Development of hybrid alphavirus-influenza A, B, and D pseudovirions for rapid quantification of neutralization antibodies and antiviral drugs

The emergence and spread of highly pathogenic avian influenza H5N1 subtypes have raised global concerns due to their ability to cross species barriers and occasional spillover to humans. The viruses primarily infect wild birds and poultry, which have caused significant, sporadic outbreaks in mammals including dairy cattle. Influenza D virus is a recently identified influenza virus that mainly affects cattle with frequent spillover to other species such as swine. Despite the availability of poultry vaccines, there are no H5N1 and Influenza D vaccines for cattle or other potentially affected livestock. Given a history of frequent influenza pandemics originating from avian and mammalian hosts, there is an urgent need for enhanced surveillance, biosecurity, and the development of antivirals and vaccines. Here we describe the development of a novel hybrid alphavirus-influenza pseudovirion (Ha-IV), which is a non-replicating influenza virus-like particle composed of viral structural proteins and an RNA genome derived from a fast-expressing alphaviral vector. As a proof-of-concept, we assembled Ha-IV pseudoviruses based on influenza D and influenza A and B subtypes, and demonstrated their infectivity. In addition, we validated an influenza A pseudovirus based on the H5N1 clade 2.3.4.4b strain, A/Texas/37/2024, for rapid quantification of neutralization antibodies within 4 to 18 hours. Furthermore, we used the pseudovirus to quantify infected cow sera and performed a correlation study with the classic hemagglutinin inhibition assay (HIA). We demonstrate that the Ha-IAV pseudovirus-based assay is consistent with HIA in identifying protective antibody responses. Our results demonstrate that this new Ha-IV pseudovirus provides a rapid tool for quantifying the infectivity of emerging HA mutants and for assessing neutralizing antibody responses.