Surveillance of SARS-CoV-2 at the Huanan Seafood Market.

SARS-CoV-2, the causative agent of COVID-19, emerged in December 2019. Its origins remain uncertain. It has been reported that a number of the early human cases had a history of contact with the Huanan Seafood Market. Here we present the results of surveillance for SARS-CoV-2 within the market. From January 1st 2020, after closure of the market, 923 samples were collected from the environment. From 18th January, 457 samples were collected from 18 species of animals, comprising of unsold contents of refrigerators and freezers, swabs from stray animals, and the contents of a fish tank. Using RT-qPCR, SARS-CoV-2 was detected in 73 environmental samples, but none of the animal samples. Three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.99% to 100% with the human isolate HCoV-19/Wuhan/IVDC-HB-01/2019. SARS-CoV-2 lineage A (8782T and 28144C) was found in an environmental sample. RNA-seq analysis of SARS-CoV-2 positive and negative environmental samples showed an abundance of different vertebrate genera at the market. In summary, this study provides information about the distribution and prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stages of the COVID-19 outbreak.

The critical role of RAGE in severe influenza infection: A target for control of inflammatory response in the disease.

Controlling the excessive inflammatory response is one of the key ways to reduce the severity and mortality of severe influenza virus infections. RAGE is involved in inflammatory responses and acute lung injuries. Here, we investigated the role of RAGE and its potential application as a target for severe influenza treatment through serological correlation analysis for influenza patients, and treatment with the RAGE inhibitor FPS-ZM1 on A549 cells or mice with influenza A (H1N1) infection. The results showed high levels of RAGE were correlated with immunopathological injury and severity of influenza, and FPS-ZM1 treatment increased the viability of A549 cells with influenza A infection and decreased morbidity and mortality of influenza A virus infection in mice. The RAGE/NF-κb inflammatory signaling pathway is a major targeting pathway for FPS-ZM1 treatment in severe influenza. These findings provide further insights into the immune injury of severe influenza and a potential targeting candidate for the disease treatment.

Spleen tyrosine kinase inhibitor R406 has both antiviral and anti-inflammatory effects on severe influenza A infection.

Death due to severe influenza is usually a fatal complication of a dysregulated immune response more than the acute virulence of an infectious agent. Although spleen tyrosine kinase (SYK) as a critical immune signaling molecule and therapeutic target plays roles in airway inflammation and acute lung injury, the role of SYK in influenza virus infection is not clear. Here, we investigated the antiviral and anti-inflammatory effects of SYK inhibitor R406 on influenza infection through a coculture model of human alveolar epithelial (A549) and macrophage (THP-1) cell lines and mouse model. The results showed that R406 treatment increased the viability of A549 and decreased the pathogenicity and mortality of lethal influenza virus in mice with influenza A infection, decreased levels of intracellular signaling molecules under the condition of inflammation during influenza virus infection. Combination therapy with oseltamivir further ameliorated histopathological damage in the lungs of mice and further delayed the initial time to death compared with R406 treatment alone. This study demonstrated that phosphorylation of SYK is involved in the pathogenesis of influenza, and R406 has antiviral and anti-inflammatory effects on the treatment of the disease, which may be realized through multiple pathways, including the already reported SYK/STAT/IFNs-mediated antiviral pathway, as well as TNF-α/SYK- and SYK/Akt-based immunomodulation pathway.

A Comparison of Etiology, Pathogenesis, Vaccinal and Antiviral Drug Development between Influenza and COVID-19.

Influenza virus and coronavirus, two kinds of pathogens that exist widely in nature, are common emerging pathogens that cause respiratory tract infections in humans. In December 2019, a novel coronavirus SARS-CoV-2 emerged, causing a severe respiratory infection named COVID-19 in humans, and raising a global pandemic which has persisted in the world for almost three years. Influenza virus, a seasonally circulating respiratory pathogen, has caused four global pandemics in humans since 1918 by the emergence of novel variants. Studies have shown that there are certain similarities in transmission mode and pathogenesis between influenza and COVID-19, and vaccination and antiviral drugs are considered to have positive roles as well as several limitations in the prevention and control of both diseases. Comparative understandings would be helpful to the prevention and control of these diseases. Here, we review the study progress in the etiology, pathogenesis, vaccine and antiviral drug development for the two diseases.

Epidemiologic, Clinical, and Genetic Characteristics of Human Infections with Influenza A(H5N6) Viruses, China.

The recent rise in the frequency of influenza A(H5N6) infections in China has raised serious concerns about whether the risk for human infection has increased. We surveyed epidemiologic, clinical, and genetic data of human infections with A(H5N6) viruses. Severe disease occurred in 93.8% of cases, and the fatality rate was 55.4%. Median patient age was 51 years. Most H5N6 hemagglutinin (HA) genes in human isolates in 2021 originated from subclade 2.3.4.4b; we estimated the time to most recent common ancestor as June 16, 2020. A total of 13 genotypes with HA genes from multiple subclades in clade 2.3.4.4 were identified in human isolates. Of note, 4 new genotypes detected in 2021 were the major causes of increased H5N6 virus infections. Mammalian-adapted mutations were found in HA and internal genes. Although we found no evidence of human-to-human transmission, continuous evolution of H5N6 viruses may increase the risk for human infections.

A Comparative Study on the Clinical Features of Coronavirus 2019 (COVID-19) Pneumonia With Other Pneumonias.

BACKGROUND: A novel coronavirus (COVID-19) has raised world concern since it emerged in Wuhan, China in December 2019. The infection may result in severe pneumonia with clusters of illness onsets. Its impacts on public health make it paramount to clarify the clinical features with other pneumonias. METHODS: Nineteen COVID-19 and 15 other patients with pneumonia (non-COVID-19) in areas outside of Hubei were involved in this study. Both COVID-19 and non-COVID-19 patients were confirmed to be infected using throat swabs and/or sputa with/without COVID-2019 by real-time RT-PCR. We analyzed the demographic, epidemiological, clinical, and radiological features from those patients, and compared the differences between COVID-19 and non-COVID-19. RESULTS: All patients had a history of exposure to confirmed cases of COVID-19 or travel to Hubei before illness. The median (IQR) duration was 8 (6-11) and 5 (4-11) days from exposure to onset in COVID-19 and non-COVID-19 cases, respectively. The clinical symptoms were similar between COVID-19 and non-COVID-19. The most common symptoms were fever and cough. Fifteen (78.95%) COVID-19 but 4 (26.67%) non-COVID-19 patients had bilateral involvement while 17 COVID-19 patients (89.47%) but 1 non-COVID-19 patient (6.67%) had multiple mottling and ground-glass opacity on chest CT images. Compared with non-COVID-19, COVID-19 presents remarkably more abnormal laboratory tests, including AST, ALT, γ-GT, LDH, and α-HBDH. CONCLUSIONS: The COVID-19 infection has onsets similar to other pneumonias. CT scan may be a reliable test for screening COVID-19 cases. Liver function damage is more frequent in COVID-19 than non-COVID-19 patients. LDH and α-HBDH may be considerable markers for evaluation of COVID-19.

Biological features of novel avian influenza A (H7N9) virus.

Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1ß, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.

Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses.

Clade 2.3.4.4 highly pathogenic avian influenza viruses (H5Nx) have spread from Asia to other parts of the world. Since 2014, human infections with clade 2.3.4.4 highly pathogenic avian influenza H5N6 viruses have been continuously reported in China. To investigate the genesis of the virus, we analyzed 123 H5 or N6 environmental viruses sampled from live-poultry markets or farms from 2012 to 2015 in Mainland China. Our results indicated that clade 2.3.4.4 H5N2/N6/N8 viruses shared the same hemagglutinin gene as originated in early 2009. From 2012 to 2015, the genesis of highly pathogenic avian influenza H5N6 viruses occurred via two independent pathways. Three major reassortant H5N6 viruses (reassortants A, B, and C) were generated. Internal genes of reassortant A and B viruses and reassortant C viruses derived from clade 2.3.2.1c H5N1 and H9N2 viruses, respectively. Many mammalian adaption mutations and antigenic variations were detected among the three reassortant viruses. Considering their wide circulation and dynamic reassortment in poultry, we highly recommend close monitoring of the viruses in poultry and humans. IMPORTANCE Since 2014, clade 2.3.4.4 highly pathogenic avian influenza (H5Nx) viruses have caused many outbreaks in both wild and domestic birds globally. Severe human cases with novel H5N6 viruses in this group were also reported in China in 2014 and 2015. To investigate the genesis of the genetic diversity of these H5N6 viruses, we sequenced 123 H5 or N6 environmental viruses sampled from 2012 to 2015 in China. Sequence analysis indicated that three major reassortants of these H5N6 viruses had been generated by two independent evolutionary pathways. The H5N6 reassortant viruses had been detected in most provinces of southern China and neighboring countries. Considering the mammalian adaption mutations and antigenic variation detected, the spread of these viruses should be monitored carefully due to their pandemic potential.

Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

Epidemiology of human infections with avian influenza A(H7N9) virus in China.

BACKGROUND: The first identified cases of avian influenza A(H7N9) virus infection in humans occurred in China during February and March 2013. We analyzed data obtained from field investigations to describe the epidemiologic characteristics of H7N9 cases in China identified as of December 1, 2013. METHODS: Field investigations were conducted for each confirmed case of H7N9 virus infection. A patient was considered to have a confirmed case if the presence of the H7N9 virus was verified by means of real-time reverse-transcriptase-polymerase-chain-reaction assay (RT-PCR), viral isolation, or serologic testing. Information on demographic characteristics, exposure history, and illness timelines was obtained from patients with confirmed cases. Close contacts were monitored for 7 days for symptoms of illness. Throat swabs were obtained from contacts in whom symptoms developed and were tested for the presence of the H7N9 virus by means of real-time RT-PCR. RESULTS: Among 139 persons with confirmed H7N9 virus infection, the median age was 61 years (range, 2 to 91), 71% were male, and 73% were urban residents. Confirmed cases occurred in 12 areas of China. Nine persons were poultry workers, and of 131 persons with available data, 82% had a history of exposure to live animals, including chickens (82%). A total of 137 persons (99%) were hospitalized, 125 (90%) had pneumonia or respiratory failure, and 65 of 103 with available data (63%) were admitted to an intensive care unit. A total of 47 persons (34%) died in the hospital after a median duration of illness of 21 days, 88 were discharged from the hospital, and 2 remain hospitalized in critical condition; 2 patients were not admitted to a hospital. In four family clusters, human-to-human transmission of H7N9 virus could not be ruled out. Excluding secondary cases in clusters, 2675 close contacts of case patients completed the monitoring period; respiratory symptoms developed in 28 of them (1%); all tested negative for H7N9 virus. CONCLUSIONS: Most persons with confirmed H7N9 virus infection had severe lower respiratory tract illness, were epidemiologically unrelated, and had a history of recent exposure to poultry. However, limited, nonsustained human-to-human H7N9 virus transmission could not be ruled out in four families.

Two Outbreak Sources of Influenza A (H7N9) Viruses Have Been Established in China.

UNLABELLED: Due to enzootic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. The Yangtze River Delta region, which is located in eastern China, is well recognized as the original source for H7N9 outbreaks. Based on the evolutionary analysis of H7N9 viruses from all three outbreak waves since 2013, we identified the Pearl River Delta region as an additional H7N9 outbreak source. H7N9 viruses are repeatedly introduced from these two sources to the other areas, and the persistent circulation of H7N9 viruses occurs in poultry, causing continuous outbreak waves. Poultry movements may contribute to the geographic expansion of the virus. In addition, the AnH1 genotype, which was predominant during wave 1, was replaced by JS537, JS18828, and AnH1887 genotypes during waves 2 and 3. The establishment of a new source and the continuous evolution of the virus hamper the elimination of H7N9 viruses, thus posing a long-term threat of H7N9 infection in humans. Therefore, both surveillance of H7N9 viruses in humans and poultry and supervision of poultry movements should be strengthened. IMPORTANCE: Since its occurrence in humans in eastern China in spring 2013, the avian H7N9 viruses have been demonstrating the continuing pandemic threat posed by the current influenza ecosystem in China. As the viruses are silently circulated in poultry, with potentially severe outcomes in humans, H7N9 virus activity in humans in China is very important to understand. In this study, we identified a newly emerged H7N9 outbreak source in the Pearl River Delta region. Both sources in the Yangtze River Delta region and the Pearl River Delta region have been established and found to be responsible for the H7N9 outbreaks in mainland China.

Human infection with a novel avian-origin influenza A (H7N9) virus.

BACKGROUND: Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus. METHODS: We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses. RESULTS: A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died. CONCLUSIONS: Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study.

BACKGROUND: Human infections with different avian influenza viruses--eg, H5N1, H9N2, and H7N9--have raised concerns about pandemic potential worldwide. We report the first human infection with a novel reassortant avian influenza A H10N8 virus. METHODS: We obtained and analysed clinical, epidemiological, and virological data from a patient from Nanchang City, China. Tracheal aspirate specimens were tested for influenza virus and other possible pathogens by RT-PCR, viral culture, and sequence analyses. A maximum likelihood phylogenetic tree was constructed. FINDINGS: A woman aged 73 years presented with fever and was admitted to hospital on Nov 30, 2013. She developed multiple organ failure and died 9 days after illness onset. A novel reassortant avian influenza A H10N8 virus was isolated from the tracheal aspirate specimen obtained from the patient 7 days after onset of illness. Sequence analyses revealed that all the genes of the virus were of avian origin, with six internal genes from avian influenza A H9N2 viruses. The aminoacid motif GlnSerGly at residues 226-228 of the haemagglutinin protein indicated avian-like receptor binding preference. A mixture of glutamic acid and lysine at residue 627 in PB2 protein--which is associated with mammalian adaptation--was detected in the original tracheal aspirate samples. The virus was sensitive to neuraminidase inhibitors. Sputum and blood cultures and deep sequencing analysis indicated no co-infection with bacteria or fungi. Epidemiological investigation established that the patient had visited a live poultry market 4 days before illness onset. INTERPRETATION: The novel reassortant H10N8 virus obtained is distinct from previously reported H10N8 viruses. The virus caused human infection and could have been associated with the death of a patient. FUNDING: Emergency Research Project on human infection with avian influenza H7N9 virus, the National Basic Research Program of China, and the National Mega-projects for Infectious Diseases.

Cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza A (H1N1): role of the host immune response in pathogenesis.

Pathological studies on fatal cases caused by 2009 pandemic influenza H1N1 virus (2009 pH1N1) reported extensive diffuse alveolar damage and virus infection predominantly in the lung parenchyma. However, the host immune response after severe 2009 pH1N1 infection is poorly understood. Herein, we investigated viral load, the immune response, and apoptosis in lung tissues from 50 fatal cases with 2009 pH1N1 virus infection. The results suggested that 7 of the 27 cytokines/chemokines showed remarkably high expression, including IL-1 receptor antagonist protein, IL-6, tumor necrosis factor-α, IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-ß, and interferon-inducible protein-10 in lung tissues of 2009 pH1N1 fatal cases. Viral load, which showed the highest level on day 7 of illness onset and persisted until day 17 of illness, was positively correlated with mRNA levels of IL-1 receptor antagonist protein, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-ß, interferon-inducible protein-10, and regulated on activation normal T-cell expressed and secreted. Apoptosis was evident in lung tissues stained by the TUNEL assay. Decreased Fas and elevated FasL mRNA levels were present in lung tissues, and cleaved caspase-3 was frequently seen in pneumocytes, submucosal glands, and lymphoid tissues. The pathogenesis of the 2009 pH1N1 virus infection is associated with viral replication and production of proinflammatory mediators. FasL and caspase-3 are involved in the pathway of 2009 pH1N1 virus-induced apoptosis in lung tissues, and the disequilibrium between the Fas and FasL level in lung tissues could contribute to delayed clearance of the virus and subsequent pathological damages.

R229I substitution from oseltamivir induction in HA1 region significantly increased the fitness of a H7N9 virus bearing NA 292K.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

Oseltamivir inhibits both viral entry and release but enhances apoptosis of cells infected with influenza A H1N1.

Oseltamivir (Tamiflu) has been shown to be effective as a treatment and chemoprophylaxis for influenza virus infections. However, the safety consideration of oseltamivir should not be overlooked because adverse events associated with the drug have frequently been reported after the use of Tamiflu for the treatment of influenza patients. Here, we investigated oseltamivir's influence on viral load, apoptosis, and cytokine response of influenza A H1N1-infected A549 or THP-1 cells. In addition, we analyzed the effect of the drug on the function of influenza-infected NK cells after oseltamivir treatment. The results of our study suggest that oseltamivir may inhibit both viral entry and release but may also enhance apoptosis of infected cells and interfere with NK cell function during influenza infection.

Inactivation of the novel avian influenza A (H7N9) virus under physical conditions or chemical agents treatment.

BACKGROUND: In the spring of 2013, a novel avian-origin influenza A (H7N9) virus in Eastern China emerged causing human infections. Concerns that a new influenza pandemic could occur were raised. The potential effect of chemical agents and physical conditions on inactivation of the novel avian influenza H7N9 virus had not been assessed. METHODS: To determine the inactivation effectiveness of the novel avian influenza A (H7N9) virus under various physical conditions and chemical treatments, two H7N9 viruses A/Anhui/1/2013 and A/Shanghai/1/2013 were treated by varied temperatures, ultraviolet light, varied pHs and different disinfectants. The viruses with 107.7 EID50 were exposed to physical conditions (temperature, ultraviolet light and pH) or treated with commercial chemical agents (Sodium Hypochlorite, Virkon®-S, and Ethanol) respectively. After these treatments, the viruses were inoculated in SPF embryonated chicken eggs, the allantoic fluid was collected after 72-96 hours culture at 35°C and tested by haemagglutination assay. RESULTS: Both of the tested viruses could tolerate conditions under 56°C for 15 minutes or 60°C for 5 minutes, but their infectivity was completely lost under 56°C for 30 minutes, 65°C for 10 minutes, 70°C, 75°C and 100°C for 1 minute. It was also observed that the H7N9 viruses lost their infectivity totally after exposure of ultraviolet light irradiation for 30 minutes or longer time. Additionally, the viruses were completely inactivated at pH less than 2 for 0.5 hour or pH 3 for 24 hours, however, viruses remained infectious under pH treatment of 4-12 for 24 hours. The viruses were totally disinfected when treated with Sodium Hypochlorite, Virkon®-S and Ethanol at recommended concentrations after only 5 minutes. CONCLUSIONS: The novel avian influenza A (H7N9) virus can be inactivated under some physical conditions or with chemical treatments, but they present high tolerance to moderately acidic or higher alkali conditions. The results provided the essential information for public health intervention of novel H7N9 avian influenza outbreak.

Deficiency of C-reactive protein or human C-reactive protein transgenic treatment aggravates influenza A infection in mice.

C-reactive protein (CRP) has been shown to be a potential candidate target in the immunotherapy of severe influenza A infection. However, it is unclear on the pathogenesis associated with CRP in influenza infections. Here, we used influenza A H1N1 CA04 to infect human CRP transgenic mice (KI), CRP knockout mice (KO), and wild-type mice (WT), respectively, and compared the viral pathogenicity and associated immune response in those mice. The results showed that CA04 infection resulted in 100%, 80%, and 60% death in KO, KI, and WT mice, respectively. Compared to WT mice, CA04 infection resulted in higher TCID50 in lungs on day 3 after infection but lowered HI antibody titers in sera of survivors on day 21 after infection in KI mice. ELISA assay showed that IFN-γ concentration was significantly increased in sera of WT, KI, or KO mice on day 7 after infection, and IL-17 was remarkably increased in sera of WT mice but decreased in sera of KI mice while no significant change in sera of KO mice on day 3 or 7 after infection. Quantitative RT-PCR showed that the relative expression levels of immune checkpoint CTLA-4, LAIR-1, GITR, BTLA, TIM-3, or PD-1 mRNA in the lung presented decreased levels on day 3 or 7 after infection in KI or KO mice. The correlation analysis showed that mRNA expression levels of the 6 molecules positively correlated with viral TICD50 in WT mice but negatively correlated with viral TCID50 in KI or KO mice. However, only LAIR-1 presented a significant correlation in each lung tissue of WT, KI, or KO mice with CA07 infection statistically. IHC results showed that LAIR-1 positive cells could be found in WT, KO, or KI mice lung tissues with CA04 infection, and the positive cells were mainly distributed in an inflammatory dense area. Our results suggested that deficiency of CRP or human CRP transgenic treatment aggravates influenza A virus infection in mice. CRP is a double sword in immune regulation of influenza infection in which IL-17 and immune checkpoint may be involved.

The Epidemiological Pattern and Co-infection of Influenza A and B by Surveillance Network From 2009 to 2014 in Anhui Province, China.

Influenza-like illness (ILI) is one of the most important public health problems globally, causing an enormous disease burden. Influenza infections are the most common cause of ILI. Bacterial and virus co-infection is common yet the data of co-infection with influenza A and B viruses are scarce. To identify the epidemiological patterns of and co-infection of influenza A and B in Anhui province, China, we analyzed the surveillance data of 5 years from 2009 to 2014 collected by the Chinese National influenzas network. The results showed that the weekly ratio of ILI was 3.96 ± 1.9% (95% CI 3.73-4.2%) in outpatients and the highest affected population was children under 5 years old. The epidemic of influenza viruses was highest during 2009-2010. For the other 4 surveillance years, school-aged people (5-14 years) were the most highly affected population. Influenza B and H3N2 viruses were more prevalent than H1N1pdm09 virus after 2010. In addition, a significant co-circulation of influenza A (H1N1pdm09 and H3N2) and influenza B virus was detected with 0.057% PCR positive rate during 2009-2014 in Eastern China, yet isolated only in pediatric patients. Our data reveals school-aged population would be the main vulnerable population and a distinct seasonality for influenza. In addition, the co-infection of influenza A and B were found in Anhui Province, China. Ongoing surveillance is critical to understand the seasonality variation and make evidence-based vaccination recommendations. Information on the epidemiological patterns and co-infections of influenza A and B can help us to implement different strategies for selecting vaccine formulations and monitoring new emerging influenza strains. In addition, the identification of the susceptible population can help us to develop more precise protection measures.

Highly Prevalent SARS-CoV-2 Antigenemia in COVID-19 Patients.

Background: Many issues, such as severity assessment and antibody responses, remain to be answered eagerly for evaluation and understanding of COVID-19. Immune lesion is one of key pathogenesis of the disease. It would be helpful to understand the disease if an investigation on antigenemia and association was conducted in the patients with SARS-CoV-2 infection. Methods: A total of 156 patients admitted to the First People's Hospital of Hefei or Anhui Provincial Hospital on January to February 2020 were involved in this study. SARS-CoV-2 nucleocapsid (NP) antigen, specific IgM/IgG antibodies, and RNA were detected in sequential sera from three COVID-19 patients, and additional 153 COVID-19 patients by means of NP-antigen capture enzyme-linked immunosorbent assay, colloidal gold quick diagnosis, and real-time RT-PCR, respectively. The clinical types of COVID-19 patients were classified into asymptomatic, mild, moderate, severe, and critical, following on the Chinese guideline of COVID-19 diagnosis and treatment. The demographic and clinical data of patients were obtained for comparable analysis. Results: NP antigen was detected in 5 of 20 sequential sera collected from three COVID-19 patients with typically clinical symptoms, and 60.13% (92/153) expanded samples collected within 17 days after illness onset. No SARS-CoV-2 RNA segment was detected in these sera. The NP positive proportion reached a peak (84.85%, 28/33) on 6 to 8 days after illness onset. Both NP concentration and positive proportion were increased with the increase of clinical severity of COVID-19. Compared to NP negative patients, NP positive patients had older age [years, medians (interquartile ranges (IQR)), 49 (6) vs. 31 (11)], lower positive proportion of NP specific IgM [27.17% (25/92) vs. 59.02% (36/61)], and IgG [21.74% (20/92) vs. 59.02% (36/61)] antibodies, and longer duration [days, medians (IQR), 24 (10) vs. 21 (13)] from illness to recovery. Conclusions: SARS-CoV-2 NP antigenemia occurred in COVID-19, and presented highly prevalent at early stage of the disease. The antigenemia was related to clinical severity of the disease, and may be responsible for the delay of detectable SARS-Cov-2 IgM.