Recombinant A(H6N1)-H274Y avian influenza virus with dual drug resistance does not require permissive mutations to retain the replicative fitness in vitro and in ovo.

The possible emergence of drug-resistant avian flu raises concerns over the limited effectiveness of currently approved antivirals (neuraminidase inhibitors - NAIs) in the hypothetical event of a zoonotic spillover. Our study demonstrated that the recombinant avian A(H6N1) viruses showed reduced inhibition (RI) by multiple NAI drugs following the introduction of point mutations found predominantly in the neuraminidase gene (NA) of NAI-resistant human influenza strains (E119V, R292K and H274Y; N2 numbering). Moreover, A(H6N1)-H274Y showed increased replication efficiency in vitro, and a fitness advantage over wild-type (WT) when co-inoculated into embryonated hen's eggs. The results presented in our study together with the zoonotic potential of the A(H6N1) virus as evidenced by the human infection from 2013, highlight the need for enhanced monitoring of NAI resistance-associated signatures in circulating LPAI (low pathogenic avian influenza) globally.

Risk assessment of the newly emerged H7N9 avian influenza viruses.

Since the first human case in 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human infections with a mortality rate of approximately 40%. Despite large-scale poultry vaccination regimes across China, the H7N9 AIVs continue to persist and evolve rapidly in poultry. Recently, several strains of H7N9 AIVs have been isolated and shown the ability to escape vaccine-induced immunity. To assess the zoonotic risk of the recent H7N9 AIV isolates, we rescued viruses with hemagglutinin (HA) and neuraminidase (NA) from these H7N9 AIVs and six internal segments from PR8 virus and characterized their receptor binding, pH of fusion, thermal stability, plaque morphology and in ovo virus replication. We also assessed the cross-reactivity of the viruses with human monoclonal antibodies (mAbs) against H7N9 HA and ferret antisera against H7N9 AIV candidate vaccines. The H7N9 AIVs from the early epidemic waves had dual sialic acid receptor binding characteristics, whereas the more recent H7N9 AIVs completely lost or retained only weak human sialic acid receptor binding. Compared with the H7N9 AIVs from the first epidemic wave, the 2020/21 viruses formed larger plaques in Madin-Darby canine kidney (MDCK) cells and replicated to higher titres in ovo, demonstrating increased acid stability but reduced thermal stability. Further analysis showed that these recent H7N9 AIVs had poor cross-reactivity with the human mAbs and ferret antisera, highlighting the need to update the vaccine candidates. To conclude, the newly emerged H7N9 AIVs showed characteristics of typical AIVs, posing reduced zoonotic risk but a heightened threat for poultry.

Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Reverse genetics based H5N2 vaccine provides clinical protection against H5N1, H5N8 and H9N2 avian influenza infection in chickens.

The current study aimed to develop broadly protective vaccines for avian influenza. In an earlier study, HA stalk (universal flu vaccine) was found to be broadly protective against different subtypes of influenza virus in mice. Hence, we were interested to know its breadth of protective efficacy either alone or combined with inactivated rgH5N2 (clade 2.3.2.1a) vaccine against challenge viruses of homologous H5N1, heterologous H5N8 (clade 2.3.4.4) and heterosubtypic H9N2 virus in specific pathogen-free chickens. The rgH5N2 vaccine alone or in combination with HA stalk elicited sufficient pre-challenge immunity in the form of haemagglutination inhibiting (HI) antibodies and neutralizing antibodies (MNT) against H5N1, H5N8, and H9N2 in chickens. The rgH5N2 vaccine alone or in combination with HA stalk also attenuated the shedding of H5N1, H5N8 and H9N2 in chickens and protected against the lethal challenge of H5N1 or H5N8. In contrast, all HA stalk immunised chickens died upon H5N1 or H5N8 challenge and H9N2 challenged chickens survived. Our study suggests that the rgH5N2 vaccine can provide clinical protection against H5N1, H5N8 and can attenuate the viral shedding of H9N2 in chickens.

The Origin of Internal Genes Contributes to the Replication and Transmission Fitness of H7N9 Avian Influenza Virus.

H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate A/Anhui/1/13 (H7N9) and three reassortant viruses (2:6 H7N9) which contained the hemagglutinin and neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs belonging to (i) G1 subgroup 2, (ii) G1 subgroup 3, or (iii) BJ94 lineages, enzootic in different regions throughout Asia. Infection of chickens with the 2:6 H7N9 containing G1-like H9N2 internal genes conferred attenuation in vivo, with reduced shedding and transmission to contact chickens. However, possession of BJ94-like H9N2 internal genes resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis showed that the 2:6 H7N9 with BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo coinfection experiments followed, where chickens were coinfected with pairs of Anhui/13 H7N9 and a 2:6 H7N9 reassortant. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 AIVs with internal gene segments of BJ94-like or G1-like H9N2 viruses. Coinfection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens, and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential. IMPORTANCE H9N2 avian influenza viruses (AIVs) are enzootic in poultry in different geographical regions. The internal genes of these viruses can be exchanged with other zoonotic AIVs, most notably the A/Anhui/1/2013-lineage H7N9, which can give rise to new virus genotypes with increased veterinary, economic and public health threats to both poultry and humans. We investigated the propensity of the internal genes of H9N2 viruses (G1 or BJ94) in the generation of novel reassortant H7N9 AIVs. We observed that the internal genes of H7N9 which were derivative of BJ94-like H9N2 virus have a fitness advantage compared to those from the G1-like H9N2 viruses for efficient transmission among chickens. We also observed the generation of novel reassortant viruses during chicken transmission which infected and replicated efficiently in human cells. Therefore, such emergent reassortant genotypes may pose an elevated zoonotic threat.

Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential.

An H7N9 low-pathogenicity avian influenza virus (LPAIV) emerged in 2013 through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. To examine a natural reassortment scenario between H7N9 and G1 lineage H9N2 viruses predominant in the Indian subcontinent, we performed an experimental coinfection of chickens with A/Anhui/1/2013/H7N9 (Anhui/13) virus and A/Chicken/Pakistan/UDL-01/2008/H9N2 (UDL/08) virus. Plaque purification and genotyping of the reassortant viruses shed via the oropharynx of contact chickens showed H9N2 and H9N9 as predominant subtypes. The reassortant viruses shed by contact chickens also showed selective enrichment of polymerase genes from H9N2 virus. The viable "6+2" reassortant H9N9 (having nucleoprotein [NP] and neuraminidase [NA] from H7N9 and the remaining genes from H9N2) was successfully shed from the oropharynx of contact chickens, plus it showed an increased replication rate in human A549 cells and a significantly higher receptor binding to α2,6 and α2,3 sialoglycans compared to H9N2. The reassortant H9N9 virus also had a lower fusion pH, replicated in directly infected ferrets at similar levels compared to H7N9 and transmitted via direct contact. Ferrets exposed to H9N9 via aerosol contact were also found to be seropositive, compared to H7N9 aerosol contact ferrets. To the best of our knowledge, this is the first study demonstrating that cocirculation of H7N9 and G1 lineage H9N2 viruses could represent a threat for the generation of novel reassortant H9N9 viruses with greater virulence in poultry and a zoonotic potential. IMPORTANCE We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of the G1 lineage that are enzootic in poultry across the Indian subcontinent and the Middle East. Coinfection of chickens with these viruses resulted in the emergence of novel reassortant H9N9 viruses with genes derived from both H9N2 and H7N9 viruses. The "6+2" reassortant H9N9 (having NP and NA from H7N9) virus was shed from contact chickens in a significantly higher proportion compared to most of the reassortant viruses, showed significantly increased replication fitness in human A549 cells, receptor binding toward human (α2,6) and avian (α2,3) sialic acid receptor analogues, and the potential to transmit via contact among ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with zoonotic potential.

Identification and molecular characterization of H9N2 viruses carrying multiple mammalian adaptation markers in resident birds in central-western wetlands in India.

We report here a targeted risk-based study to investigate the presence of influenza A viruses at the migratory-wild-domestic bird interface across the major wetlands of central India's Maharashtra state during the winter migration season. The H9N2 viruses have been isolated and confirmed in 3.86% (33/854) of the fecal samples of resident birds. To investigate the genetic pools of H9N2 circulating in resident birds, we sequenced two isolates of H9N2 from distant wetlands. Sequence and phylogenetic analyses have shown that these viruses are triple reassortants, with HA, NA, NP, and M genes belonging to G1 sub-lineage (A/quail/Hong Kong/G1/1997), PB2, PB1, and NS genes originating from the prototype Eurasian lineage (A/mallard/France/090360/2009) and PA gene deriving from Y439/Korean-like (A/duck/Hong Kong/Y439/97) sub-lineage. It was confirmed not only that four of their gene segments had a high genetic association with the zoonotic H9N2 virus, A/Human/India/TCM2581/2019, but also that they had many molecular markers associated with mammalian adaptation and enhanced virulence in mammals including the unique multiple basic amino acids, KSKR↓GLF at the HA cleavage site, and analog N-and O-glycosylation patterns on HA with that of the zoonotic H9N2 virus. Furthermore, future experiments would be to characterize these isolates biologically to address the public health concern. Importantly, due to the identification of these viruses at a strategic geographical location in India (a major stop-over point in the Central Asian flyway), these novel viruses also pose a possible threat to be exported to other regions via migratory/resident birds. Consequently, systematic investigation and active monitoring are a prerequisite for identifying and preventing the spread of viruses of zoonotic potential by enforcing strict biosecurity measures.

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins.

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.

Adsorptive mutation and N-linked glycosylation modulate influenza virus antigenicity and fitness.

Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is "fit" and able to overcome vaccine pressure.

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19.

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential.

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.

A ligation and restriction enzyme independent cloning technique: an alternative to conventional methods for cloning hard-to-clone gene segments in the influenza reverse genetics system.

BACKGROUND: Reverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest. METHODS: Herein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation. RESULTS: Hard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4-315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued. CONCLUSION: The LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.

Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza.

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

Application of HDR-CRISPR/Cas9 and Erythrocyte Binding for Rapid Generation of Recombinant Turkey Herpesvirus-Vectored Avian Influenza Virus Vaccines.

Avian influenza viruses (AIVs) are highly contagious and have caused huge economical loss to the poultry industry. AIV vaccines remain one of the most effective methods of controlling this disease. Turkey herpesvirus (HVT) is a commonly used live attenuated vaccine against Marek's disease; it has also been used as a viral vector for recombinant AIV vaccine development. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a gene editing tool which, in vaccinology, has facilitated the development of recombinant DNA viral-vectored vaccines. Here, we utilize homology-directed repair (HDR) for the generation of a HVT-H7N9 HA bivalent vaccine; a H7N9 HA expression cassette was inserted into the intergenic region between UL45 and UL46 of HVT. To optimize the selection efficiency of our bivalent vaccine, we combined CRISPR/Cas9 with erythrocyte binding to rapidly generate recombinant HVT-H7HA candidate vaccines.

Evolution of Codon Usage Bias in Henipaviruses Is Governed by Natural Selection and Is Host-Specific.

Hendra virus (HeV) and Nipah virus (NiV) are among a group of emerging bat-borne paramyxoviruses that have crossed their species-barrier several times by infecting several hosts with a high fatality rate in human beings. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation in different hosts is lacking. A study of codon usage patterns in henipaviruses may provide some fruitful insight into their evolutionary processes of synonymous codon usage and host-adapted evolution. Here, we performed a systematic evolutionary and codon usage bias analysis of henipaviruses. We found a low codon usage bias in the coding sequences of henipaviruses and that natural selection, mutation pressure, and nucleotide compositions shapes the codon usage patterns of henipaviruses, with natural selection being more important than the others. Also, henipaviruses showed the highest level of adaptation to bats of the genus Pteropus in the codon adaptation index (CAI), relative to the codon de-optimization index (RCDI), and similarity index (SiD) analyses. Furthermore, a comparison to recently identified henipa-like viruses indicated a high tRNA adaptation index of henipaviruses for human beings, mainly due to F, G and L proteins. Consequently, the study concedes the substantial emergence of henipaviruses in human beings, particularly when paired with frequent exposure to direct/indirect bat excretions.

A two dose immunization with an inactivated reassortant H5N2 virus protects chickens against lethal challenge with homologous 2.3.2.1 clade and heterologous 2.2 clade highly pathogenic avian influenza H5N1 viruses.

The present study was aimed at generating a reassortant vaccine candidate virus with clade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 clade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.

The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus.

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte-derived dendritic cells.

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro-inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte-derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF-α, -ß and -γ were significantly upregulated in the H5N1 group. Pro-inflammatory cytokines (IL-1ß, TNF-α and IL-18) were highly upregulated in early mid (IL-1), and late (IL-6) phases of H5N1 virus infection. IL-8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus-induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV-infected cells.

Elevated level of pro inflammatory cytokine and chemokine expression in chicken bone marrow and monocyte derived dendritic cells following LPS induced maturation.

The study was designed to characterize and compare chicken bone marrow and peripheral blood monocyte derived dendritic cells (chBM-DC and chMoDC) and to evaluate inflammatory cytokine and chemokine alterations in response upon LPS stimulation. Typical morphology was observed in DCs from 48h of culture using recombinant chicken GM-CSF and IL-4. Maturation of DCs with LPS (1µg/ml) showed significant up regulation of mRNA of surface markers (CD40, CD80, CD83, CD86, MHC-II and DC-LAMP (CD208)), pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α (LITAF)), iNOS, chemokine CXCli2 and TLRs4 and 15. Basal level of TLR1 mRNA expression was higher followed by TLR15 in both DCs irrespective of their origin. Expression of iNOS and CXCLi2 mRNA in mature DCs of both origins were higher than other surface molecules and cytokines studied. Hence, its level of expression can also be used as an additional maturation marker for LPS induced chicken dendritic cell maturation along with CD83 and CD40. LPS matured DCs of both origins upregulated IL-12 and IFN-γ. Based on CD40 and CD83 mRNA expression, it was observed that LPS induced the maturation in both DCs, but chMoDCs responded better in expression of surface markers and inflammatory mediator genes.