Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 virusus

Highly pathogenic avian influenza (HPAI) viruses of H5 clade 2.3.4.4 have spread to many countries in Asia, Europe and North America by migratory wild birds, causing outbreaks on hundreds of poultry farms. Strategies to control spread by wild birds appear limited, hence timely characterization of novel viruses is important to limit the risk for the poultry sector and human health. In this study we characterize three recent viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the H5N8 group A and B viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent virus, the H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with the group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds, and between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, showing that chickens are efficiently infected by (fecal)contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector

Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene

Anguillid herpesvirus (AnHV, also known as Herpesvirus anguillae or HVA) is found in both Japanese and European eels. Based on restriction enzyme analysis a small number of differences were found between AnHV isolated from Japanese eels and from European eels. The total genome size of both is about 245 kb, which is confirmed by alternating-field electrophoresis. Using a set of degenerate primers based on conserved regions within DNA-directed DNA polymerase coding regions, a 463 base pair fragment was isolated from both Japanese and European AnHV. Nucleotide sequence analysis showed that the cloned regions of both viruses have identical sequences. Based on this part of the DNA-polymerase sequence, primers were selected and used to develop a sensitive PCR to detect AnHV DNA in eel tissue samples. To avoid false negative results and to estimate the number of AnHV genome copies found in tissues, 100 copies of an internal control plasmid were added to the tissue samples. This semi-quantitative AnHV PCR can be used for both the European and Japanese isolates of AnHV, detects as few as 10 genome copies and is 100 times more sensitive than standard virus isolation

Genetic relationship between poultry and wild bird viruses during the highly pathogenic avian influenza H5N6 epidemic in the Netherlands, 2017–2018

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017–2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016–2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm-to-farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long-term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population.</p

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021–2022 outbreak in the Netherlands included red fox (Vulpes vulpes),polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals weresubmitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal.Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness