NS1: a promising novel target antigen with strong immunogenicity and protective efficacy for avian flavivirus vaccine development.

Tembusu virus (TMUV), an avian pathogenic flavivirus, has emerged as a significant threat to the duck industry in Southeast Asia, causing substantial economic losses. Due to the antibody-dependent enhancement (ADE) effect of TMUV subneutralizing antibodies, there is a pressing need to further develop new TMUV vaccine target antigens that ensure both safety and efficacy. Here, the TMUV non-structural protein 1 (NS1) as a target for development of effective anti-TMUV vaccines was unveiled. The amino acid sequences of TMUV NS1 exhibit a high degree of conservation across different strains (92.63-100%). To investigate the potential of TMUV NS1 as a vaccine target, the TMUV NS1-based plasmids were constructed and identified the C-terminal 30 amino acids residues of TMUV E (EC30) as an effective signal peptide for promoting NS1 expression and secretion. Subsequently, the plasmid pVAX1-EC30-NS1 was employed to immunize ducks, resulting in specific anti-NS1 IgG responses being stimulated, while without inducing anti-TMUV neutralizing antibodies. Furthermore, the cellular immune responses triggered by the TMUV NS1 were evaluated, observing a notable increase in lymphocyte proliferation at 4 wk and 6 wk postinjection with the pVAX1-EC30-NS1. Additionally, there was a significant up-regulation of NS1-specific Il-4 and Ifnγ levels at these time points. Following this, ducks from different groups were challenged with TMUV, and remarkably, those immunized with the NS1 vaccine displayed significantly lower viral copies both at 3 d postinfection (dpi) and 7 dpi (P < 0.05) compared to ducks immunized with the control vector. Notably, the NS1 demonstrated remarkable protection against TMUV challenge without causing severe gross lesions. Collectively, these findings highlighted the impressive immunogenicity and protectivity of the TMUV NS1. Consequently, NS1 holds great promise as a novel antigen target for the development of efficient and safe TMUV vaccines.

Effect of previous heterologous flavivirus vaccinations on human antibody responses in tick-borne encephalitis and dengue virus infections.

Arthropod-borne flaviviruses include a number of medically relevant human pathogens such as the mosquito-borne dengue (DEN), Zika, and yellow fever (YF) viruses as well as tick-borne encephalitis virus (TBEV). All flaviviruses are antigenically related and anamnestic responses due to prior immunity can modulate antibody specificities in subsequent infections or vaccinations. In our study, we analyzed the induction of broadly flavivirus cross-reactive antibodies in tick-borne encephalitis (TBE) and DEN patients without or with prior flavivirus exposure through TBE and/or YF vaccination, and determined the contribution of these antibodies to TBE and dengue virus (DENV) neutralization. In addition, we investigated the formation of cross-reactive antibodies in TBE-vaccination breakthroughs (VBTs). A TBEV infection without prior YF or TBE vaccination induced predominantly type-specific antibodies. In contrast, high levels of broadly cross-reactive antibodies were found in samples from TBE patients prevaccinated against YF as well as in DEN patients prevaccinated against TBE and/or YF. While these cross-reactive antibodies did not neutralize TBEV, they were effective in neutralizing DENV. This discrepancy points to structural differences between the two viruses and indicates that broadly cross-reactive epitopes are less accessible in TBEV than in DENV. In TBE VBT infections, type-specific antibodies dominated the antibody response, thus revealing no difference from that of unvaccinated TBE patients. Our results emphasize significant differences in the structural properties of different flaviviruses that have an impact on the induction of broadly cross-reactive antibodies and their functional activities in virus neutralization.

A Prototype-Pathogen Approach for the Development of Flavivirus Countermeasures.

Flaviviruses are a genus within the Flaviviridae family of positive-strand RNA viruses and are transmitted principally through mosquito and tick vectors. These viruses are responsible for hundreds of millions of human infections worldwide per year that result in a range of illnesses from self-limiting febrile syndromes to severe neurotropic and viscerotropic diseases and, in some cases, death. A vaccine against the prototype flavivirus, yellow fever virus, has been deployed for 85 years and is highly effective. While vaccines against some medically important flaviviruses are available, others have proven challenging to develop. The emergence and spread of flaviviruses, including dengue virus and Zika virus, demonstrate their pandemic potential. This review highlights the gaps in knowledge that need to be addressed to allow for the rapid development of vaccines against emerging flaviviruses in the future.

Bacillus subtilis expressing duck Tembusu virus E protein induces immune protection in ducklings.

Duck Tembusu virus (DTMUV) is an infectious disease that emerged in China in 2010. It has caused serious economic losses to the poultry industry and may pose a threat to public health. We aimed to develop a new Bacillus subtilis (B. subtilis)-based oral vaccine to control DTMUV transmission among poultry; to this end, we constructed a B. subtilis strain that can secrete DTMUV E protein. Ducklings were orally immunized, and serum antibodies, mucosal antibodies, and splenic cytokines were detected. The results showed that, in addition to high levels of specific IgG, there were also high levels of specific secretory immunoglobulin A (sIgA) in ducklings orally treated with recombinant B. subtilis. In addition, the levels of IFN-γ, IL-2, IL-4, and IL-10 in spleens were significantly boosted by recombinant B. subtilis. Recombinant B. subtilis could effectively enhance ducklings resistance to DTMUV and significantly reduce viral load (p<0.01), along with pathological damage in the brain, heart, and spleen. This is the first study to apply a B. subtilis live-vector vaccine platform for DTMUV disease prevention and control, and our results suggest that B. subtilis expressing DTMUV E protein may be a candidate vaccine against DTMUV.

Study on the Protective Immunity Induced by Pseudotyped Baculovirus Expressing the E Protein of Tembusu Virus in Ducklings.

The Duck Tembusu virus (DTMUV), a pathogenic flavivirus, has been causing significant economic losses in the Chinese poultry industry since 2010. This virus can severely decrease egg production and inhibit the growth of laying ducks and ducklings. While many vaccines have been developed to prevent DTMUV infection, fresh outbreaks continue to occur, as few effective vaccines are available. The E glycoprotein of DTMUV is the primary target for inducing protective immunity in the natural host. Therefore, we conducted an investigation and successfully developed a recombinant baculovirus containing the DTMUV E gene. Ducklings were then vaccinated with the purified protein derived from this virus as a potential vaccine candidate. Our findings demonstrated that the E glycoprotein of DTMUV was highly expressed in Sf9 cells. The vaccination of ducklings with the recombinant baculovirus Bac-E resulted in the induction of strong humoral and cellular immune responses. Most significantly, we observed that the vaccine provided 100% protective immunity against lethal challenges with the DTMUV YY5 strain.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

Immunization with Usutu virus and with a chimeric West Nile virus (WNV) harboring Usutu-E protein protects immunocompetent adult mice against lethal challenges with different WNV lineage 1 and 2 strains.

West Nile virus (WNV) and Usutu virus (USUV), two antigenically related flaviviruses co-circulating in Europe, can cause severe neurological disease in animals and humans. The immune response against USUV and WNV and their immunopathogenesis are still poorly investigated. Here we present results upon sequential infections of adult immunocompetent CD-1 and BALB/c mice primed with two different doses (high dose, HD or low dose, LD) of an USUV isolate and challenged with HD or LD of three different WNV isolates. CD-1 and BALB/c LD USUV-primed mice, regardless of the dose, are largely protected from lethal WNV challenges despite showing no detectable neutralizing antibodies. Furthermore, mice immunized with a chimeric virus harboring the E protein of USUV within the WNV backbone (WNVE-USUV) are protected against a lethal challenge with WNV. We believe these findings could contribute to understanding the dynamics of the interaction during sequential infection of these two flaviviruses.

Flavivirus proteases: The viral Achilles heel to prevent future pandemics.

Flaviviruses are important human pathogens and include dengue (DENV), West Nile (WNV), Yellow fever virus (YFV), Japanese encephalitis (JEV) and Zika virus (ZIKV). DENV, transmitted by mosquitoes, causes diseases ranging in severity from mild dengue fever with non-specific flu-like symptoms to fatal dengue hemorrhagic fever and dengue shock syndrome. DENV infections are caused by four serotypes, DENV1-4, which interact differently with antibodies in blood serum. The incidence of DENV infection has increased dramatically in recent decades and the CDC estimates 400 million dengue infections occur each year, resulting in ∼25,000 deaths mostly among children and elderly people. Similarly, ZIKV infections are caused by infected mosquito bites to humans, can be transmitted sexually and through blood transfusions. If a pregnant woman is infected, the virus can cross the placental barrier and can spread to her fetus, causing severe brain malformations in the child including microcephaly and other birth defects. It is noteworthy that the neurological manifestations of ZIKV were also observed in DENV endemic regions, suggesting that pre-existing antibody response to DENV could augment ZIKV infection. WNV, previously unknown in the US (and known to cause only mild disease in Middle East), first arrived in New York city in 1999 (NY99) and spread throughout the US and Canada by Culex mosquitoes and birds. WNV is now endemic in North America. Thus, emerging and re-emerging flaviviruses are significant threat to human health. However, vaccines are available for only a limited number of flaviviruses, and antiviral therapies are not available for any flavivirus. Hence, there is an urgent need to develop therapeutics that interfere with essential enzymatic steps, such as protease in the flavivirus lifecycle as these viruses possess significant threat to future pandemics. In this review, we focus on our E. coli expression of NS2B hydrophilic domain (NS2BH) covalently linked to NS3 protease domain (NS3Pro) in their natural context which is processed by the combined action of both subunits of the NS2B-NS3Pro precursor. Biochemical activities of the viral protease such as solubility and autoproteolysis of NS2BH-NS3Pro linkage depended on the C-terminal portion of NS2BH linked to the NS3Pro domain. Since 2008, we also focus on the use of the recombinant protease in high throughput screens and characterization of small molecular compounds identified in these screens.

European College of Equine Internal Medicine consensus statement on equine flaviviridae infections in Europe.

Horses and other equids can be infected with several viruses of the family Flaviviridae, belonging to the genus Flavivirus and Hepacivirus. This consensus statement focuses on viruses with known occurrence in Europe, with the objective to summarize the current literature and formulate clinically relevant evidence-based recommendations regarding clinical disease, diagnosis, treatment, and prevention. The viruses circulating in Europe include West Nile virus, tick-borne encephalitis virus, Usutu virus, Louping ill virus and the equine hepacivirus. West Nile virus and Usutu virus are mosquito-borne, while tick-borne encephalitis virus and Louping ill virus are tick-borne. The natural route of transmission for equine hepacivirus remains speculative. West Nile virus and tick-borne encephalitis virus can induce encephalitis in infected horses. In the British Isle, rare equine cases of encephalitis associated with Louping ill virus are reported. In contrast, equine hepacivirus infections are associated with mild acute hepatitis and possibly chronic hepatitis. Diagnosis of flavivirus infections is made primarily by serology, although cross-reactivity occurs. Virus neutralization testing is considered the gold standard to differentiate between flavivirus infections in horses. Hepacivirus infection is detected by serum or liver RT-PCR. No direct antiviral treatment against flavi- or hepacivirus infections in horses is currently available and thus, treatment is supportive. Three vaccines against West Nile virus are licensed in the European Union. Geographic expansion of flaviviruses pathogenic for equids should always be considered a realistic threat, and it would be beneficial if their detection was included in surveillance programs.

A Single Mutation at Position 120 in the Envelope Protein Attenuates Tembusu Virus in Ducks.

A live attenuated duck Tembusu virus (TMUV) vaccine FX2010-180P (180P) was successfully utilized to prevent TMUV infections in ducks in China. Compared with wild-type TMUV, 180P was highly attenuated and lost transmissibility in ducks. However, the mechanism of the attenuation of 180P remains poorly understood. To explore the key molecular basis of attenuation, chimeric and site mutant viruses in the background of the wild-type TMUV-FX2010 (FX) strain were rescued, and the replication, tissue tropism, and transmissibility were characterized in ducks. The results show that the envelope (E) protein was responsible for attenuation and loss of transmission in ducks. Further studies showed that a D120N amino acid mutation located in domain II of the E protein was responsible for the attenuation and transmissibility loss of 180P in ducks. The D120N substitution resulted in an extra high-mannose type N-linked glycosylation (NLG) in the E protein of 180P compared with the wild-type TMUV, which might restrict the tissue tropism and transmissibility of TMUV in ducks. Our findings elucidate that N120 in the E protein is a key molecular basis of TMUV attenuation in ducks and provide new insight into the role of NLG in TMUV tissue tropism and transmissibility.

One antibody to treat them all.

Conserved flavivirus protein holds potential as target for versatile vaccines and therapies.

Dengue Virus Entry/Fusion Inhibition By Small Bioactive Molecules: A Critical Review.

Many flaviviruses are remarkable human pathogens that can be transmitted by mosquitoes and ticks. Despite the availability of vaccines for viral infections such as yellow fever, Japanese encephalitis, and tick-borne encephalitis, flavivirus-like dengue is still a significant life-threatening illness worldwide. To date, there is no antiviral treatment for dengue therapy. Industry and the research community have been taking ongoing steps to improve anti-flavivirus treatment to meet this clinical need. The successful activity has been involved in the inhibition of the virus entry fusion process in the last two decades. In this study, the latest understanding of the use of small molecules used as fusion inhibitors has been comprehensively presented. We summarized the structure, the process of fusion of dengue virus E protein (DENV E), and the amino acids involved in the fusion process. Special attention has been given to small molecules that allow conformational changes to DENV E protein, viz. blocking the pocket of ßOG, which is important for fusion.

West Nile Virus Vaccination Protects against Usutu Virus Disease in Mice.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1-/- mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1-/- model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.

Replication/Assembly Defective Avian Flavivirus With Internal Deletions in the Capsid Can Be Used as an Approach for Living Attenuated Vaccine.

Avian Tembusu virus (TMUV) is a novel flavivirus causing severe egg drop and fatal encephalitis in avian in Asia. In the present study, we screened the structural and functional requirements of TMUV capsid protein (CP) for viral morphogenesis using reverse genetics methods in combination with replicon packaging assays. TMUV-CP showed dramatic functional and structural flexibility, and even though 44 residues were removed from the N-terminus, it was still capable of packaging replicon RNA; in addition, 33 residues were deleted from the C-terminus (containing nearly the entire α4-helix), and infectious particles were still produced, although α4-α4' is supposedly vital for CP dimerization and nucleocapsid formation. We further analyzed two mutants (ΔC20-43 and ΔC64-96 viruses) with relatively large deletions that still replicated well in BHK-21 cells. Our data indicate that internal deletions within CP impaired viral replication or assembly, resulting in attenuated virus proliferation in cells and attenuated virulence in duck embryos, and these deletion mutations are quite stable in cell culture. An in vivo assay indicated that both ΔC20-43 virus and ΔC64-96 virus were highly attenuated in ducklings but still immunogenic. Single-dose immunization with ΔC20-43 virus or ΔC64-96 virus could protect ducklings from a lethal challenge with good antigen clearance. Together, our data shed light on replication/assembly defective TMUV with internal deletions in CP and provide an effective approach to attenuate viral virulence in live vaccines without changing the antigen composition.

Structure and function of capsid protein in flavivirus infection and its applications in the development of vaccines and therapeutics.

Flaviviruses are enveloped single positive-stranded RNA viruses. The capsid (C), a structural protein of flavivirus, is dimeric and alpha-helical, with several special structural and functional features. The functions of the C protein go far beyond a structural role in virions. It is not only responsible for encapsidation to protect the viral RNA but also able to interact with various host proteins to promote virus proliferation. Therefore, the C protein plays an important role in infected host cells and the viral life cycle. Flaviviruses have been shown to affect the health of humans and animals. Thus, there is an urgent need to effectively control flavivirus infections. The structure of the flavivirus virion has been determined, but there is relatively little information about the function of the C protein. Hence, a greater understanding of the role of the C protein in viral infections will help to discover novel antiviral strategies and provide a promising starting point for the further development of flavivirus vaccines or therapeutics.

Minocycline prevents primary duck neurons from duck Tembusu virus-induced death.

Duck Tembusu virus (DTMUV), a neurotropic flavivirus, is a causative agent of severe neurological diseases in different birds. No approved vaccines or antiviral therapeutic treatments are available to date. The poultry industry experiences significant economic losses due to DTMUV infections. Minocycline is a second-generation semi-synthetic tetracycline analogue that is commonly used as an antimicrobial treatment. Experimental studies have indicated the successful protective effects of minocycline against neuronal cell death from neurodegenerative diseases and viral encephalitis. The aim of this study was to investigate the effects of minocycline on DTMUV infection in neurons. Primary duck neurons were treated with minocycline, which exhibited neuroprotective effects via anti-apoptotic function rather than through viral replication inhibition. Minocycline might serve as a potential effective drug in DTMUV infection.

Interleukin-2 shows high adjuvanticity for an inactivated vaccine against duck Tembusu virus disease.

Currently, the widely used vaccine against duck Tembusu virus (DTMUV) disease is inactivated vaccine which, however, facing the limits of large inoculation dose, short immunization period, and incomplete effectiveness. Access to efficient adjuvants aiding for DTMUV inactivated vaccine seems to be of critical importance. Interleukin-2 (IL-2) was reported to induce a persistent expansion of effector T cells and could be a promising molecular adjuvant for many kinds of vaccines. In this study, the efficacy of duck interleukin (dIL)-2 as an adjuvant for a DTMUV inactivated vaccine was evaluated. Fifty-five Pekin ducks were divided into 5 groups and intramuscularly administered with 5 batches of vaccines at 42 D (A: DTUMV + dIL-2; B: 1/2DTUMV + dIL-2; C: DTUMV; D: 1/2DTUMV and E: PBS), respectively, and received the second vaccination 2 wk later. Fifty-six days after immunization, 6 ducks from each group were randomly selected to conduct a challenge protection test. Antibody titers and cytokine responses were detected to assess humoral and cellular immune responses in serum of inoculated ducks by hemagglutination inhibition and ELISA, respectively; virus isolation and RT-PCR method were used in immunity protective test. Our results showed that dIL-2 exerted an enhanced effect on the vaccine while reducing the dose of inoculated antigen highlighting high adjuvanticity of IL-2. The vaccines supplemented with IL-2 induced a higher level of antibodies and higher percentage of inhibition values than inactivated vaccines without IL-2 to a significant extent. The production level of IFN-α, IFN-γ, and IL-6 genes were elevated, enhancing both humoral and cellular responses. Furthermore, it provided higher protection after virus challenge. Therefore, IL-2 can be considered as a potential adjuvant for inactivated vaccine against DTMUV disease.

Anti-Zika virus and anti-Usutu virus activity of human milk and its components.

The benefits of human milk are mediated by multiple nutritional, trophic, and immunological components, able to promote infant's growth, maturation of its immature gut, and to confer protection against infections. Despite these widely recognized properties, breast-feeding represents an important mother-to-child transmission route of some viral infections. Different studies show that some flaviviruses can occasionally be detected in breast milk, but their transmission to the newborn is still controversial. The aim of this study is to investigate the antiviral activity of human milk (HM) in its different stages of maturation against two emerging flaviviruses, namely Zika virus (ZIKV) and Usutu virus (USUV) and to verify whether HM-derived extracellular vesicles (EVs) and glycosaminoglycans (GAGs) contribute to the milk protective effect. Colostrum, transitional and mature milk samples were collected from 39 healthy donors. The aqueous fractions were tested in vitro with specific antiviral assays and EVs and GAGs were derived and characterized. HM showed antiviral activity against ZIKV and USUV at all the stages of lactation with no significant differences in the activity of colostrum, transitional or mature milk. Mechanism of action studies demonstrated that colostrum does not inactivate viral particles, but it hampers the binding of both flaviviruses to cells. We also demonstrated that HM-EVs and HM-GAGs contribute, at least in part, to the anti-ZIKV and anti-USUV action of HM. This study discloses the intrinsic antiviral activity of HM against ZIKV and USUV and demonstrates the contribution of two bioactive components in mediating its protective effect. Since the potential infectivity of HM during ZIKV and USUV infection is still unclear, these data support the World Health Organization recommendations about breast-feeding during ZIKV infection and could contribute to producing new guidelines for a possible USUV epidemic.

T lymphocyte responses to flaviviruses - diverse cell populations affect tendency toward protection and disease.

Dengue virus (DENV), Yellow Fever virus, West Nile virus, Japanese encephalitis virus and Zika virus are medically important flaviviruses transmitted to humans by mosquitoes and circulate in overlapping geographic areas. Cross-reactive immune responses have been demonstrated among the flaviviruses, particularly the four DENV serotypes. The immunological imprint left by a flavivirus infection can therefore have profound effects on the responses to subsequent infections. In this review we summarize recent research focusing on T cell responses to DENV using clinical samples from prospective cohort studies in Asia. These data suggest that durability of different T cell populations after natural infection or vaccination is an important consideration for the outcome of subsequent flavivirus exposures and we argue for continued investigation in the context of longitudinal cohort studies.