Determining highly pathogenic H5 avian influenza clade 2.3.2.1c seroprevalence in ducks, Purbalingga, Central Java, Indonesia

Background and Aim: In Indonesia, highly pathogenic avian influenza (HPAI) H5N1 outbreaks in poultry are still reported. The disease causes a decrease in egg production and an increase in mortality; this has an impact on the economic losses of farmers. Several studies have considered that ducks play a role in the HPAI endemicity in the country; however, little is known about whether or not the type of duck farming is associated with HPAI H5 virus infection, particularly within clade 2.3.2.1c, which has been predominantly found in poultry since 2014. A cross-sectional study was conducted to determine the HPAI seroprevalence for H5 subtype clade 2.3.2.1c in laying ducks that are kept intensively and nomadically and to determine the associated risk factors. Materials and Methods: Forty-nine duck farmers were randomly selected from ten sub-districts in Purbalingga District, Central Java, Indonesia; a cross-sectional study was implemented to collect field data. Based on an expected HPAI prevalence level of 10%, estimated accuracy of ± 5%, and 95% confidence interval (CI), the total sample size was calculated at 36 individuals. Samples must be multiplied by 7 to reduce bias; thus, 252 ducks were taken as samples in this study. Considering that the maintenance and duck handling were uniform and farmers complained that the effect of activity to take duck samples would reduce egg production, this study only took samples from 245 ducks (oropharyngeal swabs and serum). Those samples were taken from five birds on each farm. Hemagglutination inhibition tests examined the serum samples for HPAI H5 Clade 2.3.2.1c, and pool swab samples (five swabs in one viral media transport) were examined by real-time reverse transcription-polymerase chain reaction (qRT-PCR) test for influenza Type A and H5 subtype virus. Information regarding farm management was obtained using a questionnaire; face-to-face interviews were conducted with the duck farmers using native Javanese language. Results: Serum and swabs from 245 ducks were collected in total. For individual birds, 54.69% (134/245) of serum samples were H5 seropositive. Seroprevalence among nomadic ducks was 59.28% (95% CI: 0.48-0.61), which was higher than among intensively farmed ducks (48.57%, 95% CI: 0.38-0.58). Farm-level seroprevalence was 50% (95% CI: 0.30-0.69) for nomadic ducks but only 28.57% (95% CI: 0.11-0.51) for intensively farmed ducks. The farm-level virus prevalence (proportion of flocks with at least one bird positive for influenza Type A) was 17.85% (95% CI: 0.07-0.35) for nomadic ducks and 4.76% (1/21) for intensively farmed ducks (95% CI: 0.008-0.23). All influenza Type A positive samples were negative for the H5 subtype, indicating that another HA subtype AI viruses might have been circulating in ducks in the study area. A relationship between duck farms that were H5 seropositive and their maintenance system was present; however, this relationship was not significant, the nomadic duck system detected 2 times higher H5-seropositive ducks than the intensive farming system (OR: 2.16, 95% CI: 0.33-14.31). Conclusion: This study found that the seroprevalence of HPAI in the duck population level in Purbalingga was 54.69% and demonstrated that the nomadic duck farming system was more likely to acquire HPAI H5 infection than the intensive farming duck system. Other risk factors should be further investigated as the diversity of the farming system is partially related to HPAI H5 infection.

Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity

Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

Identification the Presence of Clade 2.3.2.1c-Avian Influenza H5N1, a Highly Pathogenic Virus in Iraq, 2018

For the first time in Iraq, we identified in March, 2018 the presence of a highly virulent avian influenza virus (AIV), H5N1 (Clade 2.3.2.1c), causing highly pathogenic avian influenza (HPAI) in poultry farms, Iraq,. The identification of the virus was done using a rapid serological test, a real time-qPCR, and glycoprotein gene sequencing. Using sequencing and phylogenetic analyses, the clade 2.3.2.1c virus was recorded to be clustered, with high similarity to Asian and West African AIV, HPAI H5N1 from Ivory Coast identified in 2015. According to our knowledge, there was no previous detection of the clade 2.3.2.1c made in Iraq. Our results provide evidence that high risk of HPAI H5 outbreaks might be present in Iraq, and this needs to lead to high quality surveillance targeting of wild and domestic birds for early diagnosis of HPAI. The current work provides feasible and accurate approaches for understanding the evolution of HPAI H5 virus in different countries around the world.

Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity

Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A [A(H5N1)] viruses have evolved to clade 2.3.2.1a, b and c, and currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

First Detection of a Novel Reassortant Avian Influenza A(H5N6) Clade 2.3.2.1c Virus, Isolated from a Wild Bird in China

We report the first isolation of a reassortant clade 2.3.2.1c avian influenza A(H5N6) virus isolated from a wild bird sample in Jiangxi, China, in 2016. Sequence analyses indicated that this virus most likely evolved from Eurasia-derived H5N1 and H6N6 viruses through frequent interactions at the wild-domestic bird interface.

DESIGNING AND CLONING NA GENE OF INFLUENZA A/H5N1 VIRUS INTO pHW2000 VECTOR FOR PREPARATION OF A CANDIDATE VACCINE MASTERSEED STRAIN

The influenza A/H5N1 virus is an RNA virus belonging to the family of Orthomyxoviridae. The highly pathogenic influenza A/H5N1 virus exhibit the ability to cause high mortality in poultry and infect humans. Technology for vaccine seed strain production of influenza A virus using reverse genetics requires the creation of recombinant vectors carrying viral genomic segments. To create recombinant pHW2000 vectors containing the neuraminidase (NA) gene segment encoding an important surface antigen of influenza A virus, two N1 NA gene structures were designed based on the NA gene sequences of two subtypes of highly pathogenic influenza A/H5N1 clade (clade 1.1 and clade 2.3.2.1c) and then inserted into pHW2000 vector. These two clades of highly pathogenic avian influenza viruses that are still circulating in Vietnam, with antigen homology and genetic relationships to many strains of influenza A viruses, have been suggested to be used for producing vaccines against emerging avian influenza A/H5N1 virus. Each NA gene construct consists of 1453 nucleotides in which two ends of the gene are two non-coding regions (46 nucleotides and 57 nucleotides) containing primer binding site and cleavage site of BsaI. In the middle of each NA gene is one region of 1350 nucleotides encoding 449 amino acids, ensuring catalytic function and antigenicity of NA protein. Two NA segments corresponding to the two clades of influenza A viruses were successfully cloned into pHW2000 vectors for the generation of two recombinant vectors pHW2000-NA clade 1.1 and pHW2000-NA clade 2.3.2.1c. These recombinant vectors will be used for production of candidate avian influenza vaccine strains using reverse genetics technique.