Limited Genetic Diversity Detected in Middle East Respiratory Syndrome-Related Coronavirus Variants Circulating in Dromedary Camels in Jordan.

Middle East respiratory syndrome-related coronavirus (MERS-CoV) is a persistent zoonotic pathogen with frequent spillover from dromedary camels to humans in the Arabian Peninsula, resulting in limited outbreaks of MERS with a high case-fatality rate. Full genome sequence data from camel-derived MERS-CoV variants show diverse lineages circulating in domestic camels with frequent recombination. More than 90% of the available full MERS-CoV genome sequences derived from camels are from just two countries, the Kingdom of Saudi Arabia (KSA) and United Arab Emirates (UAE). In this study, we employ a novel method to amplify and sequence the partial MERS-CoV genome with high sensitivity from nasal swabs of infected camels. We recovered more than 99% of the MERS-CoV genome from field-collected samples with greater than 500 TCID50 equivalent per nasal swab from camel herds sampled in Jordan in May 2016. Our subsequent analyses of 14 camel-derived MERS-CoV genomes show a striking lack of genetic diversity circulating in Jordan camels relative to MERS-CoV genome sequences derived from large camel markets in KSA and UAE. The low genetic diversity detected in Jordan camels during our study is consistent with a lack of endemic circulation in these camel herds and reflective of data from MERS outbreaks in humans dominated by nosocomial transmission following a single introduction as reported during the 2015 MERS outbreak in South Korea. Our data suggest transmission of MERS-CoV among two camel herds in Jordan in 2016 following a single introduction event.

Efficacy of Bivalent Inactivated Vaccine Containing Insect Cell-Expressed Avian Influenza H5 and Egg-Based Newcastle Disease Virus (NDV) Against Dual Infection with Highly Pathogenic H5N1 and Velogenic NDV in Chickens.

The genetic evolution of highly pathogenic avian influenza (HPAI) in Egypt has developed a new clade H5N1 (2.2.1.2) since 2014. Meanwhile, the new avian influenza virus (AIV) clade mutually with the velogenic Newcastle disease virus (NDV) isolate of genotype VII in Egypt (genotype VII) has resulted in severe economic losses in the broiler industry. An inactivated bivalent vaccine containing H5 (belonging to H5N1 clade 2.3.2) recombinant baculovirus expressed by insect cell (recH5) and egg-based NDV LaSota strain (recH5/NDV vaccine) was evaluated for protection against the challenge of dual HPAIV H5N1 clade 2.2.1.2 and vNDV infection in commercial broiler chickens. Vaccination was performed when chickens were 10 days old, and then birds of the respective groups were challenged with 106 50% egg infective dose per chicken of each virus in 100 µl of allantoic fluid via the intranasal route at 21 days postvaccination in a single or sequential infection of both viruses. Results showed that the recH5/NDV vaccine was able to protect chickens against single or dual challenges of both viruses ranging up to 90%-100%. Unvaccinated chickens have demonstrated 100% mortalities to a single virus challenge. Vaccinated chickens showed significant decreases in both viruses, shedding titers up to <2 log 10 after challenge in comparison with unvaccinated ones. Cessation of viral shedding was obtained at 7 to 10 days postchallenge. The vaccinated chickens showed high hemagglutination inhibition antibody titers >6 log 2 against both H5N1 and NDV antigens at 2 wk postvaccination. The single vaccination of bivalent inactivated recH5-NDV vaccine at 10 days old in commercial chickens has provided significant clinical protective immunity against single or dual challenge with HPAI-H5N1clade 2.2.1.2 and vNDV-genotype VII.

Eficacia en pollos de una vacuna inactivada bivalente que contiene la proteína H5 del virus de influenza aviar H5 expresada en células de insecto y el virus de la enfermedad de Newcastle replicado en embrión de pollo contra la infección dual con un virus de influenza aviar H5N1 altamente patogénico y un virus de la enfermedad de Newcastle velogénico. La evolución genética de la influenza aviar altamente patógena (HPAI) en Egipto ha desarrollado un nuevo clado H5N1 (2.2.1.2) desde el año 2014. Mientras tanto, el nuevo clado del virus de la influenza aviar (AIV) junto con aislados del virus de la enfermedad de Newcastle velogénicos de genotipo VII en Egipto ha provocado graves pérdidas económicas para la industria de pollos de engorde. Una vacuna bivalente inactivada que contiene la proteína H5 (perteneciente al clado H5N1 2.3.2) expresada en un baculovirus recombinante y replicado células de insecto (recH5) junto con la cepa LaSota del virus de Newcastle replicada en embriones de pollo (vacuna recH5/NDV) fue evaluada en la protección contra el desafío doble con un virus de influenza aviar de alta patogenicidad H5N1 clado 2.2.1.2 y contra un virus virulento de la enfermedad de Newcastle en pollos de engorde comerciales. La vacunación se realizó cuando los pollos tenían diez días de edad y luego las aves de los grupos respectivos fueron desafiadas con 106 dosis infectivas para embrión de pollo al 50% por pollo de cada virus en 100 µl de líquido alantoideo a través de la vía intranasal a los 21 días posteriores a la vacunación en una sola infección o con infección secuencial con ambos virus. Los resultados mostraron que la vacuna recH5/NDV fue capaz de proteger a los pollos contra los desafíos simples o dobles con ambos virus con un rango que va del 90% al 100%. Los pollos no vacunados mostraron 100% de mortalidad ante el desafío simple con un solo virus. Los pollos vacunados mostraron disminuciones significativas en la eliminación de ambos virus, arrojando títulos menores de 2 log10 después del desafío, en comparación con las aves no vacunadas. El cese de la propagación viral se observó de los siete a los diez días posteriores al desafío. Los pollos vacunados mostraron altos títulos de anticuerpos por inhibición de la hemaglutinación con títulos mayores de 6log2 contra los antígenos H5N1 y NDV a las dos semanas posteriores a la vacunación. La inmunización única con la vacuna recH5/NDV inactivada bivalente a los 10 días de edad en pollos comerciales proporcionó una inmunidad protectora clínica significativa contra el desafío simple o doble con un virus de influenza aviar H5N1clado 2.2.1.2 y por un virus virulento de la enfermedad de Newcastle genotipo VII.

Isolation, characterization, and antibiotic sensitivity assessment of Gallibacterium anatis biovar haemolytica, from diseased Egyptian chicken flocks during the years 2013 and 2015.

Gallibacterium anatis biovar haemolytica constitutes a part of the normal microflora in the upper respiratory and genital tracts of healthy chickens, but it is also associated with different pathological conditions. In the current study, 102 commercial chicken flocks suffering from respiratory disease and/or drop in egg production were investigated for the presence of G. anatis during 2013 and 2015. These flocks comprised 8 breeder, 32 layer, and 62 broiler flocks. By culture method, 20 flocks were found positive: one isolate derived from broiler breeders, 6 isolates from layers, and 13 isolates from broilers. G. anatis biovar haemolytica was identified by phenotyping and PCR. Additionally, partial genome sequencing of 11 isolates (5 layer isolates of 2013 and 6 broiler isolates of 2015) based on 16S rRNA and 23S rRNA gene sequences was performed and revealed 96.5% to 100% genetic relatedness. Antibiotic sensitivity of these isolates revealed that the 2013 isolates were highly susceptible to florfenicol while the isolates of 2015 were highly susceptible to cefotaxime. Gallibacterium anatis biovar haemolytica is a newly introduced bacteria in Egypt causing salpingitis, peritonitis, drop in egg production, and/or respiratory signs.

High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan.

Prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) was determined in 45 dromedary camels from two geographically separated herds in Jordan. Virus shedding was only detected in swabs obtained from the respiratory tract and primarily observed in camels younger than 3 years. MERS-CoV seroprevalence increased with age of camels. Bovine and sheep sera were seronegative. Phylogenetic analysis of partial S2 clustered the Jordanian MERS-CoV strains with contemporary MERS-CoV strains associated with nosocomial outbreaks.

Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry.

Highly pathogenic avian influenza (HPAI) H5N1 (clade 2.2) was introduced into Egypt in early 2006. Despite the control measures taken, including mass vaccination of poultry, the virus rapidly spread among commercial and backyard flocks. Since the initial outbreaks, the virus in Egypt has evolved into a third order clade (clade 2.2.1) and diverged into antigenically and genetically distinct subclades. To better understand the dynamics of HPAI H5N1 evolution in countries that differ in vaccination policy, we undertook an in-depth analysis of those virus strains circulating in Egypt between 2006 and 2010, and compared countries where vaccination was adopted (Egypt and Indonesia) to those where it was not (Nigeria, Turkey and Thailand). This study incorporated 751 sequences (Egypt n=309, Indonesia n=149, Nigeria n=106, Turkey n=87, Thailand n=100) of the complete haemagglutinin (HA) open reading frame, the major antigenic determinant of influenza A virus. Our analysis revealed that two main Egyptian subclades (termed A and B) have co-circulated in domestic poultry since late 2007 and exhibit different profiles of positively selected codons and rates of nucleotide substitution. The mean evolutionary rate of subclade A H5N1 viruses was 4.07×10(-3) nucleotide substitutions per site, per year (HPD 95%, 3.23-4.91), whereas subclade B possessed a markedly higher substitution rate (8.87×10(-3); 95% HPD 7.0-10.72×10(-3)) and a stronger signature of positive selection. Although the direct association between H5N1 vaccination and virus evolution is difficult to establish, we found evidence for a difference in the evolutionary dynamics of H5N1 viruses among countries where vaccination was or was not adopted. In particular, both evolutionary rates and the number of positively selected sites were higher in virus populations circulating in countries applying avian influenza vaccination for H5N1, compared to viruses circulating in countries which had never used vaccination. We therefore urge a greater consideration of the potential consequences of inadequate vaccination on viral evolution.

Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications.

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.

Evaluation of the protection induced by avian influenza vaccines containing a 1994 Mexican H5N2 LPAI seed strain against a 2008 Egyptian H5N1 HPAI virus belonging to clade 2.2.1 by means of serological and in vivo tests.

Since 2006 Egypt has been facing an extensive epidemic of H5N1 highly pathogenic avian influenza (HPAI) with a huge number of outbreaks both in rural and intensively reared poultry areas. The use of efficacious vaccines in this country has been, and still remains, essential for the control and possible eradication of HPAI. The present study was performed to establish whether the administration of inactivated vaccines containing an H5 virus belonging to a different lineage to the Eurasian H5N1 HPAI viruses guarantees protection from clinical signs, provides significant immune response and is able to achieve a reduction of viral shedding in the face of a challenge with a contemporary H5N1 virus isolated in Egypt. Despite the genetic and antigenic differences between the vaccine strain (H5N2/Mexico) and the challenge strain (H5N1/Egypt), confirmed by molecular and serological (haemagglutination inhibition) tests, it was established that the immune response induced by these conventional vaccines is sufficient to prevent infection in the majority of birds challenged with a contemporary H5N1 Egyptian strain. The data reported in this study also indicate that there may be a low degree of correlation between haemagglutination inhibition titres, clinical protection and reduction of shedding.

Seroprevalence of, and risk factors for, peste des petits ruminants in sheep and goats in Northern Jordan.

Peste des petits ruminants (PPR) is an economically important disease that affect sheep and goat industry in Asia and Africa. In this study, we investigated the seroprevalence, and risk factors, of PPR in sheep and goat flocks from five different governorates (Irbid, Jarash, Ajloun, Mafraq and Zarka) located in Northern Jordan. Serum samples from 929 and 400 sheep and goats, respectively, corresponding to 122 sheep flock and 60 goats flock were collected. Seroprevalence was determined using PPR competitive ELISA. Health status and management information were collected using a semi-structured pre-tested questionnaire. The individual true prevalence of PPR in sheep and goats was 29 and 49%, respectively. The flock level true prevalence of PPR was 60 and 74% in sheep and goats, respectively. In both sheep and goat flocks, large flock size, visiting live animals market and inadequate veterinary services were identified as risk factors for PPR seropositivity. Mixed (sheep and goats) raising was identified as a risk factor for PPR seropositivity in sheep flocks only.

Genetic comparison of H9N2 AI viruses isolated in Jordan in 2003.

SUMMARY. During the 1990s, several outbreaks of avian influenza (AI), caused by viruses of the H9N2 subtype, were reported in poultry in various parts of the world. Currently, this infection seems to be endemic in poultry in the Middle and Far East, and the extensive circulation of H9N2 in poultry represents a risk factor for infection of humans, because viruses of this subtype have been sporadically introduced into the human population. Little is known about the gene constellation of the H9N2 viruses that are currently circulating in the Middle East; thus, gene sequences of eight IA viruses of the H9N2 subtype isolated in Jordan in 2003 from poultry were analyzed. The results of this investigation show that all eight Jordanian isolates are closely related to each other and to other H9N2 isolates from the Middle East. Seven of eight genes of the Jordanian strains show a percentage of homology not higher than 95% with the genes of two H9N2 strains, A/HK/1073/99 and A/HK/1074/99, isolated from humans in Hong Kong. The M gene is more closely related to the corresponding gene of the two H9N2 human isolates from Hong Kong (97.7-98.2% homology). This homology suggests that the M gene of the Jordanian and human Hong Kong strains could derive from a common ancestor.