Molecular Characterization of Influenza A/H3N2 Virus Isolated from Indonesian Hajj and Umrah Pilgrims 2013 to 2014.

The Hajj and Umrah are the annual mass gatherings of Muslims in Saudi Arabia and increase the transmission risk of acute respiratory infection. This study describes influenza infection among pilgrims upon arrival in Indonesia and the genetic characterization of imported influenza A/H3N2 virus. In total, 251 swab samples with influenza-like illness were tested using real-time RT-PCR for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and influenza viruses. Complete sequences of influenza A/H3N2 HA and NA genes were obtained using DNA sequencing and plotted to amino acid and antigenicity changes. Phylogenetic analysis was performed using a neighbour-joining method including the WHO vaccine strains and influenza A/H3N2 as references. The real-time RT-PCR test detected 100 (39.5%) samples positive with influenza with no positivity of MERS-CoV. Mutations in the HA gene were mainly located within the antigenic sites A, B, and D, while for the NA gene, no mutations related to oseltamivir resistance were observed. Phylogenetic analysis revealed that these viruses grouped together with clades 3C.2 and 3C.3; however, they were not closely grouped with the WHO-recommended vaccine (clades 3C.1). Sequences obtained from Hajj and Umrah pilgrims were also not grouped together with viruses from Middle East countries but clustered according to years of collection. This implies that the influenza A/H3N2 virus mutates continually across time.

Impact of Influenza in South-East Asia

Influenza infection is asymptomatic in up to 75% of cases, but outbreaks result in significant morbidity. Reports found that severe influenza complications tend to occur among the very young (<5 years) and very old (>65 years), especially those with underlying co-morbidities like diabetes mellitus and heart disease. Even with no co-morbidity, some older persons with severe influenza may require hospitalisation or intensive care, with increased risk of myocardial infarction and stroke. In South-East Asia, influenza was often seen as a mild problem and was not deemed notifiable until the appearance of the Influenza A(H1N1) pandemic in 2009. For decades the data made available were based on extrapolated estimates collected mainly from paediatric populations, resulting in inconsistent findings. Following expanded surveillance across the region using national surveillance systems for influenza-like illness (ILI) and severe acute respiratory illness (SARI), and better diagnostic methods, improved estimates of disease burden was achieved in South-East Asia. However, two studies conducted in 2008-2010 reported findings ranging from 2-3% to 11%. With regards to increased risk of complications, the estimated global annual attack rates for influenza were 5-10% in adults and 20-30% in children, resulting in 3-5 million cases of severe illness and 290,000-650,000 deaths. A study In Singapore reported that influenza is associated with annual excess mortality rates (EMR) of 11-14.8 per 100 000 person-years, especially affecting the elderly;these rates are comparable to that of the USA. As for hospitalisation rates of children under 5 years with seasonal influenza, the USA estimated a rate of 1.4 per 100,000. Comparable rates were reported in Singapore (0.7-0.9), Thailand (2.4), Viet Nam (3.9-4.7), and the Philippines (4.7). In 2018, an updated study reported a mean annual influenza-associated respiratory EMR of 4.0-8.8 per 100 000 individuals, with South-East Asia showing a high mortality rate of 3.5-9.2 per 100,000 individuals. It was already estimated in Thailand in 2004 that influenza resulted in USD23-63 million in economic costs, with the main contribution from lost productivity due to missed workdays. Thus, comparable to countries in temperate climate, the clinical and socioeconomic impact of influenza in South-East Asia appear to be just as substantial. With the emergence of the COVID-19 pandemic in 2020, global influenza incidence dropped dramatically. In South-East Asia, the trend in influenza detections was similar to the rest of the world, with numbers slightly higher than average in early 2020, followed by a quick drop-off by the end of April 2020. After April 2020, the detection rate remained low until late July 2020, when Influenza A(H3N2) predominated in Cambodia, Malaysia, the Philippines, Singapore, Thailand and Timor-Leste;influenza B in Lao People's Democratic Republic but with an upsurge in A(H3N2) activity. Following a two-year hiatus, influenza outbreaks began to re-emerge significantly since early 2022. From February through August 2022, influenza activity in the southern hemisphere remained lower than in pre-COVID-19 pandemic years, but was at the highest level compared to similar periods since the start of the COVID-19 pandemic. Reasons for the reduction during the COVID-19 pandemic include non-pharmaceutical interventions (NPIs), reduced population mixing and reduced travel, and possibly viral interference between SARS-CoV-2 and influenza virus in the same host. In general, the reduction in influenza detections however does not appear to be associated with lack of testing. The World Health Organisation (WHO) continues to recommend that vaccination is the most effective way to prevent infection and severe outcomes caused by influenza viruses. Although influenza vaccine is not commonly used in most countries in South-East Asia, its burden is similar in other parts of the world where influenza vaccine is now routinely used. Currently, the countries in South-East Asia that are providing free influenza vacc na ion for those at high risk include Thailand, Singapore, the Philippines and Lao People's Democratic Republic.Copyright © 2023

A large A(H3N2) influenza outbreak with a high attack rate in a drug user community in Italy, April 2022.

Despite the COVID-19 pandemic, influenza remains an important issue. Especially in community settings, influenza outbreaks can be difficult to control and can result in high attack rates. In April 2022, a large A(H3N2) influenza outbreak spread in the largest Italian drug-rehabilitation community. One hundred eighty-four individuals presented influenza-like symptoms (attack rate of 26.2%); 56% previously received the influenza vaccine. Sequence analyses highlighted a genetic drift from the vaccine strain, which may have caused the observed lack of protection.

Matrix-Encoding Gene Diversity of 624 Influenza A/H3N2 Genomes Does Not Show Association with Impaired Viral Detection by Commercialized qPCR Assays.

As for the case of SARS-CoV-2, genome sequencing of influenza viruses is of potential interest to raise and address virological issues. Recently, false-negativity of real-time reverse transcription-PCR (qPCR) assays that detect influenza A/H3N2 virus RNA were reported and associated with two mutations (A37T and C161T) in the Matrix-encoding (M1) gene located on viral segment 7. This triggered a national alert in France. The present study sought to assess the association between the presence of these mutations and potential false negative results of influenza A/H3N2 virus RNA detection by commercialized qPCR assays at the clinical virology laboratory of our university hospitals in southern France. This study focused on the genetic diversity in the M1 gene and segment 7 of 624 influenza A/H3N2 virus genomes obtained from respiratory samples having tested qPCR-positive with M1 gene-targeting assays in our clinical virology laboratory. A total of 585 among the 624 influenza A/H3N2 virus genomes (93.7%) were of clade 3C.2a1b.2a.2, and 39 (6.3%) were of clade 3C.2a1b.1a. M1 gene substitutions A37T and C161T were both present in 582 (93.3%) genomes, only of clade 3C.2a1b.2a.2. Substitution A37T was present in 621 (99.5%) genomes. Substitution C161T was present in 585 genomes (93.8%), all of clade 3C.2a1b.2a.2. Moreover, 21 other nucleotide positions were mutated in ≥90% of the genomes. The present study shows that A37T/C and C161T mutations, and other mutations in the M1 gene and segment 7, were widely present in influenza A/H3N2 virus genomes recovered from respiratory samples diagnosed qPCR-positive with commercialized assays.

From Clinical Specimen to Whole Genome Sequencing of A(H3N2) Influenza Viruses: A Fast and Reliable High-Throughput Protocol.

(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >105 genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

Hemophagocytic Lymphohistiocytosis after COVID-19 Vaccination: A Case Report

Aim and background: Cases of thrombotic thrombocytopenia induced by coronavirus disease 2019 (COVID-19) vaccines have been reported recently. Herein, we describe hemophagocytic lymphohistiocytosis (HLH) following COVID-19 vaccination. Case report: A 35-year-old male, chronic alcoholic, 3 years into abstinence received first dose Covishield vaccine. He started developing a fever, testicular pain, diminished sensorium requiring invasive ventilation, and decreased urine output 4 days after getting vaccinated. Initial workup for NCCT brain and HRCT chest was normal, tropical fever panel was negative, cultures for blood and endotracheal aspirate were sterile, liver and renal functions showed mild derangement, CSF study was normal. Ultrasound examination of the abdomen revealed mild hepatosplenomegaly, mild testicular swelling, and suprainguinal lymphadenopathy, with no focus of infection. Subsequently, he developed bicytopenia with haemoglobin 9.0 g/dL and platelet counts 50 × 109/L, ferritin 2130 μg/L, triglyceride 353 mg/dL, and decreased fibrinogen 1.41 g/L. Bone marrow as well as lymph node biopsy showed haemophagocytosis with engulfment of neutrophils, lymphocytes, and normoblasts making HLH a likely diagnosis. Soluble CD25 and NK cell function could not be performed. Extensive evaluation was done to look into the etiology of HLH. SARS-CoV-2 reverse transcriptase-polymerase chain reaction (RT-PCR) test was negative. RT-PCR test for Epstein-Barr virus (EBV), influenza A (H1N1, H3N2), influenza B, cytomegalovirus (CMV) performed from endotracheal aspirate (ETA) was negative. Similarly, the RT-PCR test from serum samples for EBV, Parvo B-19, CMV, and from CSF sample for EBV, Parvo B-19, CMV, and HSV-1 was negative. Hepatitis B, C, and HIV serologies were negative. Culture and sensitivity repeated from blood, ETA and urine was sterile. Autoimmune panel including complements levels were negative. Peripheral smear, bone marrow, and lymph node biopsy were normal and did not reveal abnormal or malignant cells. He had persistent fevers to 38.6°C during the first 6 days of his admission, with a rise in his ferritin to 1950 μg/L. The patient received steroids but not etoposide. By the 8th day, his fevers resolved, with improvement in his lethargy and malaise. Two weeks later, his ferritin had reduced to 510 μg/L, platelet count rose to 180 × 109/L, and repeat ultrasound abdomen demonstrated resolution of his splenomegaly. In our patient, there was no clear precipitant of HLH other than the Covishield vaccine. There was no evidence of an infection or malignancy. Due to our patient's clinical stability, resolution of symptoms, and improvement of HLH parameters he did not require HLH specific therapy. It is unclear if he had a pre-existing genetic predisposition to HLH as genetic testing is pending, however, it is unlikely as he has reached the age of 35 and suffered from previous viral infections without developing HLH.

CD8 T cell immunity after respiratory viral infection is transient, while CD8 T cell immunity after epidermal vaccination is lung focused and durable

Pandemic respiratory viral pathogens like Influenza A and SARS COV2 exhibit continuous and evasive mutations in cell surface molecules, making vaccination with the goal of antibody-mediated protection elusive. CD8 T cells mediate eradication of viral disease, and vaccination to conserved internal viral proteins to elicit CD8 T cell memory is a promising strategy. Using a mouse model, we compared pulmonary infection with H1N1 influenza with skin (epidermal) vaccination using Modified Vaccinia Ankara (MVA) expressing highly conserved NP or another conserved Ags. H1N1 influenza pulmonary infection led to recruitment and lung infiltration with Ag specific CD8 T cells by day 5-10. By day 40, abundant CD8 lung TRM and LN TCM were present. Surprisingly, by day 80, both lung TRM and systemic TCM cells were greatly diminished and were absent at day 120. These mice were protected against lethal challenge at day 40 but not day 80, suggesting built-in obsolescence of CD8 memory. In contrast, epidermal vaccination led to CD8 T cell infiltration of lung at day 5-10, measurable at day 40 and still detectable at day 80 in lung, LN and spleen. In addition, a novel intravascular lung population of CD8 T cells was present at all time points. These mice were completely protected against lethal flu challenge at day 80 and 120. Protection was observed after pulmonary challenge with either H1N1 or H3N2 influenza as well as in B cell depleted mice. We analyzed protective immunity in skin vaccinated mice. At 2 hours after pulmonary challenge, Ag specific CD8 T cells moved from the intravascular space into the lung parenchyma, were abundant at day 3 and persisted for >80 days. Single cell RNA sequencing indicated that these intravascular T cells were transcriptionally distinct from systemic TEM and TCM. We conclude that CD8 T cell immunity after pulmonary infection is powerful but short-lived, while skin vaccine induced CD8 T cell protective immunity is mediated by lung intravascular T cells is protective and durable.

Transcriptional Interaction Between RelA and cJUN

Rationale: The SARS-CoV-2 pandemic has underscored the need for novel anti-infectious strategies, including host-directed therapeutics, against existing and emerging respiratory pathogens. We have reported that an aerosolized therapeutic comprised of a Toll-like receptor (TLR)-2/6 agonist, Pam2CSK4, and a TLR-9 agonist, ODN M362, stimulate pathogen-agnostic innate immune responses in lung epithelial cells. This therapeutic (“Pam2-ODN”) promotes synergistic microbicidal activity and host survival benefit against pneumonia caused by a wide range of pathogens. Here, we study the immunomodulatory signaling mechanisms required to effect this inducible epithelial resistance. Methods: Bioinformatic analysis of transcriptional responses from human and mouse lung epithelium al cells to influenza A H1N1 or SARS-CoV-2 (GSE147507) or Pam2-ODN (GSE289984, GSE26864) were analyzed using R and IPA software to identify essential transcription factors (TFs). Lung cell population dynamics were studied for TFs related to Pam2-ODN immunomodulatory signaling using high-throughput imaging flow cytometry (IFC). Human or mouse lung epithelial cells were stimulated with PBS or Pam2-ODN and single or dual inhibitors of TFs before challeng with influenza A H3N2 (IAV) or coronavirus OC43 (CoV) to compare the epithelium-specific transcriptional control of relevant TFs using in-cell western blotting, IFC and hemagglutination for viral burdens. Results: Functional enrichment analysis revealed RelA and cJUN to be major immunomodulatory TFs of Pam2-ODN and activators of leukocyte- and epithelial-derived antiviral immune mechanisms targeting replication of influenza A and SARS-CoV-2. Cell population dynamics studied from mouse lungs confirmed activation of RelA and cJUN in CD45+, EpCAM- leukocytes and in CD45-, EpCAM+ epithelial cells, with predominant activation of the lung epithelium and none or minimal activation of structural cell populations such as fibroblasts or endothelial cells. Studies of epithelium-specific signaling in vitro revealed co-activation of RelA-(pS536) and cJun- (pS73) TFs with Pam2-ODN, and earlier onset of cJUN phosphorylation and nuclear translocation with Pam2-ODN after IAV or CoV infection. Individual or dual inhibition of RelA and/or cJUN activity in vitro disrupted the antiviral activity of Pam2-ODN of IAV infected cells. Conclusion: Pam2-ODN induces unique, pathogen-agnostic protective signaling in lung epithelial cells that involves cooperative activation of RelA and cJUN. This combined TF signaling mechanism is not observed in other structural lung cell populations after Pam2-ODN exposure. Further, the phospho-regulation dynamics of RelA and cJUN are not replicated by IAV or CoV infection alone, suggesting a novel therapeutic process that can be leveraged to protect individuals against pneumonia. (Figure Presented).

A late sharp increase in influenza detections and low interim vaccine effectiveness against the circulating A(H3N2) strain, Denmark, 2021/22 influenza season up to 25 March 2022.

We estimated interim influenza A vaccine effectiveness (VE) following a late sharp rise in cases during an influenza A(H3N2)-dominated 2021/22 season, after lifting COVID-19 restrictions. In children aged 2-6 years offered a live attenuated influenza vaccine, adjusted VE was 62.7% (95% CI: 10.9-84.4) in hospitalised and 64.2% (95% CI: 50.5-74.1) in non-hospitalised children. In non-hospitalised patients aged 7-44 years, VE was 24.8% (95% CI: 12.8-35.2); VE was non-significant in remaining age groups and hospital/non-hospital settings.

Fatal case of simply influenza A (H3N2)-associated encephalitis in immunocompetent patient.

Influenza virus-associated encephalopathy/encephalitis is a rare entity in adults that can lead to severe neurological sequelae and even death. The clinical presentation can be quite diverse. This absence of a typical presentation along with the difficulty detecting the virus in the cerebrospinal fluid represents a diagnostic challenge. We present the case of a 79-year-old male with sudden onset of decreased consciousness and signs of right hemisphere damage. The presence of influenza A (H3N2) virus in respiratory sample along with compatible findings in cranial magnetic resonance led to the diagnosis. The patient died without responding to treatment with antivirals and immunomodulators and the anatomopathological study did not detect infectious agent. Early diagnostic suspicion is essential to establish adequate treatment and improve the prognosis.