In this study we assessed the ability of Middle East respiratory syndrome coronavirus (MERSCoV) to replicate and induce innate immunity in human monocyte-derived macrophages and dendritic cells (MDDCs), and compared it with severe acute respiratory syndrome coronavirus (SARS-CoV). Assessments of viral protein and RNA levels in infected cells showed that both viruses were impaired in their ability to replicate in these cells. Some induction of IFN-l1, CXCL10 and MxA mRNAs in both macrophages and MDDCs was seen in response to MERS-CoV infection, but almost no such induction was observed in response to SARS-CoV infection. ELISA and Western blot assays showed clear production of CXCL10 and MxA in MERS-CoV-infected macrophages and MDDCs. Our data suggest that SARS-CoV and MERS-CoV replicate poorly in human macrophages and MDDCs, but MERS-CoV is nonetheless capable of inducing a readily detectable host innate immune response. Our results highlight a clear difference between the viruses in activating host innate immune responses in macrophages and MDDCs, which may contribute to the pathogenesis of infection.

Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a Influenza A and B viruses are important respiratory pathogens and cause seasonal epidemics with an estimated 250,000 to 500,000 deaths annually. Influenza A and B viruses are structurally similar: they are negative-sense RNA viruses with a singlestranded segmented genome. The genome is structured in eight viral ribonucleoprotein (vRNP) complexes where the singlestranded RNA (ssRNA) is associated with multiple nucleoprotein (NP) molecules and a polymerase complex consisting of the PB1, PB2, and PA proteins (1). The vRNP complexes are packaged in a matrix protein shell surrounded by a host-derived lipid envelope in which the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA) are embedded. Influenza viruses bind to sialic acids on cell surface glycoproteins and enter the cells mainly via clathrin-mediated endocytosis but also by macropinocytosis and clathrin-independent entry pathways (2, 3). Influenza viruses take advantage of the host endocytic pathway; a reduction of pH during the maturation of endosomes induces a conformational change in viral HA molecules and triggers fusion between viral and endosomal membranes. Fusion is followed by the uncoating of the capsid by M1 dissociation due to acidification of the virion via the M2 ion channel protein. This results in the release of vRNPs into the cytosol. The influenza virus genome is then imported into the nucleus for transcription and replication of viral genes. Primary transcription of the viral genome is triggered by the virion-associated polymerase protein complex, which leads to the translation of early viral proteins in the cell cytoplasm. Newly synthesized polymerase, NP, and NS1 proteins are transported into the nucleus, where they initiate and regulate the replication and synthesis of cRNA and viral RNA (vRNA) molecules, followed by secondary rounds of transcription. At later stages of infection, new vRNP complexes are packaged in the nucleus, followed by M1-and nuclear export protein (NEP)-regulated export of vRNPs into the cytoplasm. Here they associate with viral envelope glycoproteins HA and NA on the plasma membrane, leading to budding of the newly formed viral particles (4).

The Zika virus (ZIKV) is a member of the Flaviviridae family and an important human pathogen. Most pathogenic viruses encode proteins that interfere with the activation of host innate immune responses. Like other flaviviruses, ZIKV interferes with the expression of interferon (IFN) genes and inhibits IFN-induced antiviral responses. ZIKV infects through epithelial barriers where IFN-λ1 is an important antiviral molecule. In this study, we analyzed the effects of ZIKV proteins on the activation of IFN-λ1 promoter. All ZIKV proteins were cloned and transiently expressed. ZIKV NS5, but no other ZIKV protein, was able to interfere with the RIG-I signaling pathway. This inhibition took place upstream of interferon regulatory factor 3 (IRF3) resulting in reduced phosphorylation of IRF3 and reduced activation of IFN-λ1 promoter. Furthermore, we showed that ZIKV NS5 interacts with the protein kinase IKKε, which is likely critical to the observed inhibition of phosphorylation of IRF3.

Influenza A viruses cause recurrent epidemics and occasional global pandemics. Wild birds are the natural reservoir of influenza A virus from where the virus can be transmitted to poultry or to mammals including humans. Mortality among humans in the highly pathogenic avian influenza H5N1 virus infection is even 60%. Despite intense research, there are still open questions in the pathogenicity of the H5N1 virus in humans. To characterize the H5N1 virus infection in human monocyte-derived macrophages (Mɸs) and dendritic cells (DCs), we used human isolates of highly pathogenic H5N1/2004 and H5N1/1997 and low pathogenic H7N9/2013 avian influenza viruses in comparison with a seasonal H3N2/1989 virus. We noticed that the H5N1 viruses have an overwhelming ability to replicate and spread in primary human immune cell cultures, and even the addition of trypsin did not equalize the infectivity of H7N9 or H3N2 viruses to the level seen with H5N1 virus. H5N1 virus stocks contained more often propagation-competent viruses than the H7N9 or H3N2 viruses. The data also showed that human DCs and Mɸs maintain 1,000- and 10,000-fold increase in the production of infectious H5N1 virus, respectively. Both analyzed highly pathogenic H5N1 viruses showed multi-cycle infection in primary human DCs and Mɸs, whereas the H3N2 and H7N9 viruses were incapable of spreading in immune cells. Interestingly, H5N1 virus was able to spread extremely efficiently despite the strong induction of antiviral interferon gene expression, which may in part explain the high pathogenicity of H5N1 virus infection in humans.

Influenza A viruses (IAVs) are viral pathogens that cause epidemics and occasional pandemics of significant mortality. The generation of efficacious vaccines and antiviral drugs remains a challenge due to the rapid appearance of new influenza virus types and antigenic variants. Consequently, novel strategies for the prevention and treatment of IAV infections are needed, given the limitations of the presently available antivirals. Here, we used enzymatically produced IAV-specific double-stranded RNA (dsRNA) molecules and Giardia intestinalis Dicer for the generation of a swarm of small interfering RNA (siRNA) molecules. The siRNAs target multiple conserved genomic regions of the IAVs. In mammalian cells, the produced 25-to 27-nucleotide-long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated Dicer-substrate siRNAs (DsiRNAs). We evaluated the efficacy of the above DsiRNA swarm at preventing IAV infections in human primary monocyte-derived macrophages and dendritic cells. The replication of different IAV strains, including avian influenza H5N1 and H7N9 viruses, was significantly inhibited by pretransfection of the cells with the IAV-specific DsiRNA swarm. Up to 7 orders of magnitude inhibition of viral RNA expression was observed, which led to a dramatic inhibition of IAV protein synthesis and virus production. The IAV-specific DsiRNA swarm inhibited virus replication directly through the RNA interference pathway although a weak induction of innate interferon responses was detected. Our results provide direct evidence for the feasibility of the siRNA strategy and the potency of DsiRNA swarms in the prevention and treatment of influenza, including the highly pathogenic avian influenza viruses. IMPORTANCE In spite of the enormous amount of research, influenza virus is still one of the major challenges for medical virology due to its capacity to generate new variants, which potentially lead to severe epidemics and pandemics. We demonstrated here that a swarm of small interfering RNA (siRNA) molecules, including more than 100 different antiviral RNA molecules targeting the most conserved regions of the influenza A virus genome, could efficiently inhibit the replication of all tested avian and seasonal influenza A variants in human primary monocyte-derived macrophages and dendritic cells. The wide antiviral spectrum makes the virusspecific siRNA swarm a potentially efficient treatment modality against both avian and seasonal influenza viruses. KEYWORDSDicer-substrate siRNA, DsiRNA, RNA interference, avian influenza virus, gene silencing, human macrophage, human moDC, influenza A virus, IAV, interferon response, IFN, siRNA swarm, viral replication Citation Jiang M, Österlund P, Westenius V, Guo D, Poranen MM, Bamford DH, Julkunen I. 2019. Efficient inhibition of avian and seasonal influenza A viruses by a virus-specific Dicersubstrate small interfering RNA swarm in human monocyte-derived macrophages and dendritic cells. J Virol 93:e01916-18. https://doi .

Zika virus (ZIKV) infections in humans are considered to be mild or subclinical. However, during the recent epidemics in the Pacific Islands and the Americas, the infection was associated with Quillain-Barré syndrome and congenital infections with fetal brain abnormalities, including microcephaly. Thus, more detailed understanding of ZIKV-host cell interactions and regulation of innate immune responses by strains of differential evolutionary origin is required. Here, we characterized the infection and immune responses triggered by two epidemic Asian/American lineage viruses, including an isolate from fetal brains, and a historical, low passage 1947 African lineage virus in human monocyte-derived dendritic cells (DCs) and macrophages. The epidemic Asian/American ZIKV replicated well and induced relatively good antiviral responses in human DCs whereas the African strain replicated less efficiently and induced weaker immune responses. In macrophages both the African and Asian strains showed limited replication and relatively weak cytokine gene expression. Interestingly, in macrophages we observed host protein degradation, especially IRF3 and STAT2, at early phases of infection with both lineage viruses, suggesting an early proteasomal activation in phagocytic cells. Our data indicates that ZIKV evolution has led to significant phenotypic differences in the replication characteristics leading to differential regulation of host innate immune responses.

Avian influenza A (H9N2) viruses have occasionally been identified in humans with upper respiratory tract infections. The novel H7N9/2013 virus identified in China shows that a low pathogenic avian influenza (LPAI) virus can be highly pathogenic in humans. Therefore, it is important to understand virus-host cell interactions and immune responses triggered by LPAI viruses in humans. We found that LPAI A/Hong Kong/1073/99 (H9N2) virus replicated efficiently in human dendritic cells (DCs). The H9N2 virus induced strong IFN gene expression although with different kinetics than seasonal influenza A/Beijing/353/89 (H3N2) virus. IFN inducible antiviral proteins were produced in H9N2 virus-infected cells at the same level as in H3N2 infection. The H9N2 virus was extremely sensitive to the antiviral actions of type I IFNs. These results indicate that the avian influenza H9N2 virus is inducing a strong antiviral IFN response in human DCs.

Background: Measles is a highly contagious disease caused by measles virus (MV), which is an envelope, negative sense virus that contains a single stranded RNA genome. The vaccination of measles-containing vaccines is scheduled at 12-15 months of age for first does and at 4-6 years of age for second does. According to successful national immunization program, measles had been eliminated in the Republic of Korea. However during 2013-2014, several measles outbreaks have occurred in unvaccinated population and even in a highly vaccinated population. In this study, we investigated the seroprevalence of measles virus in children aged 0-9 years.Methods: Total 1000 serum samples were collected among Korean children 0-9 years of age in 2014. Serum samples were analyzed for the presence of measles-specific IgG antibodies by enzyme immunoassay. We excluded samples referred for the diagnosis of measles, mumps, rubella or HIV.Results: Measles-specific IgG antibodies were detected in 49.5% of Korean children aged 0-9 years. Passive transferred antibodies against measles were present in only 20.9% of infants aged 0-7 months and exhausted all infants aged 8-11 months. After onedoes vaccination, the seropositivity rate for measles was observed in 98% of children aged 2 years and declined slightly to 96% for children aged 3-4 years. All of children aged 5-6 years who were vaccinated twice had IgG-specific measles antibodies and the antibody level declined continuously in age groups.Conclusion: The results of this study showed that maternally derived measles-specific IgG antibodies declined significantly and expired at 8 months after born but the level of immunity for measles is fully occupied after second-doses of measles vaccination. However the immunity gap after exhaustion of maternal measles antibodies before one-does measles vaccination is need to solve for protection of children from the measles.