt. The anti-angiogenesis experimental was performed using chorioallantois membrane of embryonated chicken eggs induced by b-FGF. The potency of 3MPCA was verified at dosage 30 and 60 ng and compared with celecoxib 60 ng. Toxicity prediction of 3MPCA was performed by ProTox II online program. Results: The results showed that 3MPCA was achieved in good yield (89%). Anti angogenic activity was showed by endothelial cells growth in neovascular capillaries of new blood vessel of chorioallantois membrane of embryonated chicken eggs. The endothelial cells growth decreased until 41.7-83%. The prediction LD50 was 1772mg/kg. Conclusion: (E)-3-(3-methoxyphenyl)propenoic acid can be obtained through Knovenagel reaction using microwave irradiation and it has potential as anti-angiogenesis inhibitor with low toxicity.

An inactivated NDV-HXP-S COVID-19 vaccine elicits a higher proportion of neutralizing antibodies in humans than mRNA vaccination.

NDV-HXP-S is a recombinant Newcastle disease virus-based vaccine against SARS-CoV-2, which expresses an optimized (HexaPro) spike protein on its surface. The vaccine can be produced in embryonated chicken eggs using the same process as that used for the production of the vast majority of influenza virus vaccines. Here, we performed a secondary analysis of the antibody responses after vaccination with inactivated NDV-HXP-S in a phase 1 clinical study in Thailand. The SARS-CoV-2 neutralizing and spike protein binding activity of NDV-HXP-S postvaccination serum samples was compared to that of samples from mRNA BNT162b2 (Pfizer) vaccinees. Neutralizing activity of sera from NDV-HXP-S vaccinees was comparable to that of BNT162b2 vaccinees, whereas spike protein binding activity of the NDV-HXP-S vaccinee samples was lower than that of sera obtained from mRNA vaccinees. This led us to calculate ratios between binding and neutralizing antibody titers. Samples from NDV-HXP-S vaccinees had binding to neutralizing activity ratios that were lower than those of BNT162b2 sera, suggesting that NDV-HXP-S vaccination elicits a high proportion of neutralizing antibodies and low non-neutralizing antibody titers. Further analysis showed that, in contrast to mRNA vaccination, which induces strong antibody titers to the receptor binding domain (RBD), the N-terminal domain, and the S2 domain, NDV-HXP-S vaccination induced an RBD-focused antibody response with little reactivity to S2. This finding may explain the high proportion of neutralizing antibodies. In conclusion, vaccination with inactivated NDV-HXP-S induces a high proportion of neutralizing antibodies and absolute neutralizing antibody titers that are comparable to those elicited by mRNA vaccination.

Generation of a high yield vaccine backbone for influenza B virus in embryonated chicken eggs

Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens’ eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.

Evolutionary Dynamics of Avian Influenza Viruses Isolated from Wild Birds in Moscow.

Forty-five strains of AIVs were isolated from wild aquatic birds during their autumn migration through Moscow (Russia). The aim of this work is to study the dynamics of AIV genomes in their natural habitat. Viruses were isolated from fecal sample in embryonated chicken eggs; their complete genomes were sequenced, and a phylogenetic analysis was performed. The gene segments of the same lineage persisted over the years in the absence of persistence of complete viral genomes. The genes for internal proteins of the same lineage were often maintained by the viruses over few years; however, they were typically associated with the genes of novel HA and NA subtypes. Although frequent reassortment events were observed for any pair of internal genes, there was no reassortment between HA and NA segments. The differences in the persistence of phylogenetic lineages of surface and internal proteins and the different evolutionary strategy for these two types of genes of AIVs in primary hosts are discussed.

Avian paramyxovirus serotype-1 isolation from migratory birds and environmental water in southern Japan: An epidemiological survey during the 2018/19-2021/2022 winter seasons.

Newcastle disease caused by highly pathogenic viruses of avian paramyxovirus serotype-1 (APMV-1) is a highly contagious poultry disease. Although a large-scale epidemic of Newcastle disease had occurred in Japan between the 1950s and the 2000s, there have been no outbreaks anywhere since 2010. In addition, there are no reports of epidemiological surveys of APMV-1 in wild birds in Japan in the last 10 years. We conducted the first epidemiological survey of APMV-1 in the Izumi plain, Kagoshima prefecture of southern Japan from the winter of 2018 to 2022. A total of 15 APMV-1 strains were isolated, and isolation rates from roosting water and duck fecal samples were 2.51% and 0.10%, respectively. These results indicate that the isolation method from environmental water may be useful for efficient surveillance of APMV-1 in wild birds. Furthermore, this is the first report on the success of APMV-1 isolation from environmental water samples. Genetic analysis of the Fusion (F) gene showed that all APMV-1 isolates were closely related to virus strains circulating among waterfowl in Far East Asian countries. All isolates have avirulent motifs in their cleavage site of F genes, all of which were presumed to be low pathogenic viruses in poultry. However, pathogenicity test using embryonated chicken eggs demonstrated that some isolates killed all chicken embryos regardless of viral doses inoculated (102 -106 50% egg infectious dose). These results indicated that APMV-1 strains, which are potentially pathogenic to chickens, are continuously brought into the Izumi plain by migrating wild birds.

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

The Effect of Remdesivir as An Anti-COVID-19 Drug on the Secretion of Inflammatory Markers by Chicken Liver Cells: An In Vitro Study

Background: For severe cases of acute respiratory syndrome type 2 (SARS-CoV-2) infection (COVID-19), Remdesivir (RDV) is introduced as an anti-viral drug with side effects. Hepatotoxicity from prolonged exposure to RDV is associated with increased inflammatory factors. In this study, we evaluated the effect of RDV on the secretion of inflammatory markers by chicken liver cells. Methods: In this in vitro study, 20 stage X embryonated chicken eggs were incubated for 10 days at 37.5°C and 60–65% humidity Hamburger–Hamilton stages (stage HH35). Liver cells were grown in a medium containing DMEM/F12+10% fetal bovine serum (FBS). After three days, the culture media was supplemented with four doses of RDV (1.00, 2.00, 3.00, and 4.00 μM). After 24 and 48hs, the viability of the hepatocytes, gene expression of Sox17, CXC motif chemokine receptor 4 (CXCR4), Interleukin-1 (IL-1), Interleukin-6 (IL-6), and TNF-α, and hepatocellular functions (albumin and urea secretion) were assessed. Findings: Each hepatocyte had a prominent nucleus and a nucleolus with a hexagonal shape. The pink tint of the periodic acid Schiff (PAS) positive cells in the PAS staining verified the hepatocytes’ glycogen content. Up to 50% of the cells lose viability after 48 hours in the presence of 3 and 4 μM RDV (P<0.001). In the presence of 3 μM RDV, the production and secretion of both albumin (P<0.001) and urea (P<0.05) decreased. Besides, the expression of IL-1, IL-6, and TNF-α significantly increased after treatment with 3 μM RDV (P<0.001). Conclusion: We concluded that RDV therapy altered the expression and function of hepatocyte inflammatory factors.

The Effect of Infectious Bronchitis Virus In Embryonated Chicken Eggs

The Effect of Infectious Bronchitis Virus In Embryonated Chicken Eggs

Identification and characterisation of lumpy skin disease virus recently isolated from Giza, Egypt

Lumpy skin disease (LSD) is a viral disease, geographically distributed in Africa and now, vigorously spread in the Near East and also in Europe and Asia. It has a significant economic impact on cattle industry in Africa. The aim of this study was isolation and rapid identification of LSD virus circula­ting in Egypt from clinically suspected cattle based on clinical and molecular basis in a rapid and accurate way. Fifteen representative specimens (skin sitfasts) were collected in 2018 from clinically infected cattle in Giza governorate, Egypt. The virus was isolated on chorioallantoic membrane of specific pathogen free embryonated chicken eggs and Madin Darby Bovine Kidney tissue culture cells. The isolated virus was identified and confirmed by conventional polymerase chain reaction (PCR) and real-time PCR. Histopathological examination of the lesions showed a pathognomic intracytoplasmic inclusion body in dermal stroma section. The section of dermal layer revealed vasculitis with projection of its endothelial lining. It was concluded that LSD was enzootic in Egypt and still circulating among Egyptian cattle so that LSD virus could be isolated and identified by traditional and molecular diagnostic methodes. Real time PCR assay could be applied for rapid and accurate confirmation of the field isolate of LSD virus.

Development of SYBR green RT-qPCR assay for titrating bivalent live infectious bronchitis vaccines

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID50). However, this method cannot effectively distinguish or quantify different genotypic strains in a mixture of different viruses, especially in the absence of neutralizing monoclonal antibodies. In this study, quantitative real-time PCR (RT-qPCR) was applied using specific primers for the QX- and Mass-like strains to quantitate IBV infection and for comparison with the conventional virus titration quantitative method. A strong positive correlation was observed between RT-qPCR cycle threshold values and the different EID50 concentrations. This method was further used to titrate bivalent IB vaccines, and the amount of individual genotype virus was determined based on specific primers. Thus, this RT-qPCR assay may be used as a highly specific, sensitive, and rapid alternative to the EID50 assay for titering IBVs.

Propagation of avian influenza virus in embryonated ostrich eggs

Influenza A viruses (IAVs) are typically isolated and cultured by successive passages using 9- to 11-day-old embryonated chicken eggs (ECEs) and in 14-day old ECEs for virus mutational studies. Real-time reverse transcription-polymerase chain reaction tests (RT-PCRs) are commonly used for IAV diagnosis, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus. Efforts at isolating ostrich-origin IAVs from RT-PCR positive specimens using ECEs have often been unsuccessful, raising the possibility of a species bottleneck, whereby ostrich-adapted IAVs may not readily infect and replicate in ECEs, yet the capacity of an ostrich embryo to support the replication of influenza viruses has not been previously demonstrated. This study describes an optimised method for H5 and H7 subtype IAV isolation and propagation in 28-day old embryonated ostrich eggs (EOEs), the biological equivalent of 14-day old ECEs. The viability of EOEs transported from breeding sites could be maximised by pre-incubating the eggs for 12 to 14 days prior to long-distance transportation. This method applied to studies for ostrich-adapted virus isolation andin ovostudies will enable better understanding of the virus-host interaction in ostriches and the emergence of potentially zoonotic diseases.

Genetic characterization and pathogenicity of a Eurasian avian-like H1N1 swine influenza reassortant virus

BackgroundSwine influenza viruses (SIV), considered the “mixing vessels” of influenza viruses, posed a significant threat to global health systems and are dangerous pathogens. Eurasian avian-like H1N1(EA-H1N1) viruses have become predominant in swine populations in China since 2016.MethodsLung tissue samples were obtained from pregnant sows with miscarriage and respiratory disease in Heilongjiang province, and pathogens were detected by Next-generation sequencing (NGS) and PCR. The nucleic acid of isolates was extracted to detect SIV by RT-PCR. Then, SIV-positive samples were inoculated into embryonated chicken eggs. After successive generations, the isolates were identified by RT-PCR, IFA, WB and TEM. The genetic evolution and pathogenicity to mice of A/swine/Heilongjiang/GN/2020 were analyzed.ResultsThe major pathogens were influenza virus (31%), Simbu orthobunyavirus (15%) and Jingmen tick virus (8%) by NGS, while the pathogen that can cause miscarriage and respiratory disease was influenza virus. The SIV(A/swine/Heilongjiang/GN/2020) with hemagglutination activity was isolated from lung samples and was successfully identified by RT-PCR, IFA, WB and TEM. Homology and phylogenetic analysis showed that A/swine/Heilongjiang/GN/2020 is most closely related to A/swine/Henan/SN/10/2018 and belonged to EA-H1N1. Pathogenicity in mice showed that the EA-H1N1 could cause lethal or exhibit extrapulmonary virus spread and cause severe damage to respiratory tracts effectively proliferating in lung and trachea.ConclusionA/swine/Heilongjiang/GN/2020 (EA-H1N1) virus was isolated from pregnant sows with miscarriage and respiratory disease in Heilongjiang province, China. Clinical signs associated with influenza infection were observed during 14 days with A/swine/Heilongjiang/GN/2020 infected mice. These data suggest that A/swine/Heilongjiang/GN/2020 (EA-H1N1) had high pathogenicity and could be systemic spread in mice.

The First Report of a Virulent Newcastle Disease Virus of Genotype VII.2 Causing Outbreaks in Chickens in Bangladesh.

Newcastle disease (ND) is endemic in poultry in Bangladesh. We performed genotypic and pathotypic characterization of four ND virus (NDV) isolates from recent outbreaks in broiler chickens in Bangladesh during the period of 2020-2021. Phylogenetic analysis based on the complete fusion protein gene coding sequences classified the viruses into NDV class II genotype VII.2 together with viruses from Indonesia isolated between 2014 and 2021 and a single 2020 Indian isolate. Pathogenicity testing using the intracerebral pathogenicity index in day-old chickens and mean embryo death time in embryonating chicken eggs revealed that the Bangladeshi isolates are velogenic. Inoculation of 35-day-old chickens with two NDV isolates (LT67 and N5) resulted in 100% morbidity by 3 days post inoculation (DPI), and all birds succumbed to infection by 7 DPI. Massive hemorrhages, congestion and necrotic lesions were observed in different visceral organs, which were typical for infection with a velogenic viscerotropic pathotype of NDV. At microscopic examination, tracheitis, severe pneumonia, focal proventriculitis, transmural enteritis, focal myocarditis, severe congestion and necrosis in kidneys, and lymphoid depletion in lymphoid tissues were found. Our study reports the first outbreak of the panzootic genotype VII.2 NDV in poultry in Bangladesh and documents a possible recent re-introduction of this NDV genotype from Southeast or East Asia. This study further provides viral distribution and epidemiological data that can facilitate the effective control of NDV.

Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia.

A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT_Tiwi Islands/2021). Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences. Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7-96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998-2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains. This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

Delivery of Fc-fusion Protein by a Recombinant Newcastle Disease Virus Vector.

Fc-fusion proteins (FCPs), a new generation biological medicine, have revolutionized the practice of medicines that treat diseases. However, complex manufacturing techniques are required for FCP production, casting the affordability and accessibility issues in low- and middle-income economies (LMIEs). Virus-vectored system may serve as a simple and cost-effective platform for FCP delivery. As a proof-of-concept study, Newcastle disease virus (NDV), a widely-used vector for vaccine generation, was used as a vector to express and deliver a model FCP composed of the hemagglutinin (HA) and IgG Fc. A recombinant NDV expressing the HA-Fc fusion protein was generated using reverse genetics, which had comparable replication and virulence to the parental virus. High levels of expression of soluble HA-Fc were detected in cell culture and embryonated chicken eggs inoculated with the recombinant NDV. In addition, the recombinant NDV replicated in the lung of mouse, delivering the HA-Fc protein to this organ. The HA-Fc expressed by NDV specifically bound to murine FcγRI, which was dependent on the presence of the Fc tag. The recombinant NDV induced high vector-specific antibody response, whereas it failed to elicit H7N9-specific antibody immunity in mice. The absence of HA-specific antibodies may be attributed to deficient incorporation of the HA-Fc protein into NDV virion particles. Our results indicated that NDV may be potentially used as a vector for FCP expression and delivery. This strategy may help to enhance the affordability and equal accessibility of FCP biological medicines, especially in LIMEs.