Influenza B viruses have two genetically and antigenically distinct lineages, B/Victoria/2/1987-like (VIC) and B/Yamagata/16/1988-like (YAM) viruses, that emerged in the 1980s and co-circulate annually during the influenza season. During the 2016-2017 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (AA) deletions in the hemagglutinin protein. Hemagglutination inhibition assays demonstrate that these deletion variant influenza B/VIC viruses are antigenically distinct from each other and from the progenitor B/VIC virus that lacks the deletion. Therefore, there are currently four antigenically distinct HA proteins expressed by influenza B co-circulating: B/YAM, B/VIC V1A (no deletion), B/VIC V1A.1 (two-AA deletion), and B/VIC V1A.2 and V1A.3 (three-AA deletion). The prevalence of these viruses differs across geographic regions, making it critical to have a sensitive, rapid diagnostic assay(s) that detect and distinguish these Influenza B variant viruses during surveillance. Here, we present a real time RT-PCR assay that targets the influenza B/VIC deletion region in the HA gene and detects and distinguishes the influenza B/VIC V1A, B/VIC V1A.1, B/VIC V1A.2 and B/VIC V1A.3 variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost. Coupling this assay with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterization of circulating influenza B viruses. Having accurate and detailed surveillance information on these distinct Influenza B variant viruses will provide insight into the prevalence and geographic distribution and could aid in vaccine recommendations.

We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 10(2.0), influenza B virus at 10(2.2), and SARS-CoV-2 at 10(0.3) 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2.

BackgroundDuring the 2016/17 influenza season, influenza B/VIC lineage variant viruses emerged with two (K(162)N(163)) or three (K(162)N(163)D(164)) amino acid (aa) deletions in the haemagglutinin (HA) protein. There are currently five antigenically distinct HA proteins expressed by co-circulating influenza B viruses: B/YAM, B/VIC V1A (no deletion), B/VIC V1A-2DEL (2 aa deletion) and two antigenically distinguishable groups of B/VIC V1A-3DEL (3 aa deletion). The prevalence of these viruses differs across geographical regions, making it critical to have a sensitive, rapid diagnostic assay that detects and distinguishes these influenza B variant viruses during surveillance.AimOur objective was to develop a real-time RT-PCR (rRT-PCR) assay for detection and discrimination of influenza B/VIC lineage variant viruses.MethodsWe designed a diagnostic assay with one pair of conserved primers and three probes specific to each genetic group. We used propagated influenza B/VIC variant viruses and clinical specimens to assess assay performance.ResultsThis rRT-PCR assay detects and distinguishes the influenza B/VIC V1A, B/VIC V1A-2DEL, and B/VIC V1A-3DEL variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost.ConclusionCoupling this assay with the Centers for Disease Control and Prevention's Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterisation of circulating influenza B viruses. Detailed surveillance information on these distinct influenza B variant viruses will provide insight into their prevalence and geographical distribution and could aid in vaccine recommendations.

BACKGROUND: An alarming rise in reported Lassa fever cases continues in west Africa. Liberia has the largest reported per capita incidence of Lassa fever cases in the region, but genomic information on the circulating strains is scarce. The aim of this study was to substantially increase the available pool of data to help foster the generation of targeted diagnostics and therapeutics. METHODS: Clinical serum samples collected from 17 positive Lassa fever cases originating from Liberia (16 cases) and Guinea (one case) within the past decade were processed at the Liberian Institute for Biomedical Research using a targeted-enrichment sequencing approach, producing 17 near-complete genomes. An additional 17 Lassa virus sequences (two from Guinea, seven from Liberia, four from Nigeria, and four from Sierra Leone) were generated from viral stocks at the US Centers for Disease Control and Prevention (Atlanta, GA) from samples originating from the Mano River Union (Guinea, Liberia, and Sierra Leone) region and Nigeria. Sequences were compared with existing Lassa virus genomes and published Lassa virus assays. FINDINGS: The 23 new Liberian Lassa virus genomes grouped within two clades (IV.A and IV.B) and were genetically divergent from those circulating elsewhere in west Africa. A time-calibrated phylogeographic analysis incorporating the new genomes suggests Liberia was the entry point of Lassa virus into the Mano River Union region and estimates the introduction to have occurred between 300-350 years ago. A high level of diversity exists between the Liberian Lassa virus genomes. Nucleotide percent difference between Liberian Lassa virus genomes ranged up to 27% in the L segment and 18% in the S segment. The commonly used Lassa Josiah-MGB assay was up to 25% divergent across the target sites when aligned to the Liberian Lassa virus genomes. INTERPRETATION: The large amount of novel genomic diversity of Lassa virus observed in the Liberian cases emphasises the need to match deployed diagnostic capabilities with locally circulating strains and underscores the importance of evaluating cross-lineage protection in the development of vaccines and therapeutics. FUNDING: Defense Biological Product Assurance Office of the US Department of Defense and the Armed Forces Health Surveillance Branch and its Global Emerging Infections Surveillance and Response Section.

In March 2016, an outbreak of Rift Valley fever (RVF) was identified in Kabale district, southwestern Uganda. A comprehensive outbreak investigation was initiated, including human, livestock, and mosquito vector investigations. Overall, four cases of acute, nonfatal human disease were identified, three by RVF virus (RVFV) reverse transcriptase polymerase chain reaction (RT-PCR), and one by IgM and IgG serology. Investigations of cattle, sheep, and goat samples from homes and villages of confirmed and probable RVF cases and the Kabale central abattoir found that eight of 83 (10%) animals were positive for RVFV by IgG serology; one goat from the home of a confirmed case tested positive by RT-PCR. Whole genome sequencing from three clinical specimens was performed and phylogenetic analysis inferred the relatedness of 2016 RVFV with the 2006-2007 Kenya-2 clade, suggesting previous introduction of RVFV into southwestern Uganda. An entomological survey identified three of 298 pools (1%) of Aedes and Coquillettidia species that were RVFV positive by RT-PCR. This was the first identification of RVFV in Uganda in 48 years and the 10(th) independent viral hemorrhagic fever outbreak to be confirmed in Uganda since 2010.

The relationship between age, vitamin D status, expression and functionality of the vitamin D receptor (VDR), and key genes in the vitamin D pathway in immune cells is unclear. We enrolled adults 50 to 69 years old (20 subjects) and 70+ (20 subjects) and measured: 1) 25(OH)D levels by liquid chromatography/mass spectrometry; and 2) mRNA expression of VDR, 1alpha-OHase, 1,25D3-MARRS, TREM-1, cathelicidin, RIG-I, and interferon-beta by qRT-PCR. Mean serum 25(OH)D was 30 +/- 4 ng/mL and was not associated with age. Baseline expression of VDR, 1alpha-OHase, 1,25D3-MARRS, TREM-1, and RIG-I also did not differ by age; IFN-beta expression, however, was higher in the 70+ year old group. 25(OH)D3- and 1,25(OH)2D3-induced VDR, TREM-1 and cathelicidin expression were similar between age groups, as was LPS-induced expression of VDR and of 1alpha-OHase. Ligand-induced 1,25D3-MARRS expression was higher in subjects ≥ 70 years. Serum 25(OH)D was inversely associated with LPS-stimulated VDR expression and with baseline or vitamin D-induced TREM-1 expression, adjusting for age, self-rated health, and functional status. In healthy adults ≥ 50 years, the expression and functionality of the VDR, 1alpha-OHase and key vitamin D pathway genes were not consistently associated with age.

To enhance the immunogenicity of the Influenza H5N1 vaccine, we developed an oil-in-water nanoemulsion (NE) adjuvant. NE displayed good temperature stability and maintained particle size. More importantly, it significantly enhanced IL-6 and MCP-1 production to recruit innate cells, including neutrophils, monocytes/macrophages and dendritic cells to the local environment. Furthermore, NE enhanced dendritic cell function to induce robust antigen-specific T and B cell immune responses. NE-adjuvanted H5N1 vaccine not only elicited significantly higher and long-lasting antibody responses, but also conferred enhanced protection against homologous clade 1 as well as heterologous clade 2 H5N1 virus challenge in young as well as in aged mice. The pre-existing immunity to seasonal influenza did not affect the immunogenicity of NE-adjuvanted H5N1 vaccine.

The NLR protein, NLRC5, is an important regulator of MHC I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial (NHBE) cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-beta expression. Influenza virus leads to induction of IFN-beta that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus-induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in a LRR-domain-independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.

Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012-2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate.

Original antigenic sin is a phenomenon wherein sequential exposure to closely related influenza virus variants reduces antibody (Ab) response to novel antigenic determinants in the second strain and, consequently, impairs the development of immune memory. This could pose a risk to the development of immune memory in persons previously infected with or vaccinated against influenza. Here, we explored strategies to overcome original antigenic sin responses in mice sequentially exposed to two closely related hemagglutinin 1 neuraminidase 1 (H1N1) influenza strains A/PR/8/34 and A/FM/1/47. We found that dendritic cell-activating adjuvants [Bordetella pertussis toxin (PT) or CpG ODN or a squalene-based oil-in-water nanoemulsion (NE)], upon administration during the second viral exposure, completely protected mice from a lethal challenge and enhanced neutralizing-Ab titers against the second virus. Interestingly, PT and NE adjuvants when administered during the first immunization even prevented original antigenic sin in subsequent immunization without any adjuvants. As an alternative to using adjuvants, we also found that repeated immunization with the second viral strain relieved the effects of original antigenic sin. Taken together, our studies provide at least three ways of overcoming original antigenic sin.

Recognition of pathogen associated molecular patterns by pattern recognition receptors of the innate immune system is crucial for the initiation of innate and adaptive responses and for immunological memory. We investigated the role of TLR7 in the induction of adaptive immunity and long-term memory following influenza virus infection and vaccination in C57Bl/6 mice. During infection with influenza A/PR8/34 virus, the absence of either TLR7 or MyD88 leads to reduced serum IgM and fewer IgM secreting B-cells in their secondary lymphoid organs, particularly in bone marrow. In spite of this, the absence of TLR7/MyD88 signaling did not impair the production of protective antibodies. Following immunization with the 2009 pandemic inactivated split vaccine, TLR7(-/-) mice had significantly lower levels of germinal center formation, antibody secreting cells and circulating influenza-specific antibodies than control animals. Consequently, TLR7(-/-) mice failed to develop protective immunological memory upon challenge. Furthermore, the immunogenicity of the split vaccine was likely due to TLR7 recognition of virion RNA, as its removal from the split vaccine significantly reduced the levels of influenza-specific antibodies, and compromised the vaccine protective efficacy in mice. Taken together, our data demonstrate that TLR7 plays an important role in vaccine-induced humoral immune responses to influenza through the interaction with viral RNA present in the split vaccine.

Myeloid dendritic cells (mDCs) have long been thought to function as classical APCs for T cell responses. However, we demonstrate that influenza viruses induce rapid differentiation of human monocytes into mDCs. Unlike the classic mDCs, the virus-induced mDCs failed to upregulate DC maturation markers and were unable to induce allogeneic lymphoproliferation. Virus-induced mDCs secreted little, if any, proinflammatory cytokines; however, they secreted a substantial amount of chemoattractants for monocytes (MCP-1 and IP-10). Interestingly, the differentiated mDCs secreted type I IFN and upregulated the expression of IFN-stimulated genes (tetherin, IFITM3, and viperin), as well as cytosolic viral RNA sensors (RIG-I and MDA5). Additionally, culture supernatants from virus-induced mDCs suppressed the replication of virus in vitro. Furthermore, depletion of monocytes in a mouse model of influenza infection caused significant reduction of lung mDC numbers, as well as type I IFN production in the lung. Consequently, increased lung virus titer and higher mortality were observed. Taken together, our results demonstrate that the host responds to influenza virus infection by initiating rapid differentiation of circulating monocytes into IFN-producing mDCs, which contribute to innate antiviral immune responses.

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to approximately 300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-beta (IFN-beta) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-beta by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.

Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune response against influenza A virus (IAV) infection; however, the role of Toll-like receptor 7 (TLR7) during the innate immune response to IAV infection and the cell types affected by the absence of TLR7 are not clearly understood. In this study, we show that myeloid derived suppressor cells (MDSC) accumulate in the lungs of TLR7 deficient mice more so than in wild-type C57Bl/6 mice, and display increased cytokine expression. Furthermore, there is an increase in production of Th2 cytokines by TLR7(-/-) compared with wildtype CD4+ T-cells in vivo, leading to a Th2 polarized humoral response. Our findings indicate that TLR7 modulates the accumulation of MDSCs during an IAV infection in mice, and that lack of TLR7 signaling leads to a Th2-biased response.

Retinoic-acid-inducible gene-I (RIG-I) is an important component of the innate immune response to many RNA viruses that limits viral replication until adaptive immunity becomes available to clear the infection. Upon binding to the nucleic acid genomes and replication intermediates of these viruses, RIG-I undergoes a complex activation process that involves post-translational modifications and structural rearrangements. Once activated, RIG-I upregulates well-studied signal transduction pathways that lead to the production of type-I interferons (IFNs) and a large variety of antiviral IFN-stimulated genes. Thus, an effective antiviral response is dependent on the interaction between pathogen-derived ligands and RIG-I. Recent work has begun to clarify the required characteristics of RIG-I activators and is setting the stage for the identification of authentic ligands used during viral infection.

The emergence of the pandemic 2009 H1N1 influenza virus has become a world-wide health concern. As drug resistance appears, a new generation of therapeutic strategies will be required. Here, we introduce a nanotechnology approach for the therapy of pan-demic and seasonal influenza virus infections. This approach uses gold nanorods (GNRs) to deliver an innate immune activator, producing a localized therapeutic response. We demonstrated the utility of a biocompatible gold nanorod, GNR-5'PPP-ssRNA nanoplex, as an antiviral strategy against type A influenza virus. In human respiratory bronchial epithelial cells, this nanoplex activated the retinoic acid-inducible gene I (RIG-I) pathogen recognition pathway, resulting in increased expression of IFN-beta and other IFN-stimulated genes (ISGs) (e.g., PKR, MDA5, IRF1, IRF7, and MX1). This increase in type I IFN and ISGs resulted in a decrease in the replication of H1N1 influenza viruses. These findings suggest that further evaluation of biocompatible nanoplexes as unique antivirals for treatment of seasonal and pandemic influenza viruses is warranted.

BACKGROUND: Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state. RESULTS: In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice. CONCLUSIONS: Our findings suggest that 5PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.