Quantitative evaluation of viral interference among Egyptian isolates of highly pathogenic avian influenza viruses (H5N1 and H5N8) with the lentogenic and velogenic Newcastle disease virus genotype VII in specific pathogen-free embryonated chicken eggs model.
ABSTRACT: Background and Aim:Mixed infections of the highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are considered the most distressing problem of the poultry industry. The problem arises due to the influence of a hidden virus on the replication of another suspected virus. Consequently, misdiagnosis of the real cause of disease may become a source of infection for other healthy stock by transmission and dissemination of the hidden virus. This study aimed to determine the impact of HPAIV and NDV on each other in a specific pathogen-free embryonated chicken egg (SPF-ECE) model. Materials and Methods:HPAIVs (H5N1 and H5N8) and NDVs [avirulent NDV [avNDV] and velogenic NDV [vNDV]) were inoculated into the allantois cavity of SPF-ECE with graded titers (2, 3, and 4 log10 EID50) at 24 and 48 h of incubation, followed by the collection of allantoic fluid. A quantitative reverse transcription real-time polymerase chain reaction was used to determine the viral RNA copies of both viruses. Results:Obvious interference was reported on the growth of NDVs when co-inoculated with AIVs. NDV RNA titers reduction ranged from <3 to 5 log10 to complete suppression, but slight interference with the growth of AIVs occurred. H5N1 RNA titers showed <1-2 log10 reduction when co-inoculated with vNDV compared with the H5N1 control. The interference impact of H5N8 was more powerful than that of H5N1, while vNDV showed more resistance for interference than the avNDV strain. On the other hand, interference of AIVs was not observed except when vNDV was inoculated before H5N1. The interfering impact was increased after 48 h of inoculation, whereas no titer of avNDV was detectable. Conclusion:AIV strains had a powerful effect on NDV growth, regardless of which infection occurred first.
Project description:Aim:The aim of the current study was to evaluate the efficacy of a trivalent-inactivated oil-emulsion vaccine against challenge by different clades highly pathogenic avian influenza (HPAI) viruses including HPAI-H5N8 and the virulent genotype VII Newcastle disease virus (NDV) (vNDV). Materials and Methods:The vaccine studied herein is composed of reassortant AI viruses rgA/Chicken/Egypt/ME1010/2016 (clade 22.214.171.124), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 126.96.36.199), and "NDV" (LaSota NDV/CK/Egypt/11478AF/11); all used at a concentration of 108 EID50/bird and mixed with Montanide-ISA70 oil adjuvant. Two-week-old specific pathogen free (SPF) chickens were immunized subcutaneously with 0.5 ml of the vaccine, and hemagglutination inhibition (HI) antibody titers were monitored weekly. The intranasal challenge was conducted 4 weeks post-vaccination (PV) using 106 EID50/0.1 ml of the different virulent HPAI-H5N1 viruses representing clades 2.2.1, 188.8.131.52, 184.108.40.206, 220.127.116.11b-H5N8, and the vNDV. Results:The vaccine induced HI antibody titers of >6log2 against both H5N1 and NDV viruses at 2 weeks PV. Clinical protection against all HPAI H5N1 viruses and vNDV was 100%, except for HPAI H5N1 clade-2.2.1 and HPAI H5N8 clade-18.104.22.168b viruses that showed 93.3% protection. Challenged SPF chickens showed significant decreases in the virus shedding titers up to <3log10 compared to challenge control chickens. No virus shedding was detected 6 "days post-challenge" in all vaccinated challenged groups. Conclusion:Our results indicate that the trivalent H5ND vaccine provides significant clinical protection against different clades of the HPAI viruses including the newly emerging H5N8 HPAI virus. Availability of such potent multivalent oil-emulsion vaccine offers an effective tool against HPAI control in endemic countries and promises simpler vaccination programs.
Project description:BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.
Project description:The recombinant herpesvirus of turkey (rHVT) vaccines targeting Newcastle disease (ND) and H5Nx avian influenza (AI) have been demonstrated efficient in chickens when used individually at day-old. Given the practical field constraints associated with administering two vaccines separately and in the absence of a currently available bivalent rHVT vector vaccine expressing both F(ND) and H5(AI) antigens, the aim of this study was to investigate whether interference occurs between the two vaccines when simultaneously administered in a single shot. The studies have been designed to determine (i) the ND and AI-specific protection and antibody response conferred by these vaccines inoculated alone or in combination at day-old, (ii) the influence of maternally-derived antibodies (MDA), and (iii) the potential interference between the two vaccine. Our results demonstrate that their combined administration is efficient to protect chickens against clinical signs of velogenic Newcastle disease virus (vNDV) and H5-highly pathogenic avian influenza virus (HPAIV) infections. Viral shedding following co-vaccination is also markedly reduced, while slightly lower NDV- and AIV-specific antibody responses are observed. NDV- and AIV-specific MDA show negative effects on the onset of the specific antibody responses. However, if AIV-specific MDA reduce the protection against H5-HPAIV induced by rHVT-H5(AI) vaccine, it was not observed for ND.
Project description:The A(H5N8) highly pathogenic avian influenza (HPAI) epidemic occurred in 29 European countries in 2016/2017 and has been the largest ever recorded in the EU in terms of number of poultry outbreaks, geographical extent and number of dead wild birds. Multiple primary incursions temporally related with all major poultry sectors affected but secondary spread was most commonly associated with domestic waterfowl species. A massive effort of all the affected EU Member States (MSs) allowed a descriptive epidemiological overview of the cases in poultry, captive birds and wild birds, providing also information on measures applied at the individual MS level. Data on poultry population structure are required to facilitate data and risk factor analysis, hence to strengthen science-based advice to risk managers. It is suggested to promote common understanding and application of definitions related to control activities and their reporting across MSs. Despite a large number of human exposures to infected poultry occurred during the ongoing outbreaks, no transmission to humans has been identified. Monitoring the avian influenza (AI) situation in other continents indicated a potential risk of long-distance spread of HPAI virus (HPAIV) A(H5N6) from Asia to wintering grounds towards Western Europe, similarly to what happened with HPAIV A(H5N8) and HPAIV A(H5N1) in previous years. Furthermore, the HPAI situation in Africa with A(H5N8) and A(H5N1) is rapidly evolving. Strengthening collaborations at National, EU and Global levels would allow close monitoring of the AI situation, ultimately helping to increase preparedness. No human case was reported in the EU due to AIVs subtypes A(H5N1), A(H5N6), A(H7N9) and A(H9N2). Direct transmission of these viruses to humans has only been reported in areas, mainly in Asia and Egypt, with a substantial involvement of wild bird and/or poultry populations. It is suggested to improve the collection and reporting of exposure events of people to AI.
Project description:Egypt is a hotspot for avian influenza virus (AIV) due to the endemicity of H5N1 and H9N2 viruses. AIVs were isolated from 329 samples collected in 2016-2018; 48% were H9N2, 37.1% were H5N8, 7.6% were H5N1, and 7.3% were co-infections with 2 of the 3 subtypes. The 32 hemagglutinin (HA) sequences of the H5N1 viruses formed a well-defined lineage within clade 22.214.171.124. The 10 HA sequences of the H5N8 viruses belonged to a subclade within 126.96.36.199. The 11 HA of H9N2 isolates showed high sequence homology with other Egyptian G1-like H9N2 viruses. The prevalence of H5N8 viruses in ducks (2.4%) was higher than in chickens (0.94%). Genetic reassortment was detected in H9N2 viruses. Antigenic analysis showed that H9N2 viruses are homogenous, antigenic drift was detected among H5N1 viruses. AI H5N8 showed higher replication rate followed by H9N2 and H5N1, respectively. H5N8 was more common in Southern Egypt, H9N2 in the Nile Delta, and H5N1 in both areas. Ducks and chickens played a significant role in transmission of H5N1 viruses. The endemicity and co-circulation of H5N1, H5N8, and H9N2 AIV coupled with the lack of a clear control strategy continues to provide avenues for further virus evolution in Egypt.
Project description:Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are considered as the most devastating poultry infections, owing to their worldwide distribution and economical threat. Vaccines have been widely used to control these diseases in the poultry industry in endemic countries. However, vaccination policy without differentiating infected animals from vaccinated animals (DIVA) makes the virus surveillance difficult. In this study, we developed a bivalent virus-like particle (VLP) vaccine that is composed of the hemagglutinin (HA) and matrix 1 (M1) proteins of the H5N1 HPAI virus (HPAIV) and a chimeric protein containing the ectodomain of the ND virus (NDV) fusion (F) protein fused with the cytoplasmic and transmembrane domains of the HPAIV HA protein. A single immunization of chickens with the chimeric VLP vaccine induced high levels of hemagglutination inhibition (HI) antibody titers against H5N1 HPAI virus and anti-NDV antibody detected in ELISA and protected chickens against subsequent lethal HPAIV and NDV infections. Furthermore, we could easily perform DIVA test using the commercial NP-cELISA tests against HPAIV and HI assay against NDV. These results strongly suggest that utilization of chimeric VLP vaccine in poultry species would be a promising strategy for the better control of HPAI and ND simultaneously.
Project description:Highly pathogenic avian influenza virus (HPAIV) is a multihost pathogen with lineages that pose health risks for domestic birds, wild birds, and humans. One mechanism of intercontinental HPAIV spread is through wild bird reservoirs, and wild birds were the likely sources of a Eurasian (EA) lineage HPAIV into North America in 2014. The introduction resulted in several reassortment events with North American (NA) lineage low-pathogenic avian influenza viruses and the reassortant EA/NA H5N2 went on to cause one of the largest HPAIV poultry outbreaks in North America. We evaluated three hypotheses about novel HPAIV introduced into wild and domestic bird hosts: (i) transmission of novel HPAIVs in wild birds was restricted by mechanisms associated with highly pathogenic phenotypes; (ii) the HPAIV poultry outbreak was not self-sustaining and required viral input from wild birds; and (iii) reassortment of the EA H5N8 generated reassortant EA/NA AIVs with a fitness advantage over fully Eurasian lineages in North American wild birds. We used a time-rooted phylodynamic model that explicitly incorporated viral population dynamics with evolutionary dynamics to estimate the basic reproductive number (R0) and viral migration among host types in domestic and wild birds, as well as between the EA H5N8 and EA/NA H5N2 in wild birds. We did not find evidence to support hypothesis (i) or (ii) as our estimates of the transmission parameters suggested that the HPAIV outbreak met or exceeded the threshold for persistence in wild birds (R0 > 1) and poultry (R0 ≈ 1) with minimal estimated transmission among host types. There was also no evidence to support hypothesis (iii) because R0 values were similar among EA H5N8 and EA/NA H5N2 in wild birds. Our results suggest that this novel HPAIV and reassortments did not encounter any transmission barriers sufficient to prevent persistence when introduced to wild or domestic birds.
Project description:BACKGROUND:Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. RESULTS:Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. CONCLUSION:These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.
Project description:Innate antiviral immunity establishes first line of defense against invading pathogens through sensing their molecular structures such as viral RNA. This antiviral potential of innate immunity is mainly attributed to a myriad of IFN-stimulated genes (ISGs). Amongst well-characterized ISGs, we have previously shown that antiviral potential of chicken IFN-induced proteins with tetratricopeptides repeats 5 (chIFIT5) is determined by its interaction potential with 5'ppp containing viral RNA. Here, we generated transgenic chickens using avian sarcoma-leukosis virus (RCAS)-based gene transfer system that constitutively and stably express chIFIT5. The transgenic chickens infected with clinical dose (EID<sub>50</sub> 10<sup>4</sup> for HPAIV and 10<sup>5</sup> EID<sub>50</sub> for vNDV) of high pathogenicity avian influenza virus (HPAIV; H5N1) or velogenic strain of Newcastle disease virus (vNDV; Genotype VII) showed marked resistance against infections. While transgenic chickens failed to sustain a lethal dose of these viruses (EID<sub>50</sub> 10<sup>5</sup> for HPAIV and 10<sup>6</sup> EID<sub>50</sub> for vNDV), a delayed and lower level of clinical disease and mortality, reduced virus shedding and tissue damage was observed compared to non-transgenic control chickens. These observations suggest that stable expression of chIFIT5 alone is potentially insufficient in providing sterile protection against these highly virulent viruses; however, it is sufficient to ameliorate the clinical outcome of these RNA viruses. These findings propose the potential of innate immune genes in conferring genetic resistance in chickens against highly pathogenic and zoonotic viral pathogens causing sever disease in both animals and humans.
Project description:Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5Nx viruses), which belong to the H5 clade 188.8.131.52, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines have been demonstrated to be efficacious and safe in poultry. Herein, we developed an NDV-based H5 vaccine (NDV-H5) that expresses a codon-optimized ectodomain of the hemagglutinin from the A/chicken/Iowa/04-20/2015 (H5N2) virus and evaluated its efficacy in chickens. Results showed that both live and inactivated NDV-H5 vaccines induced hemagglutinin inhibition antibody titers against the H5N2 virus in immunized chickens after prime and booster, and both NDV-H5 vaccines completely protected chickens from lethal challenge with the highly pathogenic H5N2 A/turkey/Minnesota/9845-4/2015 virus. No clinical signs and only minimal virus shedding was observed in both vaccinated groups. In contrast, all mock-vaccinated, H5N2-infected chickens shed virus and died within 5 days post challenge. Furthermore, one dose of the live NDV-H5 vaccine also provided protection of 90% chickens immunized by coarse spraying; after exposure to H5N2 challenge, sera from vaccinated surviving chickens neutralized both highly pathogenic H5N1 and H5N8 viruses. Taken together, our results suggest that the NDV-based H5 vaccine is able to protect chickens against intercontinental highly pathogenic H5Nx viruses and can be used by mass application to protect the poultry industry.