Neutrophil extracellular traps promote immunopathogenesis of virus-induced COPD exacerbations.

Respiratory viruses are a major trigger of exacerbations in chronic obstructive pulmonary disease (COPD). Airway neutrophilia is a hallmark feature of stable and exacerbated COPD but roles played by neutrophil extracellular traps (NETS) in driving disease pathogenesis are unclear. Here, using human studies of experimentally-induced and naturally-occurring exacerbations we identify that rhinovirus infection induces airway NET formation which is amplified in COPD and correlates with magnitude of inflammation and clinical exacerbation severity. We show that inhibiting NETosis protects mice from immunopathology in a model of virus-exacerbated COPD. NETs drive inflammation during exacerbations through release of double stranded DNA (dsDNA) and administration of DNAse in mice has similar protective effects. Thus, NETosis, through release of dsDNA, has a functional role in the pathogenesis of COPD exacerbations. These studies open up the potential for therapeutic targeting of NETs or dsDNA as a strategy for treating virus-exacerbated COPD.

Viral and non-viral episodes of wheezing in early life and the development of asthma and respiratory phenotypes among urban children.

BACKGROUND: Viral wheezing is an important risk factor for asthma, which comprises several respiratory phenotypes. We sought to understand if the etiology of early-life wheezing illnesses relates to childhood respiratory and asthma phenotypes. METHODS: Data were collected prospectively on 429 children in the Urban Environment and Childhood Asthma (URECA) birth cohort study through age 10 years. We identified wheezing illnesses and the corresponding viral etiology (PCR testing of nasal mucus) during the first 3 years of life. Six phenotypes of respiratory health were identified at 10 years of age based on trajectories of wheezing, allergic sensitization, and lung function. We compared the etiology of early wheezing illnesses to these wheezing respiratory phenotypes and the development of asthma. RESULTS: In the first 3 years of life, at least one virus was detected in 324 (67%) of the 483 wheezing episodes documented in the study cohort. Using hierarchical partitioning we found that non-viral wheezing episodes accounted for the greatest variance in asthma diagnosed at both 7 and 10 years of age (8.0% and 5.8% respectively). Rhinovirus wheezing illnesses explained the most variance in respiratory phenotype outcome followed by non-viral wheezing episodes (4.9% and 3.9% respectively) at 10 years of age. CONCLUSION AND RELEVANCE: Within this high-risk urban-residing cohort in early life, non-viral wheezing episodes were frequently identified and associated with asthma development. Though rhinovirus wheezing illnesses had the greatest association with phenotype outcome, the specific etiology of wheezing episodes in early life provided limited information about subsequent wheezing phenotypes.

Age-specific SARS-CoV-2 transmission differed from human rhinovirus in households during the early COVID-19 pandemic.

OBJECTIVES: Children are generally considered main drivers of transmission for respiratory viruses, but the emergence of SARS-CoV-2 challenged this paradigm. Human rhinovirus (RV) continued to co-circulate throughout the pandemic, allowing for direct comparison of age-specific infectivity and susceptibility within households between these viruses during a time of low SARS-CoV-2 population immunity. METHODS: Households with children were prospectively monitored for ≥23 weeks between August 2020 and July 2021. Upon onset of respiratory symptoms in a household, an outbreak study was initiated, including questionnaires and repeated nasal self-sampling in all household members. Swabs were tested by PCR. Age-stratified within-household secondary attack rates (SARs) were compared between SARS-CoV-2 and RV. RESULTS: A total of 307 households participated, including 582 children and 627 adults. Overall, SAR was lower for SARS-CoV-2 than for RV (aOR 0.55) and age distributions differed between both viruses (p < 0.001). Following household exposure, children were significantly less likely to become infected with SARS-CoV-2 compared to RV (aOR 0.16), whereas this was opposite in adults (aOR 1.71). CONCLUSION: In households, age-specific susceptibility to SARS-CoV-2 and RV differs and drives differences in household transmission between these pathogens. This highlights the importance of characterizing age-specific transmission risks, particularly for emerging infections, to guide appropriate infection control interventions.

Circulation pattern and genetic variation of rhinovirus infection among hospitalized children on Hainan Island, before and after the dynamic zero-COVID policy, from 2021 to 2023.

Throughout the COVID-19 pandemic, rhinovirus (RV) remained notable persistence, maintaining its presence while other seasonal respiratory viruses were largely suppressed by pandemic restrictions during national lockdowns. This research explores the epidemiological dynamics of RV infections among pediatric populations on Hainan Island, China, specifically focusing on the impact before and after the zero-COVID policy was lifted. From January 2021 to December 2023, 19 680 samples were collected from pediatric patients hospitalized with acute lower respiratory tract infections (ARTIs) at the Hainan Maternal and Child Health Hospital. The infection of RV was detected by tNGS. RV species and subtypes were identified in 32 RV-positive samples representing diverse time points by analyzing the VP4/VP2 partial regions. Among the 19 680 pediatric inpatients with ARTIs analyzed, 21.55% were found to be positive for RV infection, with notable peaks observed in April 2021 and November 2022. A gradual annual decline in RV infections was observed, alongside a seasonal pattern of higher prevalence during the colder months. The highest proportion of RV infections was observed in the 0-1-year age group. Phylogenetic analysis on 32 samples indicated a trend from RV-A to RV-C in 2022. This observation suggests potential evolving dynamics within the RV species although further studies are needed due to the limited sample size. The research emphasizes the necessity for ongoing surveillance and targeted management, particularly for populations highly susceptible to severe illnesses caused by RV infections.

Recombinant Viruses from the Picornaviridae Family Occurring in Racing Pigeons.

Viruses from Picornaviridae family are known pathogens of poultry, although the information on their occurrence and pathogenicity in pigeons is scarce. In this research, efforts are made to broaden the knowledge on Megrivirus B and Pigeon picornavirus B prevalence, phylogenetic relationship with other avian picornaviruses and their possible connection with enteric disease in racing pigeons. As a result of Oxford Nanopore Sequencing, five Megrivirus and two pigeon picornavirus B-like genome sequences were recovered, among which three recombinant strains were detected. The recombinant fragments represented an average of 10.9% and 25.5% of the genome length of the Pigeon picornavirus B and Megrivirus B reference strains, respectively. The phylogenetic analysis revealed that pigeons are carriers of species-specific picornaviruses. TaqMan qPCR assays revealed 7.8% and 19.0% prevalence of Megrivirus B and 32.2% and 39.7% prevalence of Pigeon picornavirus B in the group of pigeons exhibiting signs of enteropathy and in the group of asymptomatic pigeons, respectively. In turn, digital droplet PCR showed a considerably higher number of genome copies of both viruses in sick than in asymptomatic pigeons. The results of quantitative analysis leave the role of picornaviruses in enteropathies of pigeons unclear.

Disease Severity and Cytokine Expression in the Rhinovirus-Induced First Wheezing Episode.

Wheezing children infected with rhinovirus (RV) have a markedly increased risk of subsequently developing recurrencies and asthma. No previous studies have assessed the association between cytokine response and the severity of acute illness in the first wheezing episode in children infected with RV. Forty-seven children treated both as inpatients and as outpatients infected with RV only, aged 3-23 months, with severe first wheezing episodes were recruited. During acute illness, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with anti-CD3/anti-CD28 in vitro. A multiplex ELISA was used to quantitatively identify 56 different cytokines. The mean age of the children was 17 months, 74% were males, 79% were hospitalized, and 33% were sensitized. In adjusted analyses, the inpatient group was characterized by decreased expressions of interferon gamma (IFN-γ), interleukin 10 (IL-10), macrophage inflammatory protein 1 alpha (MIP-1α), RANTES (CCL5), and tumor necrosis factor-alpha (TNF-α) and an increased expression of ENA-78 (CXCL5) compared to the outpatient group. The cytokine response profiles from the PBMCs were different between the inpatient and outpatient groups. Our results support that firmly controlled interplay between pro-inflammatory and anti-inflammatory responses are required during acute viral infection to absolve the initial infection leading, to less severe illness.

Identification and Genomic Characterization of Two Novel Hepatoviruses in Shrews from Yunnan Province, China.

Hepatitis A virus (HAV), a member of the genus Hepatovirus (Picornaviridae HepV), remains a significant viral pathogen, frequently causing enterically transmitted hepatitis worldwide. In this study, we conducted an epidemiological survey of HepVs carried by small terrestrial mammals in the wild in Yunnan Province, China. Utilizing HepV-specific broad-spectrum RT-PCR, next-generation sequencing (NGS), and QNome nanopore sequencing (QNS) techniques, we identified and characterized two novel HepVs provisionally named EpMa-HAV and EpLe-HAV, discovered in the long-tailed mountain shrew (Episoriculus macrurus) and long-tailed brown-toothed shrew (Episoriculus leucops), respectively. Our sequence and phylogenetic analyses of EpMa-HAV and EpLe-HAV indicated that they belong to the species Hepatovirus I (HepV-I) clade II, also known as the Chinese shrew HepV clade. Notably, the codon usage bias pattern of novel shrew HepVs is consistent with that of previously identified Chinese shrew HepV. Furthermore, our structural analysis demonstrated that shrew HepVs differ from other mammalian HepVs in RNA secondary structure and exhibit variances in key protein sites. Overall, the discovery of two novel HepVs in shrews expands the host range of HepV and underscores the existence of genetically diverse animal homologs of human HAV within the genus HepV.

Interfering factors in the diagnosis of Senecavirus A.

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A.

BACKGROUND: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. METHODS: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. RESULTS: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. CONCLUSIONS: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Measuring immunocompetence in the natural population and captive individuals of noble pen shell Pinna nobilis affected by Pinna nobilis Picornavirus (PnPV).

Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7-9 x 104 vs 2-5 x 105 cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion.

HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

First parechovirus reported case in Saudi Arabia in hospitalized immunocompromised adult patient.

Human parechovirus, a member of the Picornaviridae family (PeVs), can lead to severe infections, including severe meningitis, meningoencephalitis, and sepsis-like syndrome. We report a case of human parechovirus-related encephalitis in a 52-year-old woman diagnosed with glioblastoma multiforme. She underwent surgical resection in June 2022. Unfortunately, her disease recurred, and she underwent a second resection in August 2022, followed by radiation therapy and Temozolomide therapy. She presented to the hospital with acute confusion followed by seizures, necessitating intubation for airway support. A cerebrospinal fluid (CSF) sample was obtained and processed using the Biofire FilmArray, which reported the detection of HSV-1. Despite being on Acyclovir, the patient did not show signs of improvement. Consequently, a second CSF sample was obtained and sent for next-generation sequencing (NGS), which returned a positive result for Parechovirus. In this presented case, the patient exhibited symptoms of an unknown infectious cause. The utilization of NGS and metagenomic analysis helped identify Parechovirus as the primary pathogen present, in addition to previously identified HSV. This comprehensive approach facilitated a thorough assessment of the underlying infection and guided targeted treatment. In conclusion, the application of NGS techniques and metagenomic analysis proved instrumental in identifying the root cause of the infection.

Porcine kobuvirus enhances porcine epidemic diarrhea virus pathogenicity and alters the number of intestinal lymphocytes in piglets.

Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections.

Evolutionary dynamics of canine kobuvirus in Vietnam and Thailand reveal the evidence of viral ability to evade host immunity.

Canine kobuvirus (CaKoV) is a pathogen associated with canine gastrointestinal disease (GID). This study examined 327 rectal swabs (RS), including 113 from Vietnam (46 healthy, 67 with GID) and 214 from Thailand (107 healthy and 107 with GID). CaKoV was detected in both countries, with prevalences of 28.3% (33/113) in Vietnam and 7.9% (17/214) in Thailand. Additionally, CaKoV was found in both dogs with diarrhea and healthy dogs. CaKoV was mainly found in puppies under six months of age (30.8%). Co-detection with other canine viruses were also observed. The complete coding sequence (CDS) of nine Vietnamese and four Thai CaKoV strains were characterized. Phylogenetic analysis revealed a close genetic relationship between Vietnamese and Thai CaKoV strains, which were related to the Chinese strains. CDS analysis indicated a distinct lineage for two Vietnamese CaKoV strains. Selective pressure analysis on the viral capsid (VP1) region showed negative selection, with potential positive selection sites on B-cell epitopes. This study, the first of its kind in Vietnam, provides insights into CaKoV prevalence in dogs of different ages and healthy statuses, updates CaKoV occurrence in Thailand, and sheds light on its molecular characteristics and immune evasion strategies.

Coinfection with respiratory syncytial virus and rhinovirus increases IFN-λ1 and CXCL10 expression in human primary bronchial epithelial cells.

Acute respiratory tract infection (ARTI) is common in all age groups, especially in children and the elderly. About 85% of children who present with bronchiolitis are infected with respiratory syncytial virus (RSV); however, nearly one-third are coinfected with another respiratory virus, such as human rhinovirus (HRV). Therefore, it is necessary to explore the immune response to coinfection to better understand the molecular and cellular pathways involving virus-virus interactions that might be modulated by innate immunity and additional host cell response mechanisms. This study aims to investigate the host innate immune response against RSV-HRV coinfection compared with monoinfection. Human primary bronchial/tracheal epithelial cells (HPECs) were infected with RSV, HRV, or coinfected with both viruses, and the infected cells were collected at 48 and 72 hours. Gene expression profiles of IL-6, CCL5, TNF-α, IFN-ß, IFN-λ1, CXCL10, IL-10, IL-13, IRF3, and IRF7 were investigated using real-time quantitative PCR, which revealed that RSV-infected cells exhibited increased expression of IL-10, whereas HRV infection increased the expression of CXCL10, IL-10, and CCL5. IFN-λ1 and CXCL10 expression was significantly different between the coinfection and monoinfection groups. In conclusion, our study revealed that two important cytokines, IFN-λ1 and CXCL10, exhibited increased expression during coinfection.

Detection and genetic diversity of parechoviruses in children with acute flaccid paralysis in Cameroon.

Human Parechoviruses (HPeVs) have rarely been considered in the virological investigation of Acute Flacid Paralysis (AFP) cases in Africa, where enteric infections are very common. This study investigated the prevalence and genetic diversity of HPeV in 200 children aged ≤ 15 years with AFP in Cameroon from 2018 to 2019. HPeVs were detected in their faecal RNA using 5'-untranslated real-time RT-PCR. Detected HPeVs were typed by phylogenetic comparison with homologous sequences from homotypic reference strains. Overall, HPeV RNA was detected in 11.0% (22/200) of the 200 stool samples tested. Twelve HPeVs were successfully sequenced and reliably assigned to HPeV-A1, A4, A5, A10, A14, A15, A17 and A18 genotypes. Phylogenetic analyses revealed a high genetic variability among the studied HPeVs, as well as between the studied HPeVs and their previously reported counterparts from Cameroon in 2014. These findings suggest that different HPeV genotypes co-circulate in Cameroon without documented epidemics.

mRNA and miRNA expression profiles reveal the potential roles of RLRs signaling pathway and mitophagy in duck hepatitis A virus type 1 infection.

Duck hepatitis A virus 1 (DHAV-1) is the primary cause of duck viral hepatitis, leading to sudden mortality in ducklings and significant economic losses in the duck industry. However, little is known about how DHAV-1 affects duckling liver at the molecular level. We conducted an analysis comparing the expression patterns of mRNAs and miRNAs in DHAV-1-infected duckling livers to understand the underlying mechanisms and dynamic changes. We identified 6,818 differentially expressed mRNAs (DEGs) and 144 differentially expressed microRNAs (DEMs) during DHAV-1 infection. Functional enrichment analysis of DEGs and miRNA target genes using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed their potential involvement in innate antiviral immunity, mitophagy, and pyroptosis. We constructed coexpression networks of mRNA-miRNA interactions and confirmed key DEMs (novel-mir333, novel-mir288, novel-mir197, and novel-mir71) using RT-qPCR. Further investigation demonstrated that DHAV-1 activates the RLRs signaling pathway, disrupts mitophagy, and induces pyroptosis. In conclusion, DHAV-1-induced antiviral immunity is closely linked to mitophagy, suggesting it could be a promising therapeutic target.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.