Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Background: Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. Aim: The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. Methods: In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. Results: The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. Conclusion: In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

Genetic and virological characteristics of a reassortant avian influenza A H6N1 virus isolated from wild birds at a live-bird market in Egypt.

The first detection of a human infection with avian influenza A/H6N1 virus in Taiwan in 2013 has raised concerns about this virus. During our routine surveillance of avian influenza viruses (AIVs) in live-bird markets in Egypt, an H6N1 virus was isolated from a garganey duck and was characterized. Phylogenetic analysis indicated that the Egyptian H6N1 strain A/Garganey/Egypt/20869C/2022(H6N1) has a unique genomic constellation, with gene segments inherited from different subtypes (H5N1, H3N8, H7N3, H6N1, and H10N1) that have been detected previously in AIVs from Egypt and some Eurasian countries. We examined the replication of kinetics of this virus in different mammalian cell lines (A549, MDCK, and Vero cells) and compared its pathogenicity to that of the ancestral H6N1 virus A/Quail/HK/421/2002(H6N1). The Egyptian H6N1 virus replicated efficiently in C57BL/6 mice without prior adaptation and grew faster and reached higher titers than in A549 cells than the ancestral strain. These results show that reassortant H6 AIVs might pose a potential threat to human health and highlight the need to continue surveillance of H6 AIVs circulating in nature.

Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

Monophosphoryl lipid A and poly I:C combination enhances immune responses of equine influenza virus vaccine.

Equine influenza is a contagious respiratory disease caused by H3N8 type A influenza virus. Vaccination against equine influenza is conducted regularly; however, infection still occurs globally because of the short immunity duration and suboptimal efficacy of current vaccines. Hence the objective of this study was to investigate whether an adjuvant combination can improve immune responses to equine influenza virus (EIV) vaccines. Seventy-two mice were immunized with an EIV vaccine only or with monophosphoryl lipid A (MPL), polyinosinic-polycytidylic acid (Poly I:C), or MPL + Poly I:C. Prime immunization was followed by boost immunization after 2 weeks. Mice were euthanized at 4, 8, and 32 weeks post-prime immunization, respectively. Sera were collected to determine humoral response. Bone marrow, spleen, and lung samples were harvested to determine memory cell responses, antigen-specific T-cell proliferation, and lung viral titers. MPL + Poly I:C resulted in the highest IgG, IgG1, and IgG2a antibodies and hemagglutination inhibition titers among the groups and sustained their levels until 32 weeks post-prime immunization. The combination enhanced memory B cell responses in the bone marrow and spleen. At 8 weeks post-prime immunization, the combination induced higher CD8+ central memory T cell frequencies in the lungs and CD8+ central memory T cells in the spleen. In addition, the combination group exhibited enhanced antigen-specific T cell proliferation, except for CD4+ T cells in the lungs. Our results demonstrated improved immune responses when using MPL + Poly I:C in EIV vaccines by inducing enhanced humoral responses, memory cell responses, and antigen-specific T cell proliferation.

Characterization of a human H3N8 influenza virus.

BACKGROUND: In 2022 and 2023, novel reassortant H3N8 influenza viruses infected three people, marking the first human infections with viruses of this subtype. METHODS: Here, we generated one of these viruses (A/Henan/4-10CNIC/2022; hereafter called A/Henan/2022 virus) by using reverse genetics and characterized it. FINDINGS: In intranasally infected mice, reverse genetics-generated A/Henan/2022 virus caused weight loss in all five animals (one of which had to be euthanized) and replicated efficiently in the respiratory tract. Intranasal infection of ferrets resulted in minor weight loss and moderate fever but no mortality. Reverse genetics-generated A/Henan/2022 virus replicated efficiently in the upper respiratory tract of ferrets but was not detected in the lungs. Virus transmission via respiratory droplets occurred in one of four pairs of ferrets. Deep-sequencing of nasal swab samples from inoculated and exposed ferrets revealed sequence polymorphisms in the haemagglutinin protein that may affect receptor-binding specificity. We also tested 90 human sera for neutralizing antibodies against reverse genetics-generated A/Henan/2022 virus and found that some of them possessed neutralizing antibody titres, especially sera from older donors with likely exposure to earlier human H3N2 viruses. INTERPRETATION: Our data demonstrate that reverse genetics-generated A/Henan/2022 virus is a low pathogenic influenza virus (of avian influenza virus descent) with some antigenic resemblance to older human H3N2 viruses and limited respiratory droplet transmissibility in ferrets. FUNDING: This work was supported by the Japan Program for Infectious Diseases Research and Infrastructure (JP23wm0125002), and the Japan Initiative for World-leading Vaccine Research and Development Centers (JP233fa627001) from the Japan Agency for Medical Research and Development (AMED).

First Known Human Death After Infection With the Avian Influenza A/H3N8 Virus: Guangdong Province, China, March 2023.

Here, we report on a case of human infection with the H3N8 avian influenza virus. The patient had multiple myeloma and died of severe infection. Genome analysis showed multiple gene mutations and reassortments without mammalian-adaptive mutations. This suggests that avian influenza (A/H3N8) virus infection could be lethal for immunocompromised persons.

Equine influenza outbreak in Eastern of Algeria in 2021: The first introduction of Florida Clade 1 to Maghreb area.

We have performed an equine influenza (EI) serological study of the equine population in Algeria by testing 298 serum samples collected between February and August 2021 from 5 provinces. The results were obtained performing an NP-ELISA. Our results revealed that 49.3% (147/298) samples positive for antibodies to EI (H3N8). During this study and after a gap of one decade an outbreak of EI was reported in Algeria in the first week of March 2021. The disease was confirmed by virus detection from the nasal swabs (n = 39) by qRT-PCR and by identifying 5 EI seroconversion. The virus sequences were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 1 of the Florida sublineage in the American lineage. This study indicate the first detection of FC1 strain of EIV in Maghreb area.

Systematic Review of Equine Influenza A Virus Vaccine Studies and Meta-Analysis of Vaccine Efficacy.

Vaccines against equine influenza have been available since the late 1960s, but outbreaks continue to occur periodically, affecting both vaccinated and unvaccinated animals. The aim of this study was to systematically evaluate the efficacy of vaccines against influenza A virus in horses (equine IAV). For this, PubMed, CAB abstracts, and Web of Science were searched for controlled trials of equine IAV vaccines published up to December 2020. Forty-three articles reporting equine IAV vaccination and challenge studies in previously naïve equids using an appropriate comparison group were included in a qualitative analysis of vaccine efficacy. A value for vaccine efficacy (VE) was calculated as the percentage reduction in nasopharyngeal virus shedding detected by virus isolation in embryonated hens' eggs from 38 articles. Among 21 studies involving commercial vaccines, the mean VE was 50.03% (95% CI: 23.35-76.71%), ranging from 0 to 100%. Among 17 studies reporting the use of experimental vaccines, the mean VE was 40.37% (95% CI: 19.64-62.44), and the range was again 0-100%. Overall, complete protection from virus shedding was achieved in five studies. In conclusion, although commercially available vaccines can, in some circumstances, offer complete protection from infection, the requirement for frequent vaccination in the field to limit virus shedding and hence transmission is apparent. Although most studies were conducted by a few centres, a lack of consistent study design made comparisons difficult.

Assessment of Equine Influenza Virus Status in the Republic of Korea from 2020 to 2022.

Equine influenza virus (EIV) causes acute respiratory disease in horses and belongs to the influenza A virus family Orthomyxoviridae, genus Orthomyxovirus. This virus may have severe financial implications for the horse industry owing to its highly contagious nature and rapid transmission. In the Republic of Korea, vaccination against EIV has been practiced with the active involvement of the Korea Racing Authority since 1974. In this study, we monitored the viral RNA for EIV using PCR, as well as the antibody levels against 'A/equine/South Africa/4/03 (H3N8, clade 1)', from 2020 to 2022. EIV was not detected using RT-PCR. The seropositivity rates detected using a hemagglutination inhibition assay were 90.3% in 2020, 96.7% in 2021, and 91.8% in 2022. The geometric mean of antibody titer (GMT) was 83.4 in 2020, 135.7 in 2021, and 95.6 in 2022. Yearlings and two-year-olds in training exhibited lower positive rates (59.1% in 2020, 38.9% in 2021, and 44.1% in 2022) than the average. These younger horses may require more attention for vaccination and vaccine responses against EIV. Continuous surveillance of EIV should be performed to monitor the prevalence and spread of this disease.

Performance of a microfluidic immunofluorescence assay kit for equine influenza virus antigen detection.

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field.

Analysis of avian influenza A (H3N8) viruses in poultry and their zoonotic potential, China, September 2021 to May 2022.

BackgroundTwo human cases of avian influenza A (H3N8) virus infection were reported in China in 2022.AimTo characterise H3N8 viruses circulating in China in September 2021-May 2022.MethodsWe sampled poultry and poultry-related environments in 25 Chinese provinces. After isolating H3N8 viruses, whole genome sequences were obtained for molecular and phylogenetic analyses. The specificity of H3N8 viruses towards human or avian receptors was assessed in vitro. Their ability to replicate in chicken and mice, and to transmit between guinea pigs was also investigated.ResultsIn total, 98 H3N8 avian influenza virus isolates were retrieved from 38,639 samples; genetic analysis of 31 representative isolates revealed 17 genotypes. Viruses belonging to 10 of these genotypes had six internal genes originating from influenza A (H9N2) viruses. These reassorted viruses could be found in live poultry markets and comprised the strains responsible for the two human infections. A subset of nine H3N8 viruses (including six reassorted) that replicated efficiently in mice bound to both avian-type and human-type receptors in vitro. Three reassorted viruses were shed by chickens for up to 9 days, replicating efficiently in their upper respiratory tract. Five reassorted viruses tested on guinea pigs were transmissible among these by respiratory droplets.ConclusionAvian H3N8 viruses with H9N2 virus internal genes, causing two human infections, occurred in live poultry markets in China. The low pathogenicity of H3N8 viruses in poultry allows their continuous circulation with potential for reassortment. Careful monitoring of spill-over infections in humans is important to strengthen early-warning systems and maintain influenza pandemic preparedness.

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex.

Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2.

LIMITED ACCUMULATION AND PERSISTENCE OF AN INFLUENZA A VIRUS IN TADPOLE SNAILS (PHYSA SPP.).

Waterfowl infected with avian influenza A viruses (IAVs) shed infectious virus into aquatic environments, providing a mechanism for transmission among waterfowl, while also exposing the entire aquatic ecosystem to the virus. Aquatic invertebrates such as freshwater snails are likely exposed to IAVs in the water column and sediment. Freshwater snails comprise a significant portion of some waterfowl species' diets, so this trophic interaction may serve as a novel route of IAV transmission. In these experiments, tadpole snails (Physa spp.) were exposed to a low-pathogenicity IAV (H3N8) to determine whether snails can accumulate the virus and, if so, how long virus persists in snail tissues. Snail tissues were destructively sampled and tested by reverse-transcription quantitative real-time PCR. Our experiments demonstrated that tadpole snails do accumulate IAV RNA in their tissues, although at low titers, for at least 96 h. These results indicate that it may be possible for IAV transmission to occur between waterfowl via ingestion of a natural invertebrate prey item; however, the time frame for transmission may be limited.

Airborne transmission of human-isolated avian H3N8 influenza virus between ferrets.

H3N8 avian influenza viruses (AIVs) in China caused two confirmed human infections in 2022, followed by a fatal case reported in 2023. H3N8 viruses are widespread in chicken flocks; however, the zoonotic features of H3N8 viruses are poorly understood. Here, we demonstrate that H3N8 viruses were able to infect and replicate efficiently in organotypic normal human bronchial epithelial (NHBE) cells and lung epithelial (Calu-3) cells. Human isolates of H3N8 virus were more virulent and caused severe pathology in mice and ferrets, relative to chicken isolates. Importantly, H3N8 virus isolated from a patient with severe pneumonia was transmissible between ferrets through respiratory droplets; it had acquired human-receptor-binding preference and amino acid substitution PB2-E627K necessary for airborne transmission. Human populations, even when vaccinated against human H3N2 virus, appear immunologically naive to emerging mammalian-adapted H3N8 AIVs and could be vulnerable to infection at epidemic or pandemic proportion.

Looking beyond the H5 avian influenza viruses.

Avian influenza viruses continue to cross the species barrier and infect mammals. In this issue of Cell, Sun and colleagues demonstrate that viruses obtained from humans infected with an emergent avian H3N8 viruses exhibit increasing accumulation of mutations that promote respiratory droplet transmission and disease in mammals.

Molecular characterization and phylogenetic analysis of avian influenza H3N8 virus isolated from imported waterfowl in Malaysia.

Wild aquatic birds are natural reservoirs of influenza A viruses and H3 subtype is one of the most prevalent subtypes in waterfowl. Two H3N8 viruses of low pathogenic avian influenza (LPAI) were isolated via egg inoculation technique from the fecal swab specimens from imported barnacle goose and paradise shelduck in Veterinary Research Institute Ipoh, Malaysia. The full length of eight gene segments of the two viruses were amplified and sequenced with specific primers. The sequences were molecularly characterized, and the sequence identity were assessed with other published sequences. The two viruses are identical and they possess the same amino acid sequences for all the eight gene segments. The viruses were highly similar to the H3 virus from Netherlands and N8 virus from Belgium respectively. Phylogenetic analysis revealed that all the eight gene segments were grouped in the Eurasian lineage, and genetic reassortment may occur between the internal genes of the H3 viruses and other AI subtypes. Though four amino acid substitutions were identified in the hemagglutinin gene, the viruses retained most of the avian-type receptor binding preference. Few amino acid substitutions were observed in all internal genes. Most of the neuraminidase inhibitors and adamantine resistance related mutation were not seen in the viruses. The replicative capacity, cross species transmissibility, and potential zoonotic risk of the viruses are worth further investigation. As H3 virus poses potential threats to both human and animals, and with the increase in the international trade of birds; strict quarantine practice at the entry point and good laboratory diagnostic capabilities is crucial to prevent the introduction of new AI virus into our country.

Biological features of human influenza A(H3N8) viruses in China.

Influenza A(H3N8) viruses first emerged in humans in 2022, but their public health risk has not been evaluated. Here, we systematically investigated the biological features of avian and human isolated H3N8 viruses. The human-origin H3N8 viruses exhibited dual receptor binding profiles but avian-origin H3N8 viruses bound to avian type (sialic acid α2, 3) receptors only. All H3N8 viruses were sensitive to the antiviral drug oseltamivir. Although H3N8 viruses showed lower virulence than the 2009 pandemic H1N1 (09pdmH1N1) viruses, they induced comparable infectivity in mice. More importantly, the human population is naïve to H3N8 virus infection and current seasonal vaccination is not protective. Therefore, the threat of influenza A(H3N8) viruses should not be underestimated. Any variations should be monitored closely and their effect should be studied in time for the pandemic potential preparedness purpose.

Characterization of an Emergent Chicken H3N8 Influenza Virus in Southern China: a Potential Threat to Public Health.

Although influenza A viruses of several subtypes have occasionally infected humans, to date only those of the H1, H2, and H3 subtypes have led to pandemics and become established in humans. The detection of two human infections by avian H3N8 viruses in April and May of 2022 raised pandemic concerns. Recent studies have shown the H3N8 viruses were introduced into humans from poultry, although their genesis, prevalence, and transmissibility in mammals have not been fully elucidated. Findings generated from our systematic influenza surveillance showed that this H3N8 influenza virus was first detected in chickens in July 2021 and then disseminated and became established in chickens over wider regions of China. Phylogenetic analyses revealed that the H3 HA and N8 NA were derived from avian viruses prevalent in domestic ducks in the Guangxi-Guangdong region, while all internal genes were from enzootic poultry H9N2 viruses. The novel H3N8 viruses form independent lineages in the glycoprotein gene trees, but their internal genes are mixed with those of H9N2 viruses, indicating continuous gene exchange among these viruses. Experimental infection of ferrets with three chicken H3N8 viruses showed transmission through direct contact and inefficient transmission by airborne exposure. Examination of contemporary human sera detected only very limited antibody cross-reaction to these viruses. The continuing evolution of these viruses in poultry could pose an ongoing pandemic threat. IMPORTANCE A novel H3N8 virus with demonstrated zoonotic potential has emerged and disseminated in chickens in China. It was generated by reassortment between avian H3 and N8 virus(es) and long-term enzootic H9N2 viruses present in southern China. This H3N8 virus has maintained independent H3 and N8 gene lineages but continues to exchange internal genes with other H9N2 viruses to form novel variants. Our experimental studies showed that these H3N8 viruses were transmissible in ferrets, and serological data suggest that the human population lacks effective immunological protection against it. With its wide geographical distribution and continuing evolution in chickens, other spillovers to humans can be expected and might lead to more efficient transmission in humans.