Efficacy of a Turkey Herpesvirus Vectored Newcastle Disease Vaccine against Genotype VII.1.1 Virus: Challenge Route Affects Shedding Pattern.

The control of Newcastle disease (ND) highly relies on vaccination. Immunity provided by a ND vaccine can be characterized by measuring the level of clinical protection and reduction in challenge virus shedding. The extent of shedding depends a lot on the characteristics of vaccine used and the quality of vaccination, but influenced also by the genotype of the challenge virus. We demonstrated that vaccination of SPF chicks with recombinant herpesvirus of turkey expressing the F-gene of genotype I ND virus (rHVT-ND) provided complete clinical protection against heterologous genotype VII.1.1 ND virus strain and reduced challenge virus shedding significantly. 100% of clinical protection was achieved already by 3 weeks of age, irrespective of the challenge route (intra-muscular or intra-nasal) and vaccination blocked cloacal shedding almost completely. Interestingly, oro-nasal shedding was different in the two challenge routes: less efficiently controlled following intra-nasal than intra-muscular challenge. Differences in the shedding pattern between the two challenge routes indicate that rHVT-ND vaccine induces strong systemic immunity, that is capable to control challenge virus dissemination in the body (no cloacal shedding), even when it is a heterologous strain, but less efficiently, although highly significantly (p < 0.001) suppresses the local replication of the challenge virus in the upper respiratory mucosa and consequent oro-nasal shedding.

Combined H5ND inactivated vaccine protects chickens against challenge by different clades of highly pathogenic avian influenza viruses subtype H5 and virulent Newcastle disease virus.

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 2.2.1.1), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 2.2.1.2), 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, 2.2.1.1, 2.2.1.2, 2.3.4.4b-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-2.3.4.4b 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.

Potential Biological and Climatic Factors That Influence the Incidence and Persistence of Highly Pathogenic H5N1 Avian Influenza Virus in Egypt.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

Multiple introductions of reassorted highly pathogenic avian influenza viruses (H5N8) clade 2.3.4.4b causing outbreaks in wild birds and poultry in Egypt.

Recently, an increased incidence of outbreaks of highly pathogenic avian influenza (HPAI) H5N8 in poultry linked to infected migratory birds has been reported from different European, Asian and African countries. In Egypt, incursion of HPAI H5N8 virus of clade 2.3.4.4b has been recently registered. Full genomic characterization of 3 virus isolates from wild birds and poultry (backyard and commercial farm sectors) showed high nucleotide similarity among the HA, NA, M, and NS gene segments of the three Egyptian HPAI H5N8 viruses, indicating that they are descendants of a common ancestral virus. However, the analyzed Egyptian H5N8 viruses revealed distinct genotypes involving different origins of the PB2, PB1, PA and/or NP segments. In genotype-1 represented by strain A/common-coot/Egypt/CA285/2016 the PB2 and NP segments showed closest relationship to H5N6 and H6N2 viruses, recently detected in Italy. The second is replacement of PB1 and NP genes A novel reassortant, represented by strain A/duck/Egypt/SS19/2017, showed an exchange of PB1 and NP genes which might have originated from H6N8 or H1N1 and H6N2 viruses. Finally, replacement of PA and NP genes characterized strain A/duck/Egypt/F446/2017. Bayesian phylogeographic analyses revealed that Egyptian H5N8 viruses are highly likely derived from Russian 2016 HPAI H5N8 virus (A/great_crested_grebe/Uvs-Nuur_Lake/341/2016 (H5N8)) and the reassortment likely occurred before incursion to Egypt.

Highly Pathogenic Avian Influenza Virus (H5N8) Clade 2.3.4.4 Infection in Migratory Birds, Egypt.

We isolated highly pathogenic avian influenza virus (H5N8) of clade 2.3.4.4 from the common coot (Fulica atra) in Egypt, documenting its introduction into Africa through migratory birds. This virus has a close genetic relationship with subtype H5N8 viruses circulating in Europe. Enhanced surveillance to detect newly emerging viruses is warranted.

Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt.

In Egypt, since 2006, descendants of the highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 continue to cause sharp losses in poultry production and seriously threaten public health. Potentially zoonotic H9N2 viruses established an endemic status in poultry in Egypt as well and co-circulate with HP AIV H5N1 rising concerns of reassortments between H9N2 and H5N1 viruses along with an increase of mixed infections of poultry. Nucleotide sequences of whole genomes of 15 different isolates (H5N1: 7; H9N2: 8), and of the hemagglutinin (HA) and neuraminidase (NA) encoding segments of nine further clinical samples (H5N1: 2; H9N2: 7) from 2013 and 2014 were generated and analysed. The HA of H5N1 viruses clustered with clade 2.2.1 while the H9 HA formed three distinguishable subgroups within cluster B viruses. BEAST analysis revealed that H9N2 viruses are likely present in Egypt since 2009. Several previously undescribed substituting mutations putatively associated with host tropism and virulence modulation were detected in different proteins of the analysed H9N2 and H5N1 viruses. Reassortment between HP AIV H5N1 and H9N2 is anticipated in Egypt, and timely detection of such events is of public health concern. As a rapid tool for detection of such reassortants discriminative SYBR-Green reverse transcription real-time PCR assays (SG-RT-qPCR), targeting the internal genes of the Egyptian H5N1 and H9N2 viruses were developed for the rapid screening of viral RNAs from both virus isolates and clinical samples. However, in accordance to Sanger sequencing, no reassortants were found by SG-RT-qPCR. Nevertheless, the complex epidemiology of avian influenza in poultry in Egypt will require sustained close observation. Further development and continuing adaptation of rapid and cost-effective screening assays such as the SG-RT-qPCR protocol developed here are at the basis of efforts for improvement the currently critical situation.