New hemagglutinin dual-receptor-binding pattern of a human-infecting influenza A (H7N9) virus isolated after fifth epidemic wave.

Since 2013, influenza H7N9 virus has caused five epidemic waves of human infection. The virus evolved from low pathogenic to highly pathogenic in wave 5, 2017, while the prevalence of host receptor-binding tropism in human-infecting viruses maintained dual-receptor-binding property with preference for avian receptor. A human-infecting H7N9 virus was isolated after the fifth epidemic wave and possessed an avian and human dual-receptor specificity, with a moderately higher affinity for human receptor binding. A V186I (H3 numbering) substitution in the receptor-binding site of the hemagglutinin (HA) molecule is responsible for the alteration of the dual-receptor-binding tropism. Viral strains which contain I186 amino acid of avian- and human-infecting H7N9 viruses were all isolated during or after wave 5, and their HA genes clustered in a same phylogenetic clade together with 2018-9 H7N9 isolates, highlights a new evolutionary path for human adaption of natural H7N9 viruses.

A Novel Neutralizing Antibody Specific to the DE Loop of VP1 Can Inhibit EV-D68 Infection in Mice.

Enterovirus D68 (EV-D68) belongs to the picornavirus family and was first isolated in CA, USA, in 1962. EV-D68 can cause severe cranial nerve system damage such as flaccid paralysis and acute respiratory diseases such as pneumonia. There are currently no efficient therapeutic methods or effective prophylactics. In this study, we isolated the mAb A6-1 from an EV-D68-infected rhesus macaque (Macaca mulatta) and found that the Ab provided effective protection in EV-D68 intranasally infected suckling mice. We observed that A6-1 bound to the DE loop of EV-D68 VP1 and interfered with the interaction between the EV-D68 virus and α2,6-linked sialic acids of the host cell. The production of A6-1 and its Ab properties present a bridging study for EV-D68 vaccine design and provide a tool for analyzing the process by which Abs can inhibit EV-D68 infection.

A family cluster of two fatal cases infected with influenza A (H7N9) virus in Kunming China, 2017.

Two imported family cases (mother and daughter) of fatal H7N9 infection in Kunming, China were reported in 2017. Epidemiological investigation showed that the two family members had both been exposed to sick chickens in a poultry market. The onset of illness and death of the mother was 7 days later than her daughter, raising concerns about human-to-human transmission of H7N9 in the locality. Sequence alignment and phylogenetic analysis of the virus strains isolated from the two patients revealed high sequence similarity (≥ 99%) and homology to each other. The two virus strains shared a PEIPKGR/G cleavage motif and the same key amino acid mutations across 8 viral genes except for a R292K mutation in the neuraminidase (NA) gene isolated from the mother who had been treated with oseltamivir in the clinic. Moreover, the isolated H7N9 virus possesses avian and human dual-receptor specificity and is able to efficiently proliferate in human cell lines in vitro. Further epidemiological study demonstrated that five family members who had close contacted with the patients were free of illness and negative for the H7N9 genomic test. Collectively, the H7N9 virus described here is still limited to transmit efficiently from human-to-human.

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1 -/-mice infected with influenza virus (H1N1).

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI). The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo. IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation. We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice. Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6, TNF-α, G-CSF, KC, and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1 -/- mice in comparison with that of wild type infected mice. The adaptive immune response against the H1N1 virus in IL-1R1 -/- mice was impaired with downregulated anti-viral Th1 cell, CD8+ cell, and antibody functions, which contributes to attenuated viral clearance. Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1 -/- mice compared with that in WT infected mice. Moreover, the infected IL-1R1 -/- mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung. Together, these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury, particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

Different microRNA profiles reveal the diverse outcomes induced by EV71 and CA16 infection in human umbilical vein endothelial cells using high-throughput sequencing.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) remain the predominant pathogens in hand, foot, and mouth disease (HFMD), but the factors underlying the pathogenesis of EV71 and CA16 infections have not been elucidated. Recently, the functions of microRNAs (miRNAs) in pathogen-host interactions have been highlighted. In the present study, we performed comprehensive miRNA profiling in EV71- and CA16-infected human umbilical vein endothelial cells (HUVECs) at multiple time points using high-throughput sequencing. The results showed that 135 known miRNAs exhibited remarkable differences in expression. Of these, 30 differentially expressed miRNAs presented opposite trends in EV71- and CA16-infected samples. Subsequently, we mainly focused on the 30 key differentially expressed miRNAs through further screening to predict targets. Gene ontology (GO) and pathway analysis of the predicted targets showed the enrichment of 14 biological processes, 9 molecular functions, 8 cellular components, and 85 pathways. The regulatory networks of these miRNAs with predicted targets, GOs, pathways, and co-expression genes were determined, suggesting that miRNAs display intricate regulatory mechanisms during the infection phase. Consequently, we specifically analyzed the hierarchical GO categories of the predicted targets involved in biological adhesion. The results indicated that the distinct changes induced by EV71 and CA16 infection may be partly linked to the function of the blood-brain barrier. Taken together, this is the first report describing miRNA expression profiles in HUVECs with EV71 and CA16 infections using high-throughput sequencing. Our data provide useful insights that may help to elucidate the different host-pathogen interactions following EV71 and CA16 infection and offer novel therapeutic targets for these infections.

Functional effector memory T cells contribute to protection from superinfection with heterologous simian immunodeficiency virus or simian-human immunodeficiency virus isolates in Chinese rhesus macaques.

Many studies have revealed a protective effect of infection of an individual with an immunodeficiency virus against subsequent infection with a heterologous strain. However, the extent of protection against superinfection conferred by the first infection and the biological consequences of superinfection are not well understood. Here, we report that a rhesus monkey model of mucosal superinfection was established to investigate the protective immune response. Protection against superinfection was shown to correlate with the extent of the polyfunctionality of CD4+ effector memory T cells, whereas neutralizing antibody responses did not protect against superinfection in this model. Notably, immunodeficiency-virus-associated effector memory T-cell responses might significantly contribute to the suppression of virus superinfection. This provides a potential theoretical basis for the development of an HIV/AIDS vaccine.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus.

[Expression of Type-Ι Interferon Production Pathway-Related Genes Induced by Infection Due to Enterovirus 71 or Coxsackievirus A16 in Normal Human Airway Epithelial Cells].

Hand, foot, and mouth disease(HFMD)is caused by mainly enterovirus 71(EV-A71)and coxsackievirus A16(CV-A16),and is a serious healthcare problem worldwide.EV-A71 infection is thought to progress readily to serious complications whereas CV-A16 infection, in general, results in mild symptoms and presents repeatedly. However, the underlying mechanisms leading to these differences are not known. We compared changes in expression of type-I interferon(IFN-I)-related genes in normal human bronchial epithelial(16HBE) cells. Gene-expression levels of TLR3,MAVS,MDA5,MyD88,IRF7,IFNαand IFNßwere elevated significantly after EVA71 infection.MDA5expression was increased markedly, and that of TLR3 and IRF3was decreased obviously after CV-A16 infection, but that of MAVS,MyD88,IFNαand IFNßdid not show significant differences. Viral copy number and viral titers suggested that CV-A16 replicates more efficiently than EV-A71 in 16HBE.These results suggest that IFN-I production pathway-related genes in response to infection by EV-A71 and CV-A16 have notable discrepancies. Such information could shine a light on the different manifestations caused by EV-A71 and CV-A16,and the mechanism of repeat infection by CV-A16.