The influenza D virus (IDV) was first identified and characterized in 2011. Considering the virus’ zoonotic potential, its genome nature (segmented RNA virus), its worldwide circulation in livestock ...and its role in bovine respiratory disease, an increased interest is given to IDV. However, few data are available on drivers of emergence of IDV. We first listed fifty possible drivers of emergence of IDV in ruminants and swine. As recently carried out for COVID‐19 in pets (Transboundary and Emerging Diseases, 2020), a scoring system was developed per driver and scientific experts (N = 28) were elicited to (a) allocate a score to each driver, (b) weight the drivers’ scores within each domain and (c) weight the different domains among themselves. An overall weighted score was calculated per driver, and drivers were ranked in decreasing order. Drivers with comparable likelihoods to play a role in the emergence of IDV in ruminants and swine in Europe were grouped using a regression tree analysis. Finally, the robustness of the expert elicitation was verified. Eight drivers were ranked with the highest probability to play a key role in the emergence of IDV: current species specificity of the causing agent of the disease; influence of (il)legal movements of live animals (ruminants, swine) from neighbouring/European Union member states and from third countries for the disease to (re‐)emerge in a given country; detection of emergence; current knowledge of the pathogen; vaccine availability; animal density; and transport vehicles of live animals. As there is still limited scientific knowledge on the topic, expert elicitation of knowledge and multi‐criteria decision analysis, in addition to clustering and sensitivity analyses, are very important to prioritize future studies, starting from the top eight drivers. The present methodology could be applied to other emerging animal diseases.
Sub-Saharan Africa was historically considered an animal influenza cold spot, with only sporadic highly pathogenic H5 outbreaks detected over the last 20 years. However, in 2017, low pathogenic avian ...influenza A(H9N2) viruses were detected in poultry in Sub-Saharan Africa. Molecular, phylogenetic, and antigenic characterization of isolates from Benin, Togo, and Uganda showed that they belonged to the G1 lineage. Isolates from Benin and Togo clustered with viruses previously described in Western Africa, whereas viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Viruses from Benin exhibited decreased cross-reactivity with those from Togo and Uganda, suggesting antigenic drift associated with reduced replication in Calu-3 cells. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2). Therefore, viral genetic and antigenic surveillance in Africa is of paramount importance to detect further evolution or emergence of new zoonotic strains.
Genetic analysis of circulating avian influenza viruses (AIVs) in wild birds at different geographical regions during the same period could improve our knowledge about virus transmission dynamics in ...natural hosts, virus evolution as well as zoonotic potential. Here, we report the genetic and molecular characterization of H6N2 influenza viruses isolated from migratory birds in Turkey, Egypt, and Uganda during 2017-2018. The Egyptian and Turkish isolates were genetically closer to each other than they were to the virus isolated from Uganda. Our results also suggest that multiple reassortment events were involved in the genesis of the isolated viruses. All viruses contained molecular markers previously associated with increased replication and/or pathogenicity in mammals. The results of this study indicate that H6N2 viruses carried by migratory birds on the West Asian/East African and Mediterranean/Black Sea flyways have the potential to transmit to mammals including humans. Additionally, adaptation markers in these viruses indicate the potential risk for poultry, which also increases the possibility of human exposure to these viruses.
Influenza D virus (IDV) is a novel orthomyxovirus that was first isolated in 2011 in the United States from a swine exhibiting influenza‐like disease. To date, its detection is extended to all ...continents and in a broad host range: IDV is circulating in cattle, swine, feral swine, camelids, small ruminants and horses. Evidence also suggests a possible species jump to humans, underlining the issue of zoonotic potential. In Europe, serological investigations in cattle have partially allowed the understanding of the virus diffusion in different countries such as Italy, France, Luxembourg and Ireland. The infection is widespread in cattle but limited in other investigated species, consolidating the assumption of cattle as IDV primary host. We hypothesize that commercial livestock trade could play a role in the observed differences in IDV seroprevalence among these areas. Indeed, the overall level of exposure in cattle and swine in destination countries (e.g. Italy) is higher than in origin countries (e.g. France), leading to the hypothesis of a viral shedding following the transportation of young cattle abroad and thus contributing to larger diffusion at countries of destination. IDV large geographic circulation in cattle from Northern to more Southern European countries also supports the hypothesis of a viral spread through livestock trade. This review summarizes available data on IDV seroprevalence in Europe collected so far and integrates unpublished data from IDV European surveillance framework of the last decade. In addition, the possible role of livestock trade and biosecurity measures in this pathogen's spread is discussed.
Avian influenza vaccines are commonly used in the poultry industry. The objective of this study was to compare, under experimental conditions, the protective efficacy of four imported commercial ...inactivated H9N2 vaccines (A, B, C, and D) in broiler chickens. A total of 150 one-day-old chicks were divided into six groups: four experimental groups, each containing 30 chicks, received one of the vaccines (A, B, C, or D) delivered in a 0.3-ml dose subcutaneously at 1 day of age, whereas the control, Group T, was not vaccinated but challenged and Group E was kept unvaccinated and unchallenged. At 21 days postvaccination, Groups A, B, C, D, and T were challenged with 10
embryo infective dose 50% of A/Chicken/Morocco/01/2016 (H9N2). All chicks were observed daily for clinical signs during the 12 days postchallenge (dpc). At 5 and 12 dpc, chicks were euthanatized for necropsy examination. Blood samples were collected weekly for serologic analysis and oropharyngeal swabs were collected for virus detection by real-time RT-PCR. Respiratory signs started at 48 hr pc and maximum severity was observed on 9 dpc. Chiefly, the birds vaccinated with vaccine B showed significantly more respiratory signs than did their counterparts. Serologic analysis revealed that the sera of Groups A and D birds showed a decrease in antibody (Ab) levels up to day 26; then a slight increase of Ab level was observed until day 31, while Group B and C birds showed a stabilization of the titers from day 21 until the end of the experiment. The viral shedding rate was significantly lower in Groups C and A (40%-50% of the birds shed virus for <7 days) compared with other challenged groups (60%-75% of the birds shed virus for ≥9 days). This experiment illustrated that vaccination applied on the first day in the hatchery with the four vaccines tested did not provide an acceptable protection against H9N2 in comparison with the controls that did not receive any vaccine. However, at first glance, we might favor vaccines A and C for their ability to reduce and shorten viral shedding as compared with vaccines B and D.
A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic ...evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.
Reservoirs for the continuing influenza (H5N1) outbreaks in Egypt are ill-defined. Through active surveillance, we detected highly pathogenic influenza subtype H5 viruses in all poultry sectors; ...incidence was 5%. No other subtypes were found. Continued circulation of influenza (H5N1) viruses in various regions and poultry sectors perpetuates human exposure in Egypt.
Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has ...resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses' vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.
The influenza D virus (IDV) was discovered less than ten years ago. Increased interest in this virus is due to its nature (RNA virus with high mutation rate), its worldwide circulation in livestock ...species, its probable role in bovine respiratory disease and its zoonotic potential. Until currently, the establishment of positivity cut‐off of the hemagglutination inhibition (HI) assay was not formalized in field conditions for the detection of antibodies directed against IDV in cattle (i.e. the proposed reservoir). In this study, the positivity cut‐off of the HI assays was formally established (titre = 10) using a receiver operating characteristic (ROC) curve. This information was used to estimate the sensitivity (68.04 to 73.20%) and the specificity (94.17 to 96.12%) of two different HI assays (HI1 and HI2, with two different IDV antigens) relatively to virus micro‐neutralization test (VNT) as reference test. Based on the above characteristics, the true prevalence of IDV was then estimated in Morocco using a stochastic approach. Irrespective of the HI assays used, the estimation of the true prevalence was statistically equivalent (between 48.44% and 48.73%). In addition, the Spearman rank correlation between HI titres and VNT titres was statistically good (0.76 and 0.81 for HA1 and HA2, respectively). The positive (0.82 and 0.79 for HA1 and HA2, respectively) and the negative (0.86 and 0.85 for HA1 and HA2, respectively) agreement indices between results of HI assays and VNT were good and similar. This study allowed for a formal establishment of a positivity cut‐off in HI assays for the detection of antibodies directed against IDV. This information is of prime importance to estimate the diagnostic sensitivity and specificity of the test relatively to the VNT (i.e. the reference test). Using these characteristics, the true prevalence of IDV should be determined in a country.
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