Publications

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza viruses cause annual, seasonal infection across the globe. Vaccination represents the most effective strategy to prevent such infections and/or to reduce viral disease. Two major types of influenza vaccines are approved for human use: inactivated influenza vaccines (IIVs) and live attenuated influenza vaccines (LAIVs). Two Master Donor Virus (MDV) backbones have been used to create LAIVs against influenza A virus (IAV): the United States (US) A/Ann Arbor/6/60 (AA) and the Russian A/Leningrad/134/17/57 (Len) H2N2 viruses. The mutations responsible for the temperature sensitive (ts), cold-adapted (ca) and attenuated (att) phenotypes of the two MDVs have been previously identified and genetically mapped. However, a direct comparison of the contribution of these residues to viral attenuation, immunogenicity and protection efficacy has not been conducted. Here, we compared the In vitro and in vivo phenotype of recombinant influenza A/Puerto Rico/8/34 H1N1 (PR8) viruses containing the ts, ca and att mutations of the US (PR8/AA) and the Russian (PR8/Len) MDVs. Our results show that PR8/Len is more attenuated in vivo than PR8/AA, although both viruses induced similar levels of humoral and cellular responses, and protection against homologous and heterologous viral challenges. Our findings support the feasibility of using a different virus backbone as MDV for the development of improved LAIVs for the prevention of IAV infections.

Vaccination remains the most effective approach for preventing and controlling equine influenza virus (EIV) in horses. However, the ongoing evolution of EIV has increased the genetic and antigenic differences between currently available vaccines and circulating strains, resulting in suboptimal vaccine efficacy. As recommended by the World Organization for Animal Health (OIE), the inclusion of representative strains from clade 1 and clade 2 Florida sublineages of EIV in vaccines may maximize the protection against presently circulating viral strains. In this study, we used reverse genetics technologies to generate a bivalent EIV live-attenuated influenza vaccine (LAIV). We combined our previously described clade 1 EIV LAIV A/equine/Ohio/2003 H3N8 (Ohio/03 LAIV) with a newly generated clade 2 EIV LAIV that contains the six internal genes of Ohio/03 LAIV and the HA and NA of A/equine/Richmond/1/2007 H3N8 (Rich/07 LAIV). The safety profile, immunogenicity, and protection efficacy of this bivalent EIV LAIV was tested in the natural host, horses. Vaccination of horses with the bivalent EIV LAIV, following a prime-boost regimen, was safe and able to confer protection against challenge with clade 1 (A/equine/Kentucky/2014 H3N8) and clade 2 (A/equine/Richmond/2007) wild-type (WT) EIVs, as evidenced by a reduction of clinical signs, fever, and virus excretion. This is the first description of a bivalent LAIV for the prevention of EIV in horses that follows OIE recommendations. In addition, since our bivalent EIV LAIV is based on the use of reverse genetics approaches, our results demonstrate the feasibility of using the backbone of clade 1 Ohio/03 LAIV as a master donor virus (MDV) for the production and rapid update of LAIVs for the control and protection against other EIV strains of epidemiological relevance to horses.

Zika virus (ZIKV) infection is currently one of the major concerns in human public health due to its association with neurological disorders. Intensive effort has been implemented for the treatment of ZIKV, however there are not currently approved vaccines or antivirals available to combat ZIKV infection. In this sense, the identification of virulence factors associated with changes in ZIKV virulence could help to develop safe and effective countermeasures to treat ZIKV or to prevent future outbreaks. Here, we have compared the virulence of two related ZIKV strains from the recent outbreak in Brazil (2015), Rio Grande do Norte Natal (RGN) and Paraiba. In spite of both viruses being identified in the same period of time and region, significant differences in virulence and replication were observed using a validated mouse model of ZIKV infection. While ZIKV-RGN has a 50% mouse lethal dose (MLD50) of  105 focus forming units (FFUs), ZIKV-Paraiba infection resulted in 100% of lethality with less than 10 FFUs. Combining deep-sequencing analysis and our previously described infectious ZIKV-RGN cDNA clone, we identified a natural polymorphism in the non-structural protein 2 A (NS2A) that increase the virulence of ZIKV. Moreover, results demonstrate that the single amino acid alanine to valine substitution at position 117 (A117V) in the NS2A was sufficient to convert the attenuated rZIKV-RGN in a virulent Paraiba-like virus (MLD50 < 10 FFU). The mechanism of action was also evaluated and data indicate that substitution A117V in ZIKV NS2A protein reduces host innate immune responses and viral-induced apoptosis in vitro. Therefore, amino acid substitution A117V in ZIKV NS2A could be used as a genetic risk-assessment marker for future ZIKV outbreaks.

Infectious bursal disease virus (IBDV) infection triggers the induction of type I IFN, which is mediated by melanoma differentiation-associated protein 5 recognition of the viral genomic double-stranded RNA (dsRNA). However, the mechanism of IBDV overcoming the type I IFN antiviral response remains poorly characterized. Here, we show that IBDV genomic dsRNA selectively binds to the host cellular RNA binding protein Staufen1 (STAU1) in vitro and in vivo. The viral dsRNA binding region was mapped to the N-terminal moiety of STAU1 (residues 1-468). Down-regulation of STAU1 impaired IBDV replication and enhanced IFN-β transcription in response to IBDV infection, while having little effect on the viral attachment to the host cells and cellular entry. Conversely, overexpression of STAU1 but not the IBDV dsRNA-binding deficient STAU1 mutant (469-702) led to a suppression of IBDV dsRNA-induced IFN-β promoter activity. Moreover, we found that the binding of STAU1 to IBDV dsRNA decreased the association of melanoma differentiation-associated protein 5 but not VP3 with the IBDV dsRNA in vitro. Finally, we showed that STAU1 and VP3 suppressed IFN-β gene transcription in response to IBDV infection in an additive manner. Collectively, these findings provide a novel insight into the evasive strategies used by IBDV to escape the host IFN antiviral response.-Ye, C., Yu, Z., Xiong, Y., Wang, Y., Ruan, Y., Guo, Y., Chen, M., Luan, S., Zhang, E., Liu, H. STAU1 binds to IBDV genomic double-stranded RNA and promotes viral replication via attenuation of MDA5-dependent β interferon induction.

Zika virus (ZIKV) is an emerging mosquito-borne member of the Flaviviridae family that has historically been known to cause sporadic outbreaks, associated with a mild febrile illness, in Africa and Southeast Asia [...].

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

Seasonal influenza infections are associated with an estimated 250-500 000 deaths annually. Resistance to the antiviral M2 ion-channel inhibitors has largely invalidated their clinical utility. Resistance to neuraminidase inhibitors has also been observed in several influenza A virus (IAV) strains. These data have prompted research on inhibitors that target the cap-snatching endonuclease activity of the polymerase acidic protein (PA). Baloxavir marboxil (Xofluza®), recently approved for clinical use, inhibits cap-snatching endonuclease. Resistance to Xofluza® has been reported in both in vitro systems and in the clinic. An X-ray crystallographic screening campaign of a fragment library targeting IAV endonuclease identified 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating agent at the active site. We have reported the structure-activity relationships for 3-hydroxypyridin-2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones as endonuclease inhibitors. These studies identified two distinct binding modes associated with inhibition of this enzyme that are influenced by the presence of substituents at the 5- and 6-positions of 3-hydroxypyridin-2(1H)-ones. Herein we report the structure-activity relationships associated with various para-substituted 5-phenyl derivatives of 6-(p-fluorophenyl)-3-hydroxypyridin-2(1H)-ones and the effect of using naphthyl, benzyl, and naphthylmethyl groups as alternatives to the p-fluorophenyl substituent on their activity as endonuclease inhibitors.

The influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals.

Influenza A viruses have regularly jumped to new host species to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here, we examine the processes of influenza A virus infection and evolution in mice by comparing single nucleotide variations of a human H1N1 pandemic virus, a seasonal H3N2 virus, and an H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with those seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full-genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear evidence for adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.IMPORTANCE Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts, we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and that the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza virus evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza virus pathology, host passage evolution will vary depending on the specific virus tested.