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This study aimed to evaluate the potential antiviral effects of honey extract Camelyn against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). The baby hamster kidney cell line 21 (BHK‐21), bone marrow-derived hematopoietic stem cells (HSCs), and splenic cells were used for Camelyn cytotoxicity assay. After the isolation procedures, cell viability was assessed by trypan blue dye exclusion under a microscope using a hemocytometer. The in vitro cell growth rate was carried out using the cell counting Kit 8 (CCK-8) assay. The cells were seeded in growth media with various Camelyn concentrations (35 μg, 50 μg, 70 μg, 100 μg, 150 μg, and 200 μg). The absorbance at 450 nm was determined by the multiplate reader. The antiviral effect was assessed by plaque reduction assay for the determination of drug susceptibility against SARS-CoV-2. Serial dilution of the selected compounds was pre-incubated with 40 to 100 plaque-forming units (PFUs) of SARS-CoV-2. The pre-incubated mix of Camelyn and SARS-CoV-2 was then added to the confluent Vero E6 cells After incubation cells were fixed and stained and the number of PFUs was counted under an inverted microscope and plotted against the logarithm of antiviral concentrations. Our study showed that Camelyn is not cytotoxic, has a stimulatory effect on cell proliferation, and has an inhibitory effect against SARS-CoV-2 with EC50 (half-maximal effective concentration) from 85.7 μg/mL to 192.4 μg/mL depending on product concentration and viral plaque per cell. [ J Adv Biotechnol Exp Ther 2021; 4(3.000): 290-297]

A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.

ImportancePublic health vaccination recommendations for COVID-19 primary series and boosters in previously infected individuals differ worldwide. As infection with SARS-CoV-2 is often asymptomatic, it remains to be determined if vaccine immunogenicity is comparable in all previously infected subjects. We present detailed immunological evidence to clarify the requirements for one-or two-dose primary vaccination series for naturally primed individuals.ObjectiveEvaluate the immune response to COVID-19 mRNA vaccines in healthcare workers (HCWs) who recovered from a SARS-CoV-2 infection.DesignMulticentric observational prospective cohort study of HCWs with a PCR-confirmed SARS-CoV-2 infection designed to evaluate the dynamics of T and B cells immune responses to primary infection and COVID-19 mRNA vaccination over 12 months.ParticipantsUnvaccinated HCWs with PCR-confirmed SARS-CoV-2 infection were selected based on the presence or absence of symptoms at infection and serostatus at enrollment. Age- and sex-matched adults not infected with SARS-CoV-2 prior to vaccination were included as naïve controls.ExposureVaccination with Pfizer BioNTech BNT162b2 mRNA vaccine.Main Outcome(s) and Measure(s)Immunity score (zero to three), before and after vaccination, based on anti-RBD IgG ratio, serum capacity to neutralize live virus and IFN-γ secretion capacity in response to SARS-CoV-2 peptide pools above the positivity threshold for each of the three assays. We compared the immunity score between groups based on subjects’ symptoms at diagnosis and/or serostatus prior to vaccination.ResultsNone of the naïve participants (n=14) showed a maximal immunity score of three following one dose of vaccine compared to 84% of the previously infected participants (n=55). All recovered individuals who did not have an immunity score of three were seronegative prior to vaccination, and 67% had not reported symptoms resulting from their initial infection. Following one dose of vaccine, their immune responses were comparable to naïve individuals, with significantly weaker responses than those who were symptomatic during infection.Conclusions and RelevanceIndividuals who did not develop symptoms during their initial SARS-CoV-2 infection and were seronegative prior to vaccination present immune responses comparable to that of naïve individuals. These findings highlight the importance of administering the complete two-dose primary regimen and following boosters of mRNA vaccines to individuals who experienced asymptomatic SARS-CoV-2 infection.KEY POINTSQuestionIs a single dose of COVID-19 mRNA vaccine sufficient to induce robust immune responses in individuals with prior SARS-CoV-2 infection?FindingsIn this cohort of 55 health care workers previously infected with SARS-CoV-2, we show that the absence of symptoms during initial infection and negative serostatus prior to vaccination predict the strength of immune responses to COVID-19 mRNA vaccine. Lack of symptoms and a negative serostatus prior to vaccination leads to immune responses comparable to naïve individuals.MeaningOur results support a two-dose primary series requirement for any individual with prior history of asymptomatic SARS-CoV-2 infection.

Funder: Department of Health, Australian Government, This communication summarizes the presentations given at the 1st international conference of the World Society for Virology (WSV) held virtually during 16-18 June 2021, under the theme of tackling global viral epidemics. The purpose of this biennial meeting is to foster international collaborations and address important viral epidemics in different hosts. The first day included two sessions exclusively on SARS-CoV-2 and COVID-19. The other two days included one plenary and three parallel sessions each. Last not least, 16 sessions covered 140 on-demand submitted talks. In total, 270 scientists from 49 countries attended the meeting, including 40 invited keynote speakers.

The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

A total of 3425 influenza B viruses collected from the Asia-Pacific region were tested against the four registered neuraminidase inhibitors (NAIs) (oseltamivir carboxylate, zanamivir, peramivir and laninamivir) as part of the routine surveillance work at the WHO Collaborating Centre for Research and Reference on Influenza, Melbourne between 2016 and 2020. Forty-five influenza B viruses with reduced susceptibility to one or more NAIs were identified. While the majority of these had neuraminidase (NA) mutations that were known to confer NAIs resistance, fifteen had NA mutations that had not been confirmed as being responsible for reduced NAIs susceptibility. Eleven of these NA mutations of concern were investigated using reverse genetics (RG) techniques to verify that these mutations were the cause of the reduced NAI susceptibility. All mutations were introduced separately into the NA of B/Brisbane/27/2016 (a B Victoria-lineage virus) or B/Yamanashi/166/98 (a B Yamagata-lineage virus) and the effects of these were analysed by an in vitro NAI assay. The T146K substitution in the NA of B Victoria and Yamagata-lineages resulted in a large increase in the IC

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml−1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

Background Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. Methods Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. Results I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. Conclusion The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

We performed viral culture of respiratory specimens in 118 severe acute respiratory coronavirus virus 2 (SARS-CoV-2)–infected healthcare workers (HCWs), ∼2 weeks after symptom onset. Only 1 HCW (0.8%) had a positive culture. No factors for prolonged viral shedding were identified. Infectivity is resolved in nearly all HCWs ∼2 weeks after symptom onset.

The prolonged treatment of immunosuppressed (IS) individuals with anti-influenza monotherapies may lead to the emergence of drug-resistant variants. Herein, we evaluated oseltamivir and polymerase inhibitors combinations against influenza A/H3N2 infections in an IS mouse model. Mice were IS with cyclophosphamide and infected with 3 &times, 103 PFU of a mouse-adapted A/Switzerland/9715293/2013 (H3N2) virus. Forty-eight hours post-infection, the animals started oseltamivir, favipiravir or baloxavir marboxil (BXM) as single or combined therapies for 10 days. Weight losses, survival rates and lung viral titers (LVTs) were determined. The neuraminidase (NA) and polymerase genes from lung viral samples were sequenced. All untreated animals died. Oseltamivir and favipiravir monotherapies only delayed mortality (the mean day to death (MDD) of 21.4 and 24 compared to 11.4 days for those untreated) while a synergistic improvement in survival (80%) and LVT reduction was observed in the oseltamivir/favipiravir group compared to the oseltamivir group. BXM alone or in double/triple combination provided a complete protection and significantly reduced LVTs. Oseltamivir and BXM monotherapies induced the E119V (NA) and I38T (PA) substitutions, respectively, while no resistance mutation was detected with combinations. We found that the multiple dose regimen of BXM alone provided superior benefits compared to oseltamivir and favipiravir monotherapies. Moreover, we suggest the potential for drug combinations to reduce the incidence of resistance.

International audience; Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

As part of its role in the World Health Organization’s (WHO) Global Influenza Surveillance and Response System (GISRS), the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a total of 2,393 human influenza positive samples between 1 January 2020 and 31 December 2021 (2020: n = 2,021 samples; 2021: n = 372 samples). Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties. Selected viruses were propagated in qualified cells or embryonated hen’s eggs for potential use in seasonal influenza virus vaccines. During 2020–2021, influenza A viruses (A(H1N1)pdm09 in 2020 and A(H3N2) in 2021) predominated over influenza B viruses. In 2020, the majority of A(H1N1)pdm09, A(H3N2) and influenza B viruses analysed at the Centre were found to be antigenically similar to the respective WHO recommended vaccine strains for the southern hemisphere in 2020. In 2021, the majority of A(H1N1)pdm09 and A(H3N2) viruses were found to be antigenically distinct relative to the WHO recommended vaccine strains for the southern hemisphere in 2021. Of the influenza B viruses analysed at the Centre, 46.7% were found to be antigenically distinct to the respective WHO recommended vaccine strains. Of 1,538 samples tested for susceptibility to the neuraminidase inhibitors oseltamivir and zanamivir (in 2020, n = 1,374; in 2021, n = 164), two A(H1N1)pdm09 viruses showed highly reduced inhibition against oseltamivir, and one A(H1N1)pdm09 virus showed highly reduced inhibition against zanamivir. All of these samples were received in 2020.

Zika virus (ZIKV) is an important pathogen transmitted to humans by the mosquito vector Aedes aegypti. ZIKV is able to infect several tissues and organs and, importantly, has been associated with microcephaly and central nervous system abnormalities in fetuses and newborn babies of mothers exposed to ZIKV during pregnancy, as well as neurological diseases such as Guillain-Barré syndrome in adults. There is currently no vaccine or drug licensed to prevent or treat ZIKV infections. The use of ZIKV isolation in disease diagnosis has been largely replaced by new techniques. However, virus isolation is still considered as a gold standard for the detection of ZIKV and is usually performed in research and reference laboratories for characterization, sequencing, and a variety of research experiments including pathogenesis, drug susceptibility, and vaccine efficacy. The experimental procedures presented here describe the most common techniques used for ZIKV isolation, propagation, purification, and quantification.

Zika virus (ZIKV) is an important pathogen transmitted to humans by the mosquito vector Aedes aegypti. ZIKV is able to infect several tissues and organs and, importantly, has been associated with microcephaly and central nervous system abnormalities in fetuses and newborn babies of mothers exposed to ZIKV during pregnancy, as well as neurological diseases such as Guillain-Barre syndrome in adults. There is currently no vaccine or drug licensed to prevent or treat ZIKV infections. The use of ZIKV isolation in disease diagnosis has been largely replaced by new techniques. However, virus isolation is still considered as a gold standard for the detection of ZIKV and is usually performed in research and reference laboratories for characterization, sequencing, and a variety of research experiments including pathogenesis, drug susceptibility, and vaccine efficacy. The experimental procedures presented here describe the most common techniques used for ZIKV isolation, propagation, purification, and quantification.

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) are expected to occur at framework or catalytic residues of the NA enzyme. Numerous clinical and in vitro reports already described NAI-resistant A(H1N1)pdm09 variants harboring various framework NA substitutions. By contrast, variants with NA catalytic changes remain poorly documented. Herein, we investigated the effect of R152K and R368K NA catalytic mutations on the NA enzyme properties, in vitro replicative capacity and virulence of A(H1N1)pdm09 recombinant viruses. In NA inhibition assays, the R152K and R368K substitutions resulted in reduced inhibition [10- to 100-fold increases in IC50 vs the wild-type (WT)] or highly reduced inhibition (>100-fold increases in IC50) to at least 3 approved NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Such resistance phenotype correlated with a significant reduction of affinity observed for the mutants in enzyme kinetics experiments [increased Km from 20 ± 1.77 for the WT to 200.8 ± 10.54 and 565.2 ± 135 μM (P

Influenza A(H1N1)pdm09 virus continues to circulate worldwide without evidence of significant antigenic drift between 2009 and 2016. By using escape mutants, we previously identified six haemagglutinin (HA) changes (T80R, G143E, G158E, N159D, K166E and A198E) that were located within antigenic sites. Combinations of these mutations were introduced into the A(H1N1)pdm09 HA plasmid by mutagenesis. Reassortant 6 : 2 viruses containing both the HA and NA genes of the A(H1N1)pdm09 and the six internal gene segments of A/PR/8/34 were rescued by reverse genetics. In vitro, HA inhibition and microneutralization assays showed that the HA hexa-mutant reassortant virus (RG1) escaped A(H1N1)pdm09 hyper-immune ferret antiserum recognition. C57Black/6 mice that received the vaccine formulated with A/California/07/09 were challenged with 2×104 p.f.u. of either the 6 : 2 wild-type (WT) or RG1 viruses. Reductions in body weight loss, mortality rate and lung viral titre were observed in immunized animals challenged with the 6 : 2 WT virus compared to non-immunized mice. However, immunization did not protect mice challenged with RG1 virus. To further characterize the mutations causing this antigenic change, 11 additional RG viruses whose HA gene contained single or combinations of mutations were evaluated in vitro. Although the RG1 virus was still the least reactive against hyper-immune serum by HAI testing, mutations G158E and N159D within the Sa antigenic site appeared to play the major role in the altered antigenicity of the A(H1N1)pdm09 virus. These results show that the Sa antigenic site contains the most prominent epitopes susceptible to cause an antigenic drift, escaping actual vaccine protection.

Zika virus (ZIKV) is an emerging mosquito-borne virus recently linked to intrauterine growth restriction including abnormal fetal brain development. The recent outbreak of ZIKV reached pandemic level resulting in an alarming public health emergency. At present, there is limited understanding of the infectious mechanism and no approved therapy. Nonstructural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase domain. Here we used molecular modeling to obtain the structure of ZIKV MTase and molecular docking to identify the additional hydrophobic region uniquely conserved in flavivirus MTase that can be used as a druggable site. Subsequently, a virtual screening with a library of 28 341 compounds identified 10 best hits showing decisive contacts with the MTase. In vitro efficacy analysis of these compounds against ZIKV, by plaque reduction assay, has confirmed four of the top scored ligands (Life Chemicals ID: F3043-0013, F0922-0796, F1609-0442, and F1750-0048) having EC50 (50% effective concentration) values of 4.8 ± 2.3, 12.5 ± 7.4, 17.5 ± 8.4, and 17.6 ± 3.1 μM respectively, identifying lead compounds for anti-ZIKV drug development.

Background Zika virus, a previously neglected mosquito-borne virus, is prompting worldwide concern because of its connection with congenital defects, Guillain-Barré syndrome, meningoencephalitis and myelitis in infected individuals. However, no specific antiviral therapy is available at present. In this study, we investigated the in vitro susceptibility of geographically and temporally distinct Zika viruses against the RNA polymerase inhibitors, favipiravir (T-705) and ribavirin. Methods The in vitro activity of each drug and a 1:1 mixture combination was assessed against five geographically and temporally distinct Zika strains by plaque reduction assay (PRA), the gold standard phenotypic method. Results We showed that both drugs exhibit in vitro inhibitory activity against five different Zika strains isolated in different years and continents, with mean 50% inhibitory concentration (IC50) values of 35 ±14 and 35 ±20 μM, respectively, by PRA. We did not observe a synergistic effect when both drugs were combined at the equimolar concentration (IC50 =33 ±11 μM). Conclusions These results indicate that T-705 has the potential to be used in patients with complicated diseases and/or those individuals presenting with significant comorbidities.

Background Baloxavir marboxil (BXM), a cap-dependent endonuclease inhibitor, has been recently approved in the United States for the treatment of influenza infections. It is superior to oseltamivir for reducing the time of viral shedding but is reported to have a low barrier of resistance. We sought to evaluate the viral fitness of the predominant BXM-resistant I38T PA mutant in the A/H1N1 and A/H3N2 viral backgrounds. Methods Recombinant A/Quebec/144147/2009 (H1N1) and A/Switzerland/9715293/2013 (H3N2) influenza viruses and their respective I38T PA mutants were generated by reverse genetics. Standardized inoculums (500 PFUs) of wild-type (WT) and mutant mixtures were inoculated on α2,6 MDCK cells. On day 3 post-infection (pi), the supernatants were collected and the ratios of WT/mutant viruses were determined by droplet digital PCR using specific LNA probes. Single infections and competitive experiments were also performed in C56/BL6 mice with quantification of lung viral titers on days 3 and 6 pi. Results In vitro A/H1N1 studies showed similar total copy numbers for the WT and mutant viruses on day 3 pi (1.2 × 109 and 1.3 × 109 copies/mL, respectively). The initial 50%/50% mixture became 70%/30% (WT/mutant) after one passage in cells. For A/H3N2, the total copy numbers were 8.1 × 109 and 1.0 × 109 copies/mL for the WT and mutant viruses. The initial 50%/50% mixture became 94%/6% (WT/mutant) after one passage. The I38T mutants remained stable after 4 passages in α2,6 MDCK cells. In mice, the A/H1N1 WT and I38T mutant induced similar weight loss and generated comparable lung titers on days 3 and 6 pi. In contrast, the weight loss of the A/H3N2 mutant was greater than that of the WT between days 3 and 7 pi with comparable lung titers on days 3 and 6. Following infection with 50%/50% mixtures, the mutant virus predominated over the WT on day 3 pi (73% A/H1N1 and 58% A/H3N2). Conclusion The BXM-resistant I38T PA mutant replicates well both in vitro and in vivo in the A/H1N1 and A/H3N2 backgrounds. Surveillance for the emergence and transmission of such mutant in the community is required. Disclosures All Authors: No reported Disclosures.

Influenza virus infections are a significant public threat and the best approach to prevent them is through vaccination. Because of the perpetual changes of circulating influenza strains, the efficacy of influenza vaccines rarely exceeds 50%. To improve the protection efficacy, we have designed a novel vaccine formulation that shows a broad range of protection. The formulation is made of the matrix protein 2 (M2e) and the nucleoprotein (NP) antigens. The multimerization of NP into nanoparticles improved significantly the immune response to NP. The combination of the NP nanoparticles with the PapMV-M2e nanoparticles enhances significantly the immune response directed to NP revealing the adjuvant property of the PapMV platform. The vaccine formulation combining these two types of nanoparticles protects mice from infectious challenges by two different influenza strains (H1N1 and H3N2) and is a promising influenza A vaccine capable to elicit a broad protection.

H3N8 influenza viruses are a commonly found subtype in wild birds, usually causing mild or no disease in infected birds. However, they have crossed the species barrier and have been associated with outbreaks in dogs, pigs, donkeys, and seals and therefore pose a threat to humans. A live attenuated, cold-adapted ( ca ) H3N8 vaccine virus was generated by reverse genetics using the wild-type (wt) hemagglutinin (HA) and neuraminidase (NA) genes from the A/blue-winged teal/Texas/Sg-00079/2007 (H3N8) (tl/TX/079/07) wt virus and the six internal protein gene segments from the ca influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (H2N2), the backbone of the licensed seasonal live attenuated influenza vaccine. One dose of the tl/TX/079/07 ca vaccine induced a robust neutralizing antibody response against the homologous (tl/TX/079/07) and two heterologous influenza viruses, including the recently emerged A/harbor seal/New Hampshire/179629/2011 (H3N8) and A/northern pintail/Alaska/44228-129/2006 (H3N8) viruses, and conferred robust protection against the homologous and heterologous influenza viruses. We also analyzed human sera against the tl/TX/079/07 H3N8 avian influenza virus and observed low but detectable antibody reactivity in elderly subjects, suggesting that older H3N2 influenza viruses confer some cross-reactive antibody. The latter observation was confirmed in a ferret study. The safety, immunogenicity, and efficacy of the tl/TX/079/07 ca vaccine in mice and ferrets support further evaluation of this vaccine in humans for use in the event of transmission of an H3N8 avian influenza virus to humans. The human and ferret serology data suggest that a single dose of the vaccine may be sufficient in older subjects. IMPORTANCE Although natural infection of humans with an avian H3N8 influenza virus has not yet been reported, this influenza virus subtype has already crossed the species barrier and productively infected mammals. Pandemic preparedness is an important public health priority. Therefore, we generated a live attenuated avian H3N8 vaccine candidate and demonstrated that a single dose of the vaccine was highly immunogenic and protected mice and ferrets against homologous and heterologous H3N8 avian viruses.

Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infections of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs, and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the hemagglutinin and neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein genes of the cold-adapted ( ca ) A/Ann Arbor/6/60 (H2N2) vaccine donor virus, which is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans. IMPORTANCE Equine influenza viruses have crossed the species barrier to infect other mammals such as dogs, pigs, and camels and therefore may also pose a threat to humans. We believe that it is important to develop vaccines against equine influenza viruses in the event that an EIV evolves, adapts, and spreads in humans, causing disease. We generated a live attenuated H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and protected mice and ferrets against homologous and heterologous EIV.

The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC 50 s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC 50 level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses.

We have developed pandemic live attenuated influenza vaccines (pLAIVs) against clade 1 H5N1 viruses on an Ann Arbor cold-adapted ( ca ) backbone that induced long-term immune memory. In 2015, many human infections caused by a new clade (clade 2.2.1.1) of goose/Guangdong (gs/GD) lineage H5N1 viruses were reported in Egypt, which prompted updating of the H5N1 pLAIV. We explored two strategies to generate suitable pLAIVs. The first approach was to modify the hemagglutinin gene of a highly pathogenic wild-type ( wt ) clade 2.2.1.1 virus, A/Egypt/N03434/2009 (Egy/09) (H5N1), with its unmodified neuraminidase (NA) gene; this virus was designated Egy/09 ca . The second approach was to select a low-pathogenicity avian influenza H5 virus that elicited antibodies that cross-reacted with a broad range of H5 viruses, including the Egypt H5N1 viruses, and contained a novel NA subtype for humans. We selected the low-pathogenicity A/duck/Hokkaido/69/2000 (H5N3) (dk/Hok/00) virus for this purpose. Both candidate vaccines were attenuated and immunogenic in ferrets, inducing antibodies that neutralized homologous and heterologous H5 viruses with different degrees of cross-reactivity; Egy/09 ca vaccine antisera were more specific for the gs/GD lineage viruses but did not neutralize recent North American isolates (clade 2.3.4.4), whereas antisera from dk/Hok/69 ca -vaccinated ferrets cross-reacted with clade 2.3.4.4 and 2.2.1 viruses but not clade 1 or 2.1 viruses. When vaccinated ferrets were challenged with homologous and heterologous H5 viruses, challenge virus replication was reduced in the respiratory tract. Thus, the two H5 pLAIV candidates are suitable for clinical development to protect humans from infection with different clades of H5 viruses. IMPORTANCE In response to the continuing evolution of H5N1 avian influenza viruses and human infections, new candidate H5 live attenuated vaccines were developed by using two different approaches: one targeted a specific circulating strain in Egypt, and the other was based on a virus that elicits broadly cross-reactive antibodies against a wide range of H5 viruses. Both candidate vaccines were immunogenic and exhibited protective efficacy in ferrets. Our study permits a comparison of the two approaches, and the data support the further development of both vaccine viruses to optimally prepare for the further spread of clade 2.2.1 or 2.3.4.4 viruses.

In 1947, Zika virus (ZIKV), a mosquito-borne flavivirus was identified in Uganda and subsequently spread to Asia and the Pacific regions. In 2015, it was introduced in Brazil causing an important social and sanitary alarm due to its increased virulence and rapid dissemination. Importantly, ZIKV infections have been associated with severe neurological complications such as Guillain–Barré syndrome and microcephaly in fetuses and newborns. Although enormous efforts were made by investigators in the development of effective countermeasures against ZIKV, there is still no approved specific antiviral drug for the treatment of ZIKV infections. Herein, we review several anti ZIKV candidates including drugs targeting both the virus (structural proteins and enzymes) and cellular elements.

The spread of highly pathogenic avian H5N1 influenza viruses since 1997 and their virulence for poultry and humans has raised concerns about their potential to cause an influenza pandemic. Vaccines offer the most viable means to combat a pandemic threat. However, it will be a challenge to produce, distribute and implement a new vaccine if a pandemic spreads rapidly. Therefore, efforts are being undertaken to develop pandemic vaccines that use less antigen and induce cross-protective and long-lasting responses, that can be administered as soon as a pandemic is declared or possibly even before, in order to prime the population and allow for a rapid and protective antibody response. In the last few years, several vaccine manufacturers have developed candidate pandemic and pre-pandemic vaccines, based on reverse genetics and have improved the immunogenicity by formulating these vaccines with different adjuvants. Some of the important and consistent observations from clinical studies with H5N1 vaccines are as follows: two doses of inactivated vaccine are generally necessary to elicit the level of immunity required to meet licensure criteria, less antigen can be used if an oil-in-water adjuvant is included, in general antibody titers decline rapidly but can be boosted with additional doses of vaccine and if high titers of antibody are elicited, cross-reactivity against other clades is observed. Prime-boost strategies elicit a more robust immune response. In this review, we discuss data from clinical trials with a variety of H5N1 influenza vaccines. We also describe studies conducted in animal models to explore the possibility of reassortment between pandemic live attenuated vaccine candidates and seasonal influenza viruses, since this is an important consideration for the use of live vaccines in a pandemic setting.

Immunosuppressed individuals can shed influenza virus for prolonged periods of time, leading to the frequent emergence of antiviral resistance. We evaluated the benefits of oseltamivir and favipiravir combination therapy compared to single antiviral agents and monitored the emergence of drug-resistant variants in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus. C57BL/6 mice were immunosuppressed with cyclophosphamide and infected with a lethal dose of pandemic influenza A(H1N1) virus. Forty-eight hours post-infection, mice were treated with oseltamivir (20 mg/kg), favipiravir (20 or 50 mg/kg) or both agents BID for 5 or 10 days. Body weight losses, survival rates, lung viral titers, cytokine levels and emergence of resistant viruses were evaluated. Treatment of immunosuppressed mice with high (50 mg/kg) but not low (20 mg/kg) doses of favipiravir in combination with oseltamivir (20 mg/kg) significantly delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with oseltamivir but did not prevent the emergence of oseltamivir-resistant H275Y neuraminidase variants. Combination therapy with oseltamivir and favipiravir should be considered for evaluation in clinical trials.

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines.

Influenza A/H3N2 viruses have caused the most severe epidemics since 1968 despite current immunization programs with inactivated vaccines. We undertook a side-by-side preclinical evaluation of different adjuvants (Alum, AS03, and Protollin) and routes of administration (intramuscular [i.m.] and intranasal [i.n.]) for assessing their effect on the immunogenicity and cross-reactivity of inactivated split vaccines (A/H3N2/New York/55/2004). Humoral and T cell-mediated immune responses against the homologous virus and a heterologous drifted strain (A/H3N2/Wisconsin/67/2005) were measured in BALB/c mice at 2, 6, and 19 weeks postboost. The AS03- and Alum-adjuvanted i.m. vaccines induced at least an 8-fold increase over the nonadjuvanted vaccine in functional antibody titers against both the homotypic and heterotypic strains and low IgG2a and high IgG1 levels, suggesting a mixed Th1/Th2 response with a Th2 trend. The Protollin-adjuvanted i.n. vaccine induced the lowest IgG1/IgG2a ratio, which is indicative of a mixed Th1/Th2-type profile with a Th1 trend. This adjuvanted vaccine was the only vaccine to stimulate a mucosal IgA response. Whatever the timing after the boost, both hemagglutination inhibition (HAI) and microneutralization (MN) titers were higher with the AS03-adjuvanted i.m. vaccine than with the protollin-adjuvanted i.n. vaccine. Finally, the Alum-adjuvanted i.m. vaccine and the lower-dose Protollin-adjuvanted i.n. vaccine elicited significantly higher CD4 + Th1 and Th2 responses and more gamma interferon (IFN-γ)-producing CD8 + T cells than the nonadjuvanted vaccine. Our data indicate that the adjuvanted vaccines tested in this study can elicit stronger, more persistent, and broader immune responses against A/H3N2 strains than nonadjuvanted inactivated influenza vaccines.