Remote immune processes revealed by immune-derived circulating cell-free DNA.

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here, we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N = 242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N = 92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with eosinophilic esophagitis (N = 21) and B-cell lymphoma (N = 27) showed selective elevation of eosinophil and B-cell cfDNA, respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

Diversity in the Circulation of Influenza A(H3N2) Viruses in the Northern Hemisphere in the 2018-19 Season.

While vaccination is considered the most effective means to prevent influenza infection, its seasonal effectiveness varies, depending on the circulating influenza strains. Here, we characterized the circulation of influenza strains in October-2018 and March-2019 around the world. For this, we used nasopharyngeal samples collected from outpatient and hospitalized patients in Israel and data reported in ECDC, CDC, and WHO databases. Influenza A(H3N2) was dominant in Israel, while in Europe, Asia, and USA, A(H1N1)pdm09 virus circulated first, and then the A(H3N2) virus also appeared. Phylogenetic analysis indicated that A(H3N2) viruses circulating in Israel belonged to clade-3C.3a, while in Europe, Asia, and USA, A(H3N2) viruses belonged to subclade-3C.2a1, but were later replaced by clade-3C.3a viruses in USA. The vaccine A(H3N2) components of that year, A/Singapore/INFIMH-16-0019/2016-(H3N2)-like-viruses, belonged to clade-3C.2a1. The circulation of different influenza subtypes and clades of A(H3N2) viruses in a single season highlights the need for universal influenza vaccines.

Genomic variation and epidemiology of SARS-CoV-2 importation and early circulation in Israel.

Severe acute respiratory disease coronavirus 2 (SARS-CoV-2) which causes corona virus disease (COVID-19) was first identified in Wuhan, China in December 2019 and has since led to a global pandemic. Importations of SARS-CoV-2 to Israel in late February from multiple countries initiated a rapid outbreak across the country. In this study, SARS-CoV-2 whole genomes were sequenced from 59 imported samples with a recorded country of importation and 101 early circulating samples in February to mid-March 2020 and analyzed to infer clades and mutational patterns with additional sequences identified Israel available in public databases. Recorded importations in February to mid-March, mostly from Europe, led to multiple transmissions in all districts in Israel. Although all SARS-CoV-2 defined clades were imported, clade 20C became the dominating clade in the circulating samples. Identification of novel, frequently altered mutated positions correlating with clade-defining positions provide data for surveillance of this evolving pandemic and spread of specific clades of this virus. SARS-CoV-2 continues to spread and mutate in Israel and across the globe. With economy and travel resuming, surveillance of clades and accumulating mutations is crucial for understanding its evolution and spread patterns and may aid in decision making concerning public health issues.

Transcriptomic profiling and genomic mutational analysis of Human coronavirus (HCoV)-229E -infected human cells.

Human coronaviruses (HCoVs) cause mild to severe respiratory infection. Most of the common cold illnesses are caused by one of four HCoVs, namely HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43. Several studies have applied global transcriptomic methods to understand host responses to HCoV infection, with most studies focusing on the pandemic severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and the newly emerging SARS-CoV-2. In this study, Next Generation Sequencing was used to gain new insights into cellular transcriptomic changes elicited by alphacoronavirus HCoV-229E. HCoV-229E-infected MRC-5 cells showed marked downregulation of superpathway of cholesterol biosynthesis and eIF2 signaling pathways. Moreover, upregulation of cyclins, cell cycle control of chromosomal replication, and the role of BRCA1 in DNA damage response, alongside downregulation of the cell cycle G1/S checkpoint, suggest that HCoV-229E may favors S phase for viral infection. Intriguingly, a significant portion of key factors of cell innate immunity, interferon-stimulated genes (ISGs) and other transcripts of early antiviral response genes were downregulated early in HCoV-229E infection. On the other hand, early upregulation of the antiviral response factor Apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) was observed. APOBEC3B cytidine deaminase signature (C-to-T) was previously observed in genomic analysis of SARS-CoV-2 but not HCoV-229E. Higher levels of C-to-T mutations were found in countries with high mortality rates caused by SARS-CoV-2. APOBEC activity could be a marker for new emerging CoVs. This study will enhance our understanding of commonly circulating HCoVs and hopefully provide critical information about still-emerging coronaviruses.

Altered NKp46 Recognition and Elimination of Influenza B Viruses.

Every year, millions of people worldwide are infected with influenza, causing enormous health and economic problems. The most common type of influenza is influenza A. It is known that Natural Killer (NK) cells play an important role in controlling influenza A infection, mostly through the recognition of the viral protein hemagglutinin (HA) by the activating receptor, NKp46. In contrast, little is known regarding NK cell recognition of influenza B viruses, even though they are responsible for a third of all pediatric influenza deaths and are therefore included in the seasonal vaccine each year. Here we show that NKp46 also recognizes influenza B viruses. We show that NKp46 binds the HA protein of influenza B in a sialic acid-dependent manner, and identified the glycosylated residue in NKp46, which is critical for this interaction. We discovered that this interaction has a binding affinity approximately seven times lower than NKp46 binding of influenza A's HA. Finally, we demonstrated, using mice deficient for the mouse orthologue of NKp46, named NCR1, that NKp46 is not important for influenza B elimination. These findings enable us to better understand the interactions between the different influenza viruses and NK cells that are known to be crucial for viral elimination.

Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins.

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016-2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

Comprehensive Analyses of SARS-CoV-2 Transmission in a Public Health Virology Laboratory.

SARS-CoV-2 has become a major global concern as of December 2019, particularly affecting healthcare workers. As person-to-person transmission is airborne, crowded closed spaces have high potential for rapid virus spread, especially early in the pandemic when social distancing and mask wearing were not mandatory. This retrospective study thoroughly investigates a small-scale SARS-CoV-2 outbreak in Israel's central virology laboratory (ICVL) in mid-March 2020, in which six staff members and two related family members were infected. Suspicions regarding infection by contaminated surfaces in ICVL facilities were nullified by SARS-CoV-2 negative real time polymerase chain reaction (PCR) of work surfaces swipe tests. Complete SARS-CoV-2 genomes were sequenced and mutation analyses showed inclusion of all samples to clades 20B and 20C, possessing the spike mutation D614G. Phylogenetic analysis clarified transmission events, confirming S1 as having infected at least three other staff members and refuting the association of a staff member's infected spouse with the ICVL transmission cluster. Finally, serology tests exhibited IgG and IgA antibodies in all infected individuals and revealed the occurrence of asymptomatic infections in additional staff members. This study demonstrates the advantages of molecular epidemiology in elucidating transmission events and exemplifies the importance of good laboratory practice, distancing and mask wearing in preventing SARS-CoV-2 spread, specifically in healthcare facilities.

Adenovirus load correlates with respiratory disease severity among hospitalized pediatric patients.

OBJECTIVES: Human adenoviruses (HAdVs) are common pathogens that can cause respiratory, gastrointestinal and other infections. We investigated the correlation between adenovirus viral load in clinical respiratory samples and the respiratory disease severity in pediatric patients. METHODS: Medical records of patients hospitalized in the Sheba Medical Center (SMC) with confirmed adenovirus infection were retrospectively analyzed. The possible correlation between disease severity score and Real time PCR 'cycle threshold' (Ct), a proxy of viral load, was assessed in patients aged 9 years and under. In addition, Ct values of hospitalized versus community-care patient samples, positive for various respiratory viruses including adenovirus, were compared. RESULTS: Adenovirus load in respiratory samples, as measured by Ct values, was found to be negatively correlated with respiratory disease severity in hospitalized pediatric patients aged under 9 years. Moreover, hospitalized patients presented with significantly higher Ct levels for various respiratory viruses as compared to community-care patients. CONCLUSION: In this study we found a correlation between Ct values obtained from adenovirus q-PCR analysis of respiratory clinical samples and disease severity in patients aged 9 years and under. Such finding may serve as a predictor of respiratory disease course in pediatric patients and will be beneficial for the differential diagnosis and treatment of pediatric patients.

Predominance of a Drifted Influenza A (H3N2) Clade and its Association with Age-specific Influenza Vaccine Effectiveness Variations, Influenza Season 2018-2019.

Background: Influenza A (H3N2) clade 3C.3a was the predominant influenza virus in Israel throughout the 2018-2019 season, constituting a drift from the influenza A (H3N2) vaccine. We estimated the end-of season vaccine effectiveness (VE) by age, among community patients with influenza-like illness (ILI), considering the hemagglutinin (HA) gene mutations and amino acid substitutions of influenza A (H3N2) viruses detected. Methods: Nose-throat samples were analyzed for the presence of influenza virus, type/subtype, and HA gene sequence. HA gene sequences and amino acid substitutions were compared to the influenza A/Singapore/INFIMH-16-0019/2016 (H3N2)-like 2018-2019 vaccine virus, and a phylogenetic tree was generated. Influenza VE against influenza A (H3N2) was estimated using the test-negative design. VE was estimated by age group and by 15 year moving age intervals. Results: In total, 90% of the influenza A (H3N2) viruses belonged to the 3C.3a clade, constituting a unique situation in the northern hemisphere. Adjusted all-age influenza A (H3N2) VE was -3.5% (95% CI: -51.2 to 29.1). Although adjusted VEs were very low among infants, children, and young adults, a VE of 45% (95% CI: -19.2 to 74.6) was estimated among adults aged ≥45 years old. Conclusions: The higher VE point estimates among older adults may be related to previous exposure to similar influenza viruses.

Immunogenicity and safety of vaccination against seasonal influenza vaccine in patients with psoriatic arthritis treated with secukinumab.

OBJECTIVE: To assess the immunogenicity and safety of vaccination against seasonal influenza in psoriatic arthritis (PsA) patients treated with secukinumab versus healthy controls (HC). METHODS: PsA patients administered secukinumab for ≥3 months and HC received the Sanofi Pasteur vaccine composed of 3 antigens (H3N3, H1N1, and B) and underwent clinical and laboratory assessments on the day of vaccination and 4-6 weeks later. Immunogenicity of the vaccine was evaluated by hemagglutination inhibition assay against those 3 antigens. Responders to each antigen were defined by a 4-fold increase in the antigen titer or by seroconversion in patients whose baseline level was <1/40. RESULTS: Thirty-two consecutive PsA patients treated with secukinumab for ≥3 months comprised the study group, 10 of whom received concomitant conventional synthetic disease-modifying drugs, mostly methotrexate. There were 17 age- and gender-matched HC (median age 48.5 years, 6 females). The geometric mean titers of each antigen increased significantly in both groups. The number of responders in each group was similar for H3N2 and H1N1, and significantly higher for B/Brisbane in the PsA group. The proportion of patients with a seroprotective level (a titer >1/40) was high and similar in both groups. There was no correlation between the response rate and age, gender, or selected parameters of disease activity (tender/swollen joint counts, Leeds enthesitis index, physician and patient global assessment, psoriasis area severity index, and C-reactive protein). No disease exacerbation was observed following the vaccination. No serious adverse effects were observed in both groups during the study period. CONCLUSION: Secukinumab treatment does not affect the humoral response to influenza vaccine of patients with PsA.

Forty five percent of the Israeli population were infected with the influenza B Victoria virus during the winter season 2015-16.

While infection with influenza A viruses has been extensively investigated, infections with influenza B viruses which are commonly categorized into the highly homologous Victoria and Yamagata lineages, are less studied, despite their considerable virulence. Here we used RT-PCR assays, hemagglutination inhibition assays and antibody titers to determine the levels of influenza B infection. We report of high influenza B Victoria virus prevalence in the 2015-16 winter season in Israel, affecting approximately half of the Israeli population. We further show that the Victoria B virus infected individuals of all ages and that it was present in the country throughout the entire winter season. The vaccine however included the inappropriate Yamagata virus. We propose that a quadrivalent vaccine, that includes both Yamagata and Victoria lineages, should be considered for future influenza vaccination.

Genetic divergence of Influenza A(H3N2) amino acid substitutions mark the beginning of the 2016-2017 winter season in Israel.

BACKGROUND: Influenza vaccine composition is reevaluated each year due to the frequency and accumulation of genetic changes that influenza viruses undergo. The beginning of the 2016-2017 influenza surveillance period in Israel has been marked by the dominance of influenza A(H3N2). OBJECTIVES: To evaluate the type, subtype, genetic evolution and amino acid substitutions of influenza A(H3N2) viruses detected among community patients with influenza-like illness (ILI) and hospitalized patients with respiratory illness in the first weeks of the 2016-2017 influenza season. STUDY DESIGN: Respiratory samples from community patients with influenza-like illness and from hospitalized patients underwent identification, subtyping and molecular characterization. Hemagglutinin sequences were compared to the vaccine strain, phylogenetic tree was created, and amino acid substitutions were determined. RESULTS: Influenza A(H3N2) predominated during the early stages of the 2016-2017 influenza season. Noticeably, approximately 20% of community patients and 36% of hospitalized patients, positive for influenza3), received the 2016-2017 influenza vaccine. The influenza A(H3N2) viruses demonstrated genetic divergence from the vaccine strain into three separate subgroups within the 3C.2a clade. One resembled the new 3C.2a1 subclade, one resembled the recently proposed 3C.2a2 subclade and the other was not previously described. Diversity was observed within each subgroup, in terms of additional amino acid substitutions. CONCLUSIONS: Characterization of the 2016-2017 A(H3N2) influenza viruses is imperative for determining the future influenza vaccine composition.

A(H1N1)pdm09 influenza infection: vaccine inefficiency.

The last influenza pandemic, caused by the swine A(H1N1)pdm09 influenza virus, began in North America at 2009. Since then, the World Health Organization (WHO) recommended integration of the swine-based virus A/California/07/2009 strain in yearly vaccinations. Yet, infections with A(H1N1)pdm09 have continued in subsequent years. The reasons for this are currently unknown. During the 2015-2016 influenza season, we noted an increased prevalence of A(H1N1)pdm09 influenza virus infection in Israel. Our phylogenetic analysis indicated that the circulating A(H1N1)pdm09 strains belonged to 6B.1 and 6B.2 clades and differed from the vaccinating strain, with approximately 18 amino acid differences found between the circulating strains and the immunizing A/California/07/2009 strain. Hemmaglutination inhibition (HI) assays demonstrated higher antibodies titer against the A/California/07/2009 vaccinating strain as compared to the circulating Israeli strains. We thus suggest that the current vaccination was not sufficiently effective and propose inclusion of the current circulating A(H1N1)pdm09 influenza viruses in the annual vaccine composition.

NK-cell receptors NKp46 and NCR1 control human metapneumovirus infection.

Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.

Influenza A(H1N1)pdm 2009 and influenza B virus co-infection in hospitalized and non-hospitalized patients during the 2015-2016 epidemic season in Israel.

BACKGROUND: Influenza A and B viruses co-infections are rare events and mainly occurred in immunocompromised patients. OBJECTIVES: In this study we report an unusually high occurrence of influenza A (H1N1)pdm 2009 and influenza B virus co-infections during the epidemic year 2015-2016. STUDY DESIGN: Nasopharyngeal swabs were collected from 1919 patients visiting 26 outpatient clinics distributed throughout Israel and presenting with influenza-like illness. In addition, hospitalized patient tested for influenza viruses were also included in the study. Patients samples collected between October 2015 and April 2016 were tested for the presence of influenza viruses by real-time PCR. RESULTS: Of the 1919 patient samples tested, 11 (0.6%) were co-infected with both influenza A(H1N1)pdm 2009 and influenza B/Victoria viruses. Similar observation was noted in four hospitalized patients during the same period. Patients at ages 1-72 years, and their clinical symptoms were similar to that of patients infected with either influenza A or B viruses. Of all patients, only one hospitalized patient was immunocompromised. IN CONCLUSION: Co-infection of influenza A(H1N1)pdm 2009 and influenza B viruses is an increasingly recognized phenomenon. This co-infection can occur not only in immunocompromised individuals, but also in immunocompetent patients. Although co-infection appears to be a rare event, it may still play a role in the epidemiology, pathogenicity and evolution of influenza viruses.

Suppression of human metapneumovirus (HMPV) infection by the innate sensing gene CEACAM1.

The innate sensing system is equipped with PRRs specialized in recognizing molecular structures (PAMPs) of various pathogens. This leads to the induction of anti-viral genes and inhibition of virus growth. Human Metapneumovirus (HMPV) is a major respiratory virus that causes an upper and lower respiratory tract infection in children. In this study we show that upon HMPV infection, the innate sensing system detects the viral RNA through the RIG-I sensor leading to induction of CEACAM1 expression. We further show that CEACAM1 is induced via binding of IRF3 to the CEACAM1 promoter. We demonstrate that induction of CEACAM1 suppresses the viral loads via inhibition of the translation machinery in the infected cells in an SHP2-dependent manner. In summary, we show here that HMPV-infected cells upregulates CEACAM1 to restrict HMPV infection.

A systematic view on influenza induced host shutoff.

Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the translation of genes with important housekeeping functions.

Epidemiological and Virological Characterization of Influenza B Virus Infections.

While influenza A viruses comprise a heterogeneous group of clinically relevant influenza viruses, influenza B viruses form a more homogeneous cluster, divided mainly into two lineages: Victoria and Yamagata. This divergence has complicated seasonal influenza vaccine design, which traditionally contained two seasonal influenza A virus strains and one influenza B virus strain. We examined the distribution of the two influenza B virus lineages in Israel, between 2011-2014, in hospitalized and in non-hospitalized (community) influenza B virus-infected patients. We showed that influenza B virus infections can lead to hospitalization and demonstrated that during some winter seasons, both influenza B virus lineages circulated simultaneously in Israel. We further show that the influenza B virus Yamagata lineage was dominant, circulating in the county in the last few years of the study period, consistent with the anti-Yamagata influenza B virus antibodies detected in the serum samples of affected individuals residing in Israel in the year 2014. Interestingly, we found that elderly people were particularly vulnerable to Yamagata lineage influenza B virus infections.

Transcriptome and proteome analyses and the role of atypical calpain protein and autophagy in the spliced leader silencing pathway in Trypanosoma brucei.

Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader silencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRP7.1/7.2. Co-silencing of SKCRP7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRP7.1 is involved in SLS signaling and the accompanying PCD. The role of autophagy in SLS was also investigated; eliminating autophagy through VPS34 or ATG7 silencing demonstrated that autophagy is not essential for SLS induction, but is associated with PCD. Thus, this study identified factors that are used by the parasite to cope with ER stress and to induce SLS and PCD.