Genome evolution of dengue virus serotype 1 under selection by Wolbachia pipientis in Aedes aegypti mosquitoes.

The introgression of antiviral strains of Wolbachia into Aedes aegypti mosquito populations is a public health intervention for the control of dengue. Plausibly, dengue virus (DENV) could evolve to bypass the antiviral effects of Wolbachia and undermine this approach. Here, we established a serial-passage system to investigate the evolution of DENV in Ae. aegypti mosquitoes infected with the wMel strain of Wolbachia. Using this system, we report on virus genetic outcomes after twenty passages of serotype 1 of DENV (DENV-1). An amino acid substitution, E203K, in the DENV-1 envelope protein was more frequently detected in the consensus sequence of virus populations passaged in wMel-infected Ae. aegypti than wild-type counterparts. Positive selection at residue 203 was reproducible; it occurred in passaged virus populations from independent DENV-1-infected patients and also in a second, independent experimental system. In wild-type mosquitoes and human cells, the 203K variant was rapidly replaced by the progenitor sequence. These findings provide proof of concept that wMel-associated selection of virus populations can occur in experimental conditions. Field-based studies are needed to explore whether wMel imparts selective pressure on DENV evolution in locations where wMel is established.

Using Wolbachia to Eliminate Dengue: Will the Virus Fight Back?

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.

Microbiomes in the insectivorous bat species Mops condylurus rapidly converge in captivity.

Bats are well known reservoir hosts for RNA and DNA viruses. The use of captive bats in research has intensified over the past decade as researchers aim to examine the virus-reservoir host interface. In this study, we investigated the effects of captivity on the fecal bacterial microbiome of an insectivorous microbat, Mops condylurus, a species that roosts in close proximity to humans and has likely transmitted viral infections to humans. Using amplicon 16S rRNA gene sequencing, we characterized changes in fecal bacterial community composition for individual bats directly at the time of capture and again after six weeks in captivity. We found that microbial community richness by measure of the number of observed operational taxonomic units (OTUs) in bat feces increases in captivity. Importantly, we found the similarity of microbial community structures of fecal microbiomes between different bats to converge during captivity. We propose a six week-acclimatization period prior to carrying out infection studies or other research influenced by the microbiome composition, which may be advantageous to reduce variation in microbiome composition and minimize biological variation inherent to in vivo experimental studies.

Utility of primary cells to examine NPC1 receptor expression in Mops condylurus, a potential Ebola virus reservoir.

The significance of the integral membrane protein Niemann-Pick C1 (NPC1) in the ebolavirus entry process has been determined using various cell lines derived from humans, non-human primates and fruit bats. Fruit bats have long been purported as the potential reservoir host for ebolaviruses, however several studies provide evidence that Mops condylurus, an insectivorous microbat, is also an ebolavirus reservoir. NPC1 receptor expression in the context of ebolavirus replication in microbat cells remains unstudied. In order to study Ebola virus (EBOV) cellular entry and replication in M. condylurus, we derived primary and immortalized cell cultures from 12 different organs. The NPC1 receptor expression was characterized by confocal microscopy and flow cytometry comparing the expression levels of M. condylurus primary and immortalized cells, HeLa cells, human embryonic kidney cells and cells from a European microbat species. EBOV replication kinetics was studied for four representative cell cultures using qRT-PCR. The aim was to elucidate the suitability of primary and immortalized cells from different tissues for studying NPC1 receptor expression levels and their potential influence on EBOV replication. The NPC1 receptor expression level in M. condylurus primary cells differed depending on the organ they were derived from and was for most cell types significantly lower than in human cell lines. Immortalized cells showed for most cell types higher expression levels than their corresponding primary cells. Concluding from our infection experiments with EBOV we suggest a potential correlation between NPC1 receptor expression level and virus replication rate in vitro.

Dendritic Cells Generated From Mops condylurus, a Likely Filovirus Reservoir Host, Are Susceptible to and Activated by Zaire Ebolavirus Infection.

Ebola virus infection of human dendritic cells (DCs) induces atypical adaptive immune responses and thereby exacerbates Ebola virus disease (EVD). Human DCs, infected with Ebola virus aberrantly express low levels of the DC activation markers CD80, CD86, and MHC class II. The T cell responses ensuing are commonly anergic rather than protective against EVD. We hypothesize that DCs derived from potential reservoir hosts such as bats, which do not develop disease signs in response to Ebola virus infection, would exhibit features associated with activation. In this study, we have examined Zaire ebolavirus (EBOV) infection of DCs derived from the Angolan free-tailed bat species, Mops condylurus. This species was previously identified as permissive to EBOV infection in vivo, in the absence of disease signs. M. condylurus has also been recently implicated as the reservoir host for Bombali ebolavirus, a virus species that is closely related to EBOV. Due to the absence of pre-existing M. condylurus species-specific reagents, we characterized its de novo assembled transcriptome and defined its phylogenetic similarity to other mammals, which enabled the identification of cross-reactive reagents for M. condylurus bone marrow-derived DC (bat-BMDC) differentiation and immune cell phenotyping. Our results reveal that bat-BMDCs are susceptible to EBOV infection as determined by detection of EBOV specific viral RNA (vRNA). vRNA increased significantly 72 h after EBOV-infection and was detected in both cells and in culture supernatants. Bat-BMDC infection was further confirmed by the observation of GFP expression in DC cultures infected with a recombinant GFP-EBOV. Bat-BMDCs upregulated CD80 and chemokine ligand 3 (CCL3) transcripts in response to EBOV infection, which positively correlated with the expression levels of EBOV vRNA. In contrast to the aberrant responses to EBOV infection that are typical for human-DC, our findings from bat-BMDCs provide evidence for an immunological basis of asymptomatic EBOV infection outcomes.

Inhibition of Influenza A Virus by Human Infant Saliva.

Innate antiviral factors in saliva play a role in protection against respiratory infections. We tested the anti-influenza virus activities of saliva samples taken from human infants, 1-12 months old, with no history of prior exposure to influenza. In contrast to the inhibitory activity we observed in mouse and ferret saliva, the activity of human infant saliva was complex, with both sialic acid-dependent and independent components, the proportion of which differed between individuals. Taken as a whole, we showed that the major anti-influenza activity of infant saliva is acquired over the first year of life and is associated with sialic acid-containing molecules. The activity of sialic acid-independent inhibitors was lower overall, more variable between individuals, and less dependent on age. The results show that the saliva of very young infants can provide a degree of protection against influenza, which may be critical in the absence of adaptive immunity.

Coronavirus and paramyxovirus in bats from Northwest Italy.

BACKGROUND: Bat-borne virus surveillance is necessary for determining inter-species transmission risks and is important due to the wide-range of bat species which may harbour potential pathogens. This study aimed to monitor coronaviruses (CoVs) and paramyxoviruses (PMVs) in bats roosting in northwest Italian regions. Our investigation was focused on CoVs and PMVs due to their proven ability to switch host and their zoonotic potential. Here we provide the phylogenetic characterization of the highly conserved polymerase gene fragments. RESULTS: Family-wide PCR screenings were used to test 302 bats belonging to 19 different bat species. Thirty-eight animals from 12 locations were confirmed as PCR positive, with an overall detection rate of 12.6% [95% CI: 9.3-16.8]. CoV RNA was found in 36 bats belonging to eight species, while PMV RNA in three Pipistrellus spp. Phylogenetic characterization have been obtained for 15 alpha- CoVs, 5 beta-CoVs and three PMVs; moreover one P. pipistrellus resulted co-infected with both CoV and PMV. A divergent alpha-CoV clade from Myotis nattereri SpA is also described. The compact cluster of beta-CoVs from R. ferrumequinum roosts expands the current viral sequence database, specifically for this species in Europe. To our knowledge this is the first report of CoVs in Plecotus auritus and M. oxygnathus, and of PMVs in P. kuhlii. CONCLUSIONS: This study identified alpha and beta-CoVs in new bat species and in previously unsurveyed Italian regions. To our knowledge this represents the first and unique report of PMVs in Italy. The 23 new bat genetic sequences presented will expand the current molecular bat-borne virus databases. Considering the amount of novel bat-borne PMVs associated with the emergence of zoonotic infections in animals and humans in the last years, the definition of viral diversity within European bat species is needed. Performing surveillance studies within a specific geographic area can provide awareness of viral burden where bats roost in close proximity to spillover hosts, and form the basis for the appropriate control measures against potential threats for public health and optimal management of bats and their habitats.

Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes.

UNLABELLED: Although avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease. IMPORTANCE: Risks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated liquid. We also showed that virus could spread from the respiratory tract to the brain, which was associated with end-stage disease, and very early viremia provided a marker for this. With upper respiratory tract exposure, infection of the brain was common but hard to detect, suggesting that human neurological infections might be typically undetected at autopsy. However, viral spread to systemic sites was slower after exposure to virus by this route than when virus was additionally delivered to the lungs, providing a better therapeutic window. In addition to exposure history, early parameters of infection, such as viremia, could help prioritize antiviral treatments for patients most at risk of succumbing to infection.

Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-System-Mediated Protection and Subsequent Long-Term Adaptive Immunity.

UNLABELLED: The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity. IMPORTANCE: The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system responds within hours of exposure to infectious agents, while adaptive immunity takes several days to become effective. Here we show, by using a simple lipopeptide-based TLR2 agonist, that an influenza detergent-split vaccine can be made to simultaneously stimulate and amplify both systems to provide immediate antiviral protection while giving the adaptive immune system time to implement long-term immunity. Both types of immunity induced by this approach protect against vaccine-matched as well as unrelated virus strains and potentially even against strains yet to be encountered. Conferring dual functionality to influenza vaccines is beneficial for improving community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative.

Helping themselves: optimal virus-specific CD4 T cell responses require help via CD4 T cell licensing of dendritic cells.

Although CD4(+) T cell help (Th) is critical for inducing optimal B cell and CD8(+) T cell responses, it remains unclear whether induction of CD4(+) Th responses postinfection are also dependent on CD4(+) T cell help. In this study, we show that activation of adoptively transferred Th cells during primary influenza A virus (IAV) infection enhances both the magnitude and functional breadth of endogenous primary IAV-specific CD4(+) T cell responses. This enhancement was dependent on CD154-CD40-dependent dendritic cell licensing and resulted in a greater recall capacity of IAV-specific CD4(+) and CD8(+) T memory responses after heterologous IAV infection. These data suggest that engaging pre-existing CD4 responses at the time of priming may be a strategy for improving cellular immunity after vaccination.

CD154+ CD4+ T-cell dependence for effective memory influenza virus-specific CD8+ T-cell responses.

CD40-CD154 (CD40 ligand) interactions are essential for the efficient priming of CD8(+) cytotoxic T lymphocyte (CTL) responses. This is typically via CD4(+)CD154(+) T-cell-dependent 'licensing' of CD40(+) dendritic cells (DCs); however, DCs infected with influenza A virus (IAV) upregulate CD154 expression, thus enabling efficient CTL priming in the absence of CD4(+) T activation. Therefore, it is unclear whether CD4 T cells and DCs have redundant or unique roles in the priming of primary and secondary CTL responses after infection. Here we determine the precise cellular interactions involved in CD40-CD154 regulation of both primary and secondary IAV-specific CTL responses. Infection of both CD40 KO and CD154 KO mice resulted in diminished quantitative and qualitative CTL responses after both primary and secondary infection. Adoptive transfer of CD154(+), but not CD154 KO, CD4 T cells into CD154 KO mice restored both primary and secondary IAV-specific CD8 T-cell responses. These data show that, although CD154 expression on CD4 T cells and other cell types (that is, DCs) may be redundant for the priming of primary CTL responses, CD154 expression by CD4 T cells is required for the priming memory CD8 T cells that are capable of fully responding to secondary infection.

Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses.

Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that α(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of α(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.

Influenza-induced inflammation drives pneumococcal otitis media.

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

A mouse model for the study of contact-dependent transmission of influenza A virus and the factors that govern transmissibility.

Influenza A virus transmission by direct contact is not well characterized. Here, we describe a mouse model for investigation of factors regulating contact-dependent transmission. Strains within the H3N2 but not H1N1 subtype of influenza virus were transmissible, and reverse-engineered viruses representing hybrids of these subtypes showed that the viral hemagglutinin is a determinant of the transmissible phenotype. Transmission to contact mice occurred within the first 6 to 54 h after cohousing with directly infected index mice, and the proportion of contacts infected within this period was reduced if the index mice had been preinfected with a heterologous subtype virus. A threshold level of virus present in the saliva of the index mice was identified, above which the likelihood of transmission was greatly increased. There was no correlation with transmission and viral loads in the nose or lung. This model could be useful for preclinical evaluation of antiviral and vaccine efficacy in combating contact-dependent transmission of influenza.

Intranasal administration of the TLR2 agonist Pam2Cys provides rapid protection against influenza in mice.

The protective role played by the innate immune system during early stages of infection suggests that compounds which stimulate innate responses could be used as antimicrobial or antiviral agents. In this study, we demonstrate that the Toll-like receptor-2 agonist Pam2Cys, when administered intranasally, triggers a cascade of inflammatory and innate immune signals, acting as an immunostimulant by attracting neutrophils and macrophages and inducing secretion of IL-2, IL-6, IL-10, IFN-γ, MCP-1 and TNF-α. These changes provide increased resistance against influenza A virus challenge and also reduce the potential for transmission of infection. Pam2Cys treatment also reduced weight loss and lethality associated with virulent influenza virus infection in a Toll-like receptor-2-dependent manner. Treatment did not affect the animals' ability to generate an adaptive immune response, measured by the induction of functional influenza A virus-specific CD8(+) T cells following exposure to virus. Because this compound demonstrates efficacy against distinct strains of influenza, it could be a candidate for development as an agent against influenza and possibly other respiratory pathogens.