Embryonated Chicken Eggs Research Articles

Impact of Human Adenovirus 36 on Embryonated Chicken Eggs: Insights into Growth Mechanisms.

Human adenovirus 36 (HAdV-D36) is presently the sole virus identified to be associated with an elevated risk of obesity in both humans and animals. However, its impact on embryonated chicken eggs (ECEs) remains unexplored. This study endeavoured to examine the influence of HAdV-D36 on embryonic development by utilizing embryonated chicken eggs as a dynamic model. To simulate various infection routes, the allantoic cavity and the yolk sac of ECEs were inoculated with HAdV-D36. Subsequently, embryos from both the experimental (inoculated with virus) and control (inoculated with PBS) groups were weighed and subjected to daily histological examination. The daily embryo weights were assessed and compared between groups using the Shapiro-Wilk test. Histopathological changes in tissues were examined and compared between the tested and control groups to ascertain physiological alterations induced by the virus. Our study confirmed a significant increase in the body weight of ECEs. However, this phenomenon was not attributable to adipose tissue development; rather, it was characterized by an augmented number of cells in all observed tissues compared to control subjects. We posit that HAdV-D36 may impact developing organisms through mechanisms other than enhanced adipose tissue development. Specifically, our findings indicate an increased number of cells in all tissues, a phenomenon that occurs through an as-yet-unexplored pathway.

Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b from Peru forms a monophyletic group with Chilean isolates in South America

Highly pathogenic avian Influenza virus (HPAIV) has spread in an unprecedented extent globally in recent years. Despite the large reports of cases in Asia, Europe, and North America, little is known about its circulation in South America. Here, we describe the isolation, and whole genome characterization of HPAIV obtained from sampling 26 wild bird species in Peru, representing one of the largest studies in our region following the latest HPAIV introduction in South America. Out of 147 samples analyzed, 22 were positive for detection of avian influenza virus using a qRT-PCR-based assay. Following inoculation into embryonated chicken eggs, fourteen viral isolates were obtained from which nine isolates were selected for genome characterization, based on their host relevance. Our results identified the presence of HPAIV H5N1 subtype in a highly diverse wild bird species. Phylogenetic analysis revealed that these isolates correspond to the clade 2.3.4.4b, sharing a common ancestor with North American isolates and forming a monophyletic group along with isolates from Chile. Altogether, changes at the amino acid levels compared to their closest relatives indicates the virus is evolving locally, highlighting the need for constant genomic surveillance. This data evidence the chances for spillover events increases as the virus spreads into large populations of immunologically naïve avian species and adding conditions for cross species transmission.

Isolation and molecular characterization of lumpy skin disease virus from Tamil Nadu, India during the outbreaks from 2020 to 2022.

Lumpy skin disease (LSD) caused by LSD virus is a WOAH notifiable, high-impact, transboundary poxviral disease of bovines. The first official report of LSDV in India is from Odisha state during August 2019. Since then, cases have been reported from many states including Tamil Nadu, a Southern state of India. The present study deals with isolation and molecular characterization of LSDV from Tamil Nadu during the period August 2020 to July 2022. LSDV was isolated in embryonated chicken eggs (ECE) and BHK 21 cells and was characterized based on P32, RPO30, and GPCR genes. The phylogenetic analysis revealed that Tamil Nadu isolates from India are closely related to other Indian strains, Kenyan strains and strains from neighboring countries such as Bangladesh, Nepal, and Myanmar confirming the common exotic source for the transboundary spread across borders. The presence of unique signature of amino acid (aa) at specific positions (A11, T12, T34, S99, and P199) in the GPCR sequence confirmed the identity of LSDV. A twelve nucleotide (nt94-105) insertion and corresponding aa (TILS) at 30-33 position was found in GPCR sequence and characteristic amino acid proline at 98 position (P98) in the RPO30 gene sequence of our isolates was similar to strains from Bangladesh, Nepal, and Myanmar. Further, dissimilarity of our isolates from Neethling like vaccine strains confirms the circulation of virulent filed strains responsible for the outbreaks.

First Reported Circulation of Equine Influenza H3N8 Florida Clade 1 Virus in Horses in Italy.

Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance. Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories. None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761). This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy.

Authorizing of Immunogenicity of Concentrated and Purified Newcastle Disease Virus V4 Strain using Downstream Processing.

Newcastle disease virus (NVD) from the Paramyxoviridae family is a single-stranded negative-sense RNA virus. This infection can affect both domestic poultry and almost all other bird species. It has been considered a very severe difficulty for the poultry industry all over the world. Even though it remains a potential threat to poultry industries, this virus is a powerful oncolytic virus as well. In this study, a process was accomplished to achieve concentrated and highly purified NDV V4 strain particles. Downstream processing of Newcastle virus strain V4 was characterized by amplifying virus in embryonated chicken eggs. Through a sequence of steps, harvesting allantoic fluid, clarification by centrifuge, concentration by ultrafiltration, and size exclusion separation, the reduced volume and pure virus particles were considered for the amount of ovalbumin, hemagglutinin activity, transmission electron microscopy (TEM), electrophoresis, and additionally immunogenicity of prepared antigens. The results presented a high recovery of HA activity in concentrated and purified virus with the removal of ovalbumin and the typical morphology based on TEM. Sepharose CL-4B was determined as the best media among all used resins to purify the virus. Prepared formulations as vaccines demonstrated positive hemagglutinin inhibition for 6 months and stability for 2 years. Strong evidence from organized studies supports the effectiveness of this method in concentrating and purifying intact NDV, which could be valuable in vaccine research, antiserum preparation, or even as an alternative oncotic agent to traditional methods. Despite further studies being conducted, this method can be utilized particularly on a semi-industrial scale to produce various vaccine components.

The spike protein of the apathogenic Beaudette strain of avian coronavirus can elicit a protective immune response against a virulent M41 challenge.

The avian Gammacoronavirus infectious bronchitis virus (IBV) causes major economic losses in the poultry industry as the aetiological agent of infectious bronchitis, a highly contagious respiratory disease in chickens. IBV causes major economic losses to poultry industries across the globe and is a concern for global food security. IBV vaccines are currently produced by serial passage, typically 80 to 100 times in chicken embryonated eggs (CEE) to achieve attenuation by unknown molecular mechanisms. Vaccines produced in this manner present a risk of reversion as often few consensus level changes are acquired. The process of serial passage is cumbersome, time consuming, solely dependent on the supply of CEE and does not allow for rapid vaccine development in response to newly emerging IBV strains. Both alternative rational attenuation and cell culture-based propagation methods would therefore be highly beneficial. The majority of IBV strains are however unable to be propagated in cell culture proving a significant barrier to the development of cell-based vaccines. In this study we demonstrate the incorporation of a heterologous Spike (S) gene derived from the apathogenic Beaudette strain of IBV into a pathogenic M41 genomic backbone generated a recombinant IBV denoted M41K-Beau(S) that exhibits Beaudette's unique ability to replicate in Vero cells, a cell line licenced for vaccine production. The rIBV M41K-Beau(S) additionally exhibited an attenuated in vivo phenotype which was not the consequence of the presence of a large heterologous gene demonstrating that the Beaudette S not only offers a method for virus propagation in cell culture but also a mechanism for rational attenuation. Although historical research suggested that Beaudette, and by extension the Beaudette S protein was poorly immunogenic, vaccination of chickens with M41K-Beau(S) induced a complete cross protective immune response in terms of clinical disease and tracheal ciliary activity against challenge with a virulent IBV, M41-CK, belonging to the same serogroup as Beaudette. This implies that the amino acid sequence differences between the Beaudette and M41 S proteins have not distorted important protective epitopes. The Beaudette S protein therefore offers a significant avenue for vaccine development, with the advantage of a propagation platform less reliant on CEE.

S2 subunit plays a critical role in pathogenesis of TW-like avian coronavirus infectious bronchitis virus

To investigate the critical role of the S gene in determining pathogenesis of TW-like avian infectious bronchitis virus (IBV), we generated two recombinant IBVs (rGDaGD-S1 and rGDaGD-S2) by replacing either the S1 or S2 region of GD strain with the corresponding regions from an attenuated vaccine candidate aGD strain. The virulence and pathogenicity of these recombinant viruses was assessed both in vitro and in vivo. Our results indicated the mutations in the S2 region led to decreased virulence, as evidenced by reduced virus replication in embryonated chicken eggs and chicken embryonic kidney cells as well as observed clinical symptoms, gross lesions, microscopic lesions, tracheal ciliary activity, and viral distribution in SPF chickens challenged with recombinant IBVs. These findings highlight that the S2 subunit is a key determinant of TW-like IBV pathogenicity. Our study established a foundation for future investigations into the molecular mechanisms underlying IBV virulence.

Synthetic Cell Lines for Inducible Packaging of Influenza A Virus.

Influenza A virus (IAV) is a negative-sense RNA virus that causes seasonal infections and periodic pandemics, inflicting huge economic and human costs on society. The current production of influenza virus for vaccines is initiated by generating a seed virus through the transfection of multiple plasmids in HEK293 cells followed by the infection of seed viruses into embryonated chicken eggs or cultured mammalian cells. We took a system design and synthetic biology approach to engineer cell lines that can be induced to produce all viral components except hemagglutinin (HA) and neuraminidase (NA), which are the antigens that specify the variants of IAV. Upon the transfection of HA and NA, the cell line can produce infectious IAV particles. RNA-Seq transcriptome analysis revealed inefficient synthesis of viral RNA and upregulated expression of genes involved in host response to viral infection as potential limiting factors and offered possible targets for enhancing the productivity of the synthetic cell line. Overall, we showed for the first time that it was possible to create packaging cell lines for the production of a cytopathic negative-sense RNA virus. The approach allows for the exploitation of altered kinetics of the synthesis of viral components and offers a new method for manufacturing viral vaccines.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNβ, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

In vivo label-free tissue histology through a microstructured imaging window.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.

Molecular and serological detection of Newcastle disease virus in live-bird markets, Jos, Plateau State Nigeria.

Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and it has been reported in domestic birds in Nigeria. Waterfowls and village poultry in live bird markets (LBM) acts as reservoirs, potentially reintroducing the virus to commercial poultry. This study aims at molecular and serological detection of NDV at live bird markets in Plateau State, Nigeria. A cross sectional analysis involved 309 pooled cloacal and tracheal swabs over three months were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), RT-qPCR, virus isolation and haemagglutination and haemagglutination-inhibition test (HA &HI). Virus isolation was attempted in 9 to 11 days old specific antibody negative (SAN) embryonated chicken eggs and fifteen samples showed haemagglutination. Subsequent tests confimed nine of these as NDV positive through haemagglutination and haemagglutination inhibition (HI) tests. ConventionalRT-PCR and RT-qPCR further validated five of the nine NDV positive isolates. The phylogenetic analysis of partial F gene nucleotide sequences revealed that all three isolates belonged to class II genotype XIV.2_XIVb. This finding underscores the persistent threat of NDV to local poultry, necessitating comprehensive virological surveillance to understand, isolate and characterize the virus in Nigeria. Therefore, monitoring for emerging lineages and sub-lineages in Nigeria birds is crucial for safeguarding commercial poultry production.

Effect of combined infection with Salmonella and influenza virus on their respective proliferation in chicken embryonated eggs.

Salmonella is a major food-borne bacterial pathogen that causes food poisoning related to the consumption of eggs, milk, and meat. Food safety in relation to Salmonella is particularly important for eggs because their shells as well as their contents can be a source of contamination. Chicken can also be infected with influenza virus, but it remains unclear how co-infection of Salmonella and influenza virus affect each other. The potential influence of co-infection of Salmonella and influenza virus was examined. Salmonella Abony and influenza virus were injected into chicken embryonated eggs. After incubation, proliferation of Salmonella and influenza virus was measured using a direct culture assay for bacteria and an enzyme-linked immunosorbent assay for influenza virus, respectively. Our findings indicate that the number of colony-forming units (CFUs) of Salmonella did not vary between chicken embryonated eggs co-infected with influenza A virus and Salmonella-only infected eggs. Furthermore, we found the proliferation of influenza A or B virus was not significantly influenced by co-infection of the eggs with Salmonella. These results suggest that combined infection of Salmonella with influenza virus does not affect each other, at least in terms of their proliferation.

Characterization of infectious laryngotracheitis virus isolated from commercial layer chickens in Bangladesh during the year 2021-2022.

Infectious laryngotracheitis virus (ILTV) is responsible for causing infectious laryngotracheitis (ILT), which is a rapidly spreading and extremely transmissible disease in chickens. The current research aims to isolate and characterize ILTV from layer chickens in Bangladesh. A total of 345 samples (trachea, larynx, and lungs) were collected from ILT-suspected dead and sick layer chickens of 32 ILT-suspected farms in three different outbreak districts (Gazipur, Tangail, and Mymensingh) of Bangladesh during the outbreak year 2021-2022. Rapid detection kits examined the samples for avian influenza virus (AIV) and Newcastle disease virus (NDV). ILTV-specific primers were used to screen 72 NDV- and AIV-negative samples by polymerase chain reaction (PCR). Using chorioallantoic membrane (CAM), the study isolated the ILT virus from 9 to 10-day-old seronegative embryonated chicken eggs (ECEs) using selected PCR-positive samples. The virus was confirmed using nucleotide sequencing, agar gel immunodiffusion test (AGIDT), viral neutralization test (VNT), and pathogenicity evaluations using mortality index for chicken embryos (MICEs) and intra-tracheal pathogenicity index (ITPI). The results indicated that among the PCR-positive 10 samples, only two (Alim_ILT_1001 and Alim_ILT_1,000) were found positive using ECEs. There were two field isolates of ILTVs, as shown by the amplicon size of the ICP4 gene-based PCR. A phylogenetic study of the ICP4 gene revealed that the recent isolates have a close similarity with the ILTV isolates of Turkey, Bangladesh, and Australia. AGIDT revealed strong precipitation lines due to ILTV-specific antibodies reacting with field viruses, while VNT neutralized both isolates with conventional ILTV antibodies. The pathogenicity testing indicated that Alim_ILT_1001 had MICE and ITPI values of 0.77 and 0.63, whereas Alim_ILT_1,000 had 0.71 and 0.57. Both the ILTV isolates have similarities with the isolates of Turkey, Bangladesh, and Australia, and they are highly virulent for chickens.

Characterization of the dominant strain (G-VII) of Newcastle disease viruses isolated from commercial chickens in Bangladesh during recent outbreaks.

Newcastle disease virus genotype VII (NDV-GVII), an extremely infectious pathogen, has been causing severe economic consequences for the chicken industry. The current study aimed to isolate and characterize NDV-GVII from commercial chickens in Bangladesh during a recent outbreak. From clinically suspected chickens from 70 commercial poultry farms, a total of 420 samples (trachea, lungs, and brain tissue) were collected. The samples were cultivated in 9-10 day-old seronegative embryonated chicken eggs (ECEs) after evaluating them using the rapid Newcastle disease virus (NDV) antigen detection kit. The hemagglutination (HA) inhibition test, agar gel immune diffusion (AGID) test, molecular detection by reverse transcription-polymerase chain reaction (RT-PCR), and phylogenetic studies using gene sequences of fusion (F) protein. The HA pattern of isolated NDV was determined using different avian and mammalian red blood cells (RBCs). The pathogenicity of the isolated virus was evaluated using mean death time (MDT), intravenous pathogenicity index (IVPI), and intracerebral pathogenicity index (ICPI). The study found 87 NDV samples positive using the rapid NDV Ag detection kit and then 60 positives for virus isolation in ECEs. All 60 isolates were positive for NDV by HI, AGID, and RT-PCR. Phylogenetic tree analysis indicated that recent NDV isolates belong to genotype VII and exhibit a similarity of 99.7%-98.5% with isolates from Bangladesh, Iran, and India. The new isolates, identified as velogenic strains of NDV, possess an F protein cleavage site with 112-R-T-K-R-F-117 amino acid motifs. The isolated NDV showed diversified HA activity while using RBCs from birds and mammals. The results of ICPI, IVPI, and MDT indicated that the recent NDV isolates were very virulent. This study concluded that NDV-GVII is prevalent in commercial poultry farms in Bangladesh.

Identification and genetic correlation of avian reoviruses to the currently used vaccines in Egypt

A variety of illnesses, including arthritis, tenosynovitis, stunted growth, and malabsorption syndrome, are caused by Avian Reoviruses (ARVs), which have become more prevalent in Egypt during recent years and resulted in significant economic losses. This study investigated 27 suspected samples collected from 14 broiler breeders and 13 broilers suffering from immunosuppression, decreased body weight, and diarrhea. Fourteen samples tested positive based on RT-PCR, and the virus could be isolated from ten samples in Specific Pathogen Free (SPF) embryonated chicken eggs. Ten isolates were subjected to molecular and genetic analysis of the S1 gene (sigma C) and S2 gene (sigma A). The amino acid identity of the S1 gene revealed that these viruses are closely related to the viruses that were identified in Israel during 2020 (91.8%-97.2% identity) and belonged to the genetic cluster 5 (genotype 5), which also includes some viruses that are circulating in the United States and Canada. They also showed weak similarity (48.9%-50.2%) with the available vaccine strains in the Egyptian field that belong to cluster 1, genotype 1. The S2 gene showed amino acid homology of 91.7%-98.2% with the current vaccine used in Egypt. However, the Egy-Reo-7-2021 virus had the lowest similarity (84.2%-87.6%) to the available vaccine. It is hypothesized that the difference between field and vaccine strains may have contributed to the failure of current vaccinations to produce protective immunity against current ARV strains circulated in Egypt, which made the disease a problem to the poultry industry. Developing homologous vaccines and evaluating their potency and efficacy are required in Egypt.

Isolation and molecular characterization of fowl adenovirus from inclusion body hepatitis cases in Western India during 2019-2021.

Inclusion body hepatitis (IBH) resulted in a substantial economic loss in Western India during 2019 to 2021. The study aimed to characterize fowl adenovirus (FAdV) from field outbreaks. The study was conducted on 290 liver samples from 66 poultry flocks. The samples were subjected to histopathology and molecular detection, followed by phylogenetic typing of the partial hexon gene of the virus. Spiking mortality (14%) was recorded from day 21 to day 35 with peak mortality at the 28th day of age. The necropsy showed a pale and enlarged liver with hemorrhagic and yellowish necrotic foci, accumulation of straw-colored transudate in the pericardial sac which resulted in a flabby appearance of the heart, heart enlargement, and hemorrhages on the spleen, enlarged and congested kidneys. The virus inoculation resulted in stunting and poor feathering with hepatomegaly, hemorrhages and yellowish necrotic foci on the liver as well as greenish discoloration, and kidney swelling in SPF embryonated chicken eggs. Out of 29, 16 liver samples yielded 1219 bp amplicons specific to hexon gene fragments. The sequence and phylogenetic analysis identified 14 isolates as FAdV species E serotype 11 and two as species D serotype 8b. Conclusion: The results indicated that FAdV-8b and FAdV-11 strains are involved in disease outbreaks in western India.

Characterization of Fowlpox Virus.

The complex cytoplasmic DNA virus known as the fowlpox virus (FWPV) is a member of the avipoxvirus genus, Subfamily Chordopoxvirinae, and Family Poxviridae. The large genome size of FWPV makes it a potential vector for the creation of vaccines against a range of serious veterinary and human ailments. It also allows for multiple gene insertion and the generation of abortive infection in mammalian cells. The virus, which causes fowlpox in chickens and turkeys, is mainly transmitted to poultry through aerosols or biting insects. Fowlpox is a highly contagious disease that affects both domestic and wild birds, causing cutaneous and/or diphtheritic illnesses. To control the illness, strict hygiene practices and immunization with FWPV attenuated strains or antigenically similar pigeon pox virus vaccines are employed. Recent years have seen an increase in fowlpox outbreaks in chicken flocks, primarily due to the introduction of novel forms of FWPV. It is believed that the pathogenic characteristics of these strains are enhanced by the integration of reticuloendotheliosis virus sequences of variable lengths into the FWPV genome. The standard laboratory diagnosis of FPV involves histopathological analysis, electron microscopy, virus isolation on chorioallantoic membrane (CAM) of embryonated chicken eggs or cell cultures, and serologic techniques. For quick and consistent diagnosis, polymerase chain reaction (PCR) has proven to be the most sensitive method. PCR is used in concert with restriction endonuclease enzyme analysis (REA) to identify, differentiate, and characterize the molecular makeup of isolates of the fowlpox virus. Sequencing of the amplified fragments is then done.

Isolation of Reticuloendotheliosis Virus from Chorio-allantoic Membrane of SPF Chicken Eggs inoculated with Fowl Pox Virus.

Fowl Pox Viruses (FPV) infect chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth etc. The birds, affected with FPV, also show anemia and ruffled appearance which are clinical symptoms of Reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of Reticuloendotheliosis Virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the Chorio-allantoic Membrane (CAM) of 10 days old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. But the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV virus, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Occurrence of enteric viruses causing clinical diarrhea in small ruminants in northern Indian plains: a reverse transcription PCR based molecular study.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Modifying the hemagglutinin gene with the packaging signals to improve the ability of replication in eggs of IBT-RG02 vaccine virus strain

The hemagglutinin (HA) and neuraminidase (NA) genes were derived from the popular H5N1 virus and the remaining six internal segments were derived from the A/Puerto Rico/8/34 strain (H1N1, PR8). However, some of these candidate strains have been reported to produce relatively low yields in vaccine manufacture does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8’s hemagglutinin (HA) packaging signals mediate improvement of candidate vaccine virus yield in eggs. We constructed chimeric HA genes having the coding region of IBT-RG02 HA flanked by the 50 packaging signals of PR8’s HA. The growth of candidate vaccine viruses containing the chimeric HA (RG2, RG3, RG4) was increased in embryonated chicken eggs with a nearly 1.5 to 2-fold higher titer than that of control (RG1). It is concluded that the using of chimeric HA fragment can subsequently improve the replication of reversed genetics-derived virus strains in eggs.