Research Progress on Highly Pathogenic H5N1 Avian Influenza Virus

Brief Introduction to Avian Influenza Virus

Avian influenza virus (AIV) is a common type A influenza virus, belonging to the genus influenza virus of the family Orthomyxoviridae, and is classified as a Class A epidemic by the World Organization for Animal Health (OIE). It is usually transmitted among poultry and birds, especially when accompanied by migratory birds, which will further increase its spread and cause a large-scale epidemic across regions and countries. There are two main protein protrusions on the envelope of influenza viruses, namely hemagglutinin (HA) and neuraminidase (NA), which can be used to divide influenza viruses into four types: type A (IAV), type B (IBV), type C (ICV) and type D (IDV). Among them, type A influenza virus uses a variety of birds including waterfowl and poultry as hosts, and can also infect other species such as pigs, dogs, cats, and horses. Type B influenza virus is mainly transmitted by infecting humans, and the host of type C infection is also humans. Type D influenza virus mainly uses livestock as hosts, such as pigs, cattle and sheep. There are also obvious differences in the epidemics caused by the above four influenza viruses.

Figure 1. Avian influenza transmission flow from the natural reservoir (aquatic birds) to poultry, humans, and other animal species.

Influenza A virus and type B influenza virus can both cause widespread seasonal epidemics. On this basis, influenza A virus can further cause zoonotic pandemics, while influenza B virus has not been found to be associated with pandemics. Seasonal influenza caused by influenza A virus and influenza B virus can reach 5 million cases and 500,000 deaths each year, of which infections caused by influenza B virus account for about 25%. On this basis, according to the different pathogenicity of different avian influenza viruses, they can be further divided into low pathogenic avian influenza virus (LPAIV) and highly pathogenic avian influenza virus (HPAIV). Usually, if the HA protein in the avian influenza virus has a multi-base cleavage site, it is a highly pathogenic avian influenza virus. Most avian influenza viruses are low pathogenic avian influenza viruses, and common highly pathogenic influenza viruses include two subtypes: H5N1 and H7N9. The two subtypes of H5N1 and H7N9 can infect mammals including humans, often causing severe lower respiratory tract diseases, while low pathogenic avian influenza viruses mainly infect the upper respiratory tract, usually with complications of conjunctivitis.

Structure of Avian Influenza Virus

The genetic material of avian influenza virus of the family Orthomyxoviridae is composed of 8 single-stranded negative-strand RNA (vRNA), namely, PB2, PB1, PA, HA, NA, M and NS. It is also composed of a variety of essential and non-essential proteins. The essential proteins include hemagglutinin, neuraminidase, polymerase protein (PB1, PB2, PA), nucleoprotein (NP), non-structural protein (NS) and matrix protein (M). The diameter of the avian influenza virus particles composed of the above three is 80-120nm in spherical state. The avian influenza virus isolated from clinical samples will expand from filaments to spheres after chicken embryo culture. The filamentous virus particles are 200-300nm high and 20nm in diameter at the bottom.

Pathogenicity of H5N1 Avian Influenza Virus to Animals

Avian influenza viruses are generally classified as either low pathogenic or highly pathogenic according to their pathogenicity to poultry. H5N1 avian influenza virus belongs to the latter, which is the result of the accumulation of multiple basic amino acids in the HA cleavage site. Unlike low pathogenic avian influenza, highly pathogenic avian influenza develops rapidly and has a high mortality rate. After the outbreak of H5N1 avian influenza in 1996, H5N1 avian influenza occurred in various countries around the world to varying degrees, characterized by large-scale sudden deaths of poultry, waterfowl and wild birds, which confirmed the high pathogenicity of H5N1 to poultry. H5N1 avian influenza virus can also infect mammals, but this is determined by many factors. PA-X is a fusion protein of influenza A virus (IAV). Studies have shown that the absence of PA-X protein enhances the pathogenicity and replication of highly pathogenic H5N1 avian influenza virus to mice. At the same time, analysis of viruses isolated from Vietnam in 2012 and 2013 found that two amino acid changes in the PA protein in the polymerase complex affected the activity and virulence of the viral polymerase complex in mice. In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that recombination events could lead to a highly pathogenic influenza pandemic. Studies have shown that obtaining the NS fragment from H1N1pdm09 enhances the virulence of H5N1 in mice.

Detection of Highly Pathogenic H5N1 Avian Influenza Virus

According to the regulations of the World Organization for Animal Health (OIE), chicken embryo virus isolation and culture are often used for the detection of avian influenza. In recent years, many studies have used biosensors to detect viruses, such as silver nanocluster fluorescent molecular beacons based on DNA templates. As long as the corresponding recognition sequence is embedded, the well-designed molecular beacons can be conveniently used to detect a variety of virulence genes, including the genes of H5N1 virus. Aptamer sensors can also be used for detection. Aptamers represent an alternative to antibodies as recognition agents in diagnosis and detection analysis and have advantages. In 2016, scholars proposed an analytical platform for fluorescence detection of H5N1 virus gene sequences combined with near-infrared light and 2-photon excitation. At the same time, a digital microfluidic technology immunoassay method based on surface-enhanced Raman scattering has been proposed, and a sandwich immunoassay has been designed to sensitively detect H5N1 virus.

Vaccine for Highly Pathogenic H5N1 Avian Influenza Virus

Inactivated Vaccine

Using reverse genetics technology, an inactivated vaccine strain of H5NI with extremely weak pathogenicity and well adapted to growing in chicken embryos was artificially created, and the inactivated vaccine for avian influenza was made using this strain.

Recombinant Viral Vector Vaccine

Compared with inactivated vaccines, recombinant viral vector vaccines can induce more helper T cells to produce immune responses. A protective foreign antigen is injected into the viral gene to obtain the recombinant virus, and the immune system produces the corresponding target protein, thereby inducing an immune response. The recombinant viral vector vaccines currently developed for H5N1 avian influenza mainly include fowl pox virus vector vaccine, Newcastle disease virus vector vaccine, turkey herpes virus vector vaccine, duck enteritis virus vector vaccine and infectious laryngotracheitis virus vector vaccine.

DNA Vaccine

Compared with other vaccines, DNA vaccines have more advantages. Studies have reported that optiHA was inserted into the pCAGGS vector to construct a DNA vaccine, which caused a strong immune response when injected into chickens intramuscularly. In order to improve the efficacy of DNA vaccines against H5N1 influenza virus, studies have inserted three repeated KappaB (κB) motifs upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal, which were separated by a 5 bp nucleotide spacer. Variants with κB sites in chickens stimulated stronger humoral responses against the target antigen.

Inactivated IAV H5N1 Native Antigens