Low Pathogenic Avian Influenza Strains Research Articles

Genotype Diversity, Wild Bird-to-Poultry Transmissions, and Farm-to-Farm Carryover during the Spread of the Highly Pathogenic Avian Influenza H5N1 in the Czech Republic in 2021/2022.

In 2021/2022, the re-emergence of highly pathogenic avian influenza (HPAI) occurred in Europe. The outbreak was seeded from two sources: resident and reintroduced viruses, which is unprecedented in the recorded history of avian influenza. The dominant subtype was H5N1, which replaced the H5N8 subtype that had predominated in previous seasons. In this study, we present a whole genome sequence and a phylogenetic analysis of 57 H5N1 HPAI and two low pathogenic avian influenza (LPAI) H5N1 strains collected in the Czech Republic during 2021/2022. Phylogenetic analysis revealed close relationships between H5N1 genomes from poultry and wild birds and secondary transmission in commercial geese. The genotyping showed considerable genetic heterogeneity among Czech H5N1 viruses, with six different HPAI genotypes, three of which were apparently unique. In addition, second-order reassortment relationships were observed with the direct involvement of co-circulating H5N1 LPAI strains. The genetic distance between Czech H5N1 HPAI and the closest LPAI segments available in the database illustrates the profound gaps in our knowledge of circulating LPAI strains. The changing dynamics of HPAI in the wild may increase the likelihood of future HPAI outbreaks and present new challenges in poultry management, biosecurity, and surveillance.

Major difference in clinical outcome and replication of a H3N1 avian influenza strain in young pullets and adult layers

ABSTRACTIn this study, we investigated the pathogenicity, replication and tropism of the low pathogenic avian influenza (LPAI) strain A/chicken/Belgium/460/2019(H3N1) in adult SPF layers and young SPF males. The inoculated hens showed 58% mortality and a 100% drop in egg production in the second week post inoculation. The high viral loads in the cloacal samples coincided with the period of the positive immunohistochemistry of the oviduct, acute peritonitis and time of mortality, suggesting that the replication of H3N1 in the oviduct was a major component of the onset of clinical disease and increased level of excretion of the virus. In the inoculated young birds, the clinical signs were very mild with the exception of one bird. The results suggest that the time of replication of the virus was much shorter than in the adult layers; some of the young males did not show any proof of being infected at all. To conclude, the results of the study in young birds confirmed the intravenous pathogenicity test results but also showed that the clinical signs in adult layers were very severe. Based on the mortality without a bacterial component, complete drop of egg production and post mortem findings, this H3N1 strain is a moderately virulent strain, the highest category for LPAI strains. It is important to realize that if HPAI did not exist, this moderately virulent H3N1 virus would most likely to be considered as a very virulent virus.

Time-space analysis of highly pathogenic avian influenza H5N2 outbreak in the US.

BackgroundIn early 2015, highly pathogenic avian influenza H5N2 caused outbreaks in commercial poultry farms in Minnesota and neighboring states where more than 48 million birds were affected. To date, the origin and transmission pathways of HPAI H5N2 have not been conclusively established.MethodsIn this study, we analyzed forty-six samples from turkeys and their environment that were collected at different time-points of the outbreak to identify origins and within outbreak evolutionary changes. We performed de-novo whole genome sequencing from primary samples and the most recent common ancestors of the PB2, PA, HA5, M and NS segments were traced back to Japanese HPAI H5N8 isolates. These segments appeared to have diverged from the ancestor around June and November 2014.ResultsThe time to most recent common ancestor analysis for PB1, NP and NA2 segments suggest two likely possibilities of reassortant HPAI H5N2 origin - either a reassortment in Alaska area or multiple reassortments with North American low pathogenic avian influenza strains, before the HPAI H5N2 outbreak strain emerged. Within the outbreak, viruses clustered into two and three subgroups suggesting high substitution rates of 0.702x10-2 - 1.665x10-2 (subs/site/year), over the 5-month outbreak period.ConclusionsData are suggestive of a fast evolving HPAI strain within an outbreak that should be taken into consideration in developing appropriate control strategies in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0605-4) contains supplementary material, which is available to authorized users.

Susceptibility of swine to H5 and H7 low pathogenic avian influenza viruses

BackgroundThe ability of pigs to become infected with low pathogenic avian influenza (LPAI) viruses and then generate mammalian adaptable influenza A viruses is difficult to determine. Yet, it is an important link to understanding any relationship between LPAI virus ecology and possible epidemics among swine and/or humans.ObjectivesAssess susceptibility of pigs to LPAI viruses found within the United States and their direct contact transmission potential.MethodsPigs were inoculated with one of ten H5 or H7 LPAI viruses selected from seven different bird species to test infectivity, virulence, pathogenesis, and potential to transmit virus to contact pigs through histological, RRT‐PCR and seroconversion data.ResultsAlthough pigs were susceptible to infection with each of the LPAI viruses, no clinical disease was recognized in any pig. During the acute phase of the infection, minor pulmonary lesions were found in some pigs and one or more pigs in each group were RRT‐PCR‐positive in the lower respiratory tract, but no virus was detected in upper respiratory tract (negative nasal swabs). Except for one group, one or more pigs in each LPAI group developed antibody. No LPAI viruses transmitted to contact pigs.Conclusions LPAI strains from various bird populations within the United States are capable of infecting pigs. Although adaptability and transmission of individual strains seem unlikely, the subclinical nature of the infections demonstrates the need to improve sampling and testing methods to more accurately measure incidence of LPAI virus infection in pigs, and their potential role in human‐zoonotic LPAI virus dynamics.

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China

ObjectiveTo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. MethodsRecombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses. ResultsThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. ConclusionThe 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Highly pathogenic avian influenza outbreak mitigated by seasonal low pathogenic strains: Insights from dynamic modeling

The spread of highly pathogenic avian influenza (HPAI) H5N1 remains a threat for both wild and domestic bird populations, while low pathogenic avian influenza (LPAI) strains have been reported to induce partial immunity to HPAI in poultry and some wild birds inoculated with both HPAI and LPAI strains. Here, based on the reported data and experiments, we develop a two-strain avian influenza model to examine the extent to which this partial immunity observed at the individual level can affect the outcome of the outbreaks among migratory birds in the wild at the population level during different seasons. We find a distinct mitigating effect of LPAI on the death toll induced by HPAI strain, and this effect is particularly important for populations previously exposed to and recovered from LPAI. We further investigate the effect of the dominant mode of transmission of an HPAI strain on the outcome of the epidemic. Four combinations of contact based direct transmission and indirect fecal-to-oral (or environmental) routes are examined. For a given infection peak of HPAI, indirect fecal-to-oral transmission of HPAI can lead to a higher death toll than that associated with direct transmission. The mitigating effect of LPAI can, in turn, be dependent on the route of infection of HPAI.

Achievement of avian influenza virus-like particles that could be used as a subunit vaccine against low-pathogenic avian influenza strains in ducks

Infections with H5/H7 low-pathogenic avian influenza (LPAI) viruses are now notifiable because such viruses can mutate into highly pathogenic avian influenza viruses, leading to serious problems for both animal and public health. Domestic ducks can play a crucial role in the transmission of H5 LPAI viruses to other poultry. Although prime boost vaccination using, respectively, a recombinant vaccine and an inactivated vaccine was shown to be protective in ducks against H5N1 highly pathogenic avian influenza, vaccination of domestic ducks against H5 LPAIV is poorly documented. However, substituting inactivated vaccines with subunit vaccines might be more advantageous. In this context, we generated a triple recombinant baculovirus composed of HA and NA proteins derived from a French H5N3 LPAI virus strain and the M protein derived from an Italian H7N1 LPAI virus strain. We describe a molecular construction strategy that enabled the development of virus-like particles (VLPs). Western blot analyses and neuraminidase inhibition assay of cell supernatants purified by sucrose density gradient ultracentrifugation showed that HA, NA and M1 proteins were expressed and co-released. Electron microscopy examination revealed VLPs that were morphologically identical to wild-type virus. Immunogold electron microscopy demonstrated that H5 and N3 proteins were present on the VLP surface, and haemagglutination and neuraminidase assays showed that the H and N proteins, respectively, were biologically active. In addition, VLP immunogenicity (induction of haemagglutination-inhibiting antibodies) was demonstrated in specific pathogen free Muscovy ducks. According to our successful previous experimental results of protection in ducks following vaccination with the three baculovirus-expressed proteins, the present results make feasible the reliable use of H5N3 VLPs as a subunit vaccine in this species.

Discrimination between Highly Pathogenic and Low Pathogenic H5 Avian Influenza A Viruses

To the Editor: To thoroughly investigate avian influenza outbreaks, identifying highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) is essential. Currently, determination of inserted basic amino acids within the hemagglutinin cleavage site of HPAI relies on nucleotide sequencing (1–3). Direct sequencing is relatively time-consuming and laborious and thus is not suitable for local and regional diagnostic laboratories that receive large numbers of samples that may contain HPAI or LPAI subtype H5N1. Therefore, a rapid diagnostic assay was developed to discriminate between HPAI and LPAI subtype H5 viruses by 1-step real-time reverse transcription–polymerase chain reaction (RT-PCR) with melting curve analysis. H5 primers flanking the cleavage site were designed from conserved regions among HPAI and LPAI strains by using nucleotide sequences obtained from GenBank and our previous studies (4–6). The primers consisted of a forward primer H5F3+ (nucleotides 1001–1021: 5´-AACAGATTAGTCCTTGCGACTG-3´) and a reverse primer H5R2+ (nucleotides 1124–1103: 5´-CATCTACCATTCCCTGCCATCC-3´), which yielded products of ≈124 bp and ≈112 bp, corresponding to HPAI and LPAI, respectively. Consequently, the sizes of the amplicons and percentage guanine-cytosine content were different, allowing discrimination between HPAI and LPAI by melting curve analysis (7). One-step, real-time RT-PCR with melting curve analysis was performed in an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). In each reaction, 3.0 μL of RNA sample was combined with a reaction mixture containing 10 μL 2× SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μL 40× MultiScribe (Applied Biosystems) and RNase inhibitor, each primer (at final concentration of 0.5 μmol/L), 1.5 mmol/L MgCl2, and RNase-free water in a final volume of 20 μL. The thermal profile began with incubation at 48°C for 45 min (reverse transcription), then incubation at 95°C for 10 min (predenaturation), followed by 40 cycles of amplification alternating between 94°C for 15 s (denaturation) and 68°C for 40 s (annealing/extension). The SYBR Green I fluorescent signal was obtained once per cycle at the end of the extension step. After amplification, melting curve analysis was performed by heating the sample to 95°C for 15 s, then cooling it to 70°C for 1 min, followed by a linear temperature increase to 95°C at a rate of 0.5°C/s, while continuously monitoring the fluorescent signal. Data were analyzed by the 7500 System SDS Software version 1.2 (Applied Biosystems). To develop the assay, samples previously identified as influenza A/chicken/Nakorn-Patom/Thailand/CU-K2/04 (H5N1) served as a control for HPAI, and A/duck/Hong Kong/308/78 (H5N3) served as a control for LPAI. The H5 genes (nucleotides 914–1728) of each strain were inserted into pGEM-T Easy Vector and then transcribed in vitro by using RiboMAX Large Scale RNA Production System-T7 (Promega, Madison, WI, USA). Serial 10-fold dilutions of the standard H5 RNA were subjected to a sensitivity test (8). The fluorescent signal can be detected at RNA dilutions as low as 102 copies/μL. To assess the specificity, viral RNA extracted from other subtypes of influenza A viruses (H1–H4 and H6–H15) was tested. The assay was specific for the H5 subtype, since no amplification was detected from other subtypes. Three preliminary melting curve analyses showed that this assay was effective in discriminating between the melting peaks of HPAI and LPAI (Figure). The variations of melting temperature (Tm) between runs were experimentally determined. The mean (standard deviation) of Tm values for HPAI and LPAI were 77.43°C (0.21°C) and 79.57°C (0.23°C), respectively. This assay provided high reliability and reproducibility, since the coefficients of variation were <0.30. Figure Discrimination between highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) by melting curve analysis based on real-time reverse transcription–polymerase chain reaction of the H5 HA gene with SYBR Green I fluorescent ... Seventy-eight specimens of influenza A virus were used to validate the assay. The 75 HPAI samples were isolated during the 2004 outbreak in Thailand; 3 LPAI samples, including A/avian/NY/01 (H5N2), A/Chicken/Mexico/31381-3/94 (H5N2), and A/shoveler/Egypt/03 (H5N2), were provided by the Centers for Disease Control and Prevention. The viruses were isolated in embryonated chicken eggs as described previously (9). RNA was extracted from 140 μL of allantoic fluid, and RT-PCR was performed as described above. Melting curve analysis showed that all H5N1 isolates from the 2004 outbreak in Thailand were interpreted as HPAI, whereas the 3 samples of H5N2 subtype were classified as LPAI. The Tm varied from 77.2°C to 78.1°C for HPAI and 78.75°C to 79.5°C for LPAI. The melting curve analysis results were completely concordant with the results of direct sequence analysis of the H5 gene for all samples tested. The melting curve analysis described here provides a rapid, accurate, and high-throughput approach to discriminate between HPAI and LPAI. This assay is particularly attractive for large-scale screening of suspected subtype H5 influenza A virus during outbreaks to identify candidate LPAI that could be used as vaccine strains.