Simultaneous profiling and quantification of 25 eicosanoids in human serum by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry.

The eicosanoid metabolic pathway is responsible for mediating the production of various inflammatory factors that are closely related to the development and resolution of inflammation. In biological matrices, the major quantifying obstacles were shown to be the oxidation and low quantities of eicosanoids and their metabolites. This study aimed to develop a reliable, sensitive ultrahigh-performance liquid chromatography coupled to a tandem mass spectrometry (UPLC-MS/MS) method to quantify eicosanoids in human serum. Solid-phase extraction (SPE) was used for sample preparation. The approach employed continuous ionization polarity switching. The target eicosanoids showed good linearity over the investigated concentration range (r2 > 0.99). The recovery rates were over 64.5%, and the matrix effects ranged from 73.0 to 128.0%. The limits of quantification were 0.048 ~ 0.44 ng/mL. For the broad concentration range, the CV % for accuracy and precision were less than ± 20%. We successfully applied this method to rapidly analyse 74 serum samples from severe influenza pneumonia, severe bacterial pneumonia and healthy individuals. Eicosanoid-related metabolite concentrations were quantified within a range similar to those of previously published articles. Compared to healthy individuals, our application found that 20-HETE, 14,15-EET and 11,12-EET were upregulated in severe influenza pneumonia patients, while LTB4 was downregulated. 8-HETE and 5-HETE were upregulated in severe bacterial pneumonia patients, while LTE4 was downregulated. This approach provides a means for monitoring the low quantities of eicosanoids in biological matrices, and our finding that different characteristic metabolite profiles may help discriminate the induction of severe pneumonia patients.

Differing epidemiological dynamics of influenza B virus lineages in Guangzhou, southern China, 2009-2010.

The epidemiological and evolutionary dynamics of the two cocirculating lineages of influenza B virus, Victoria and Yamagata, are poorly understood, especially in tropical or subtropical areas of Southeast Asia. We performed a phylogenetic analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of influenza B viruses isolated in Guangzhou, a southern Chinese city, during 2009 to 2010 and compared the demographic and clinical features of infected patients. We identified multiple viral introductions of Victoria strains from both Chinese and international sources, which formed two phylogenetically and antigenically distinct clades (Victoria 1 and 2), some of which persisted between seasons. We identified one dominant Yamagata introduction from outside China during 2009. Our phylogenetic analysis reveals the occurrence of reassortment events among the Victoria and Yamagata lineages and also within the Victoria lineage. We found no significant difference in clinical severity by influenza B lineage, with the exceptions that (i) the Yamagata lineage infected older people than either Victoria lineage and (ii) fewer upper respiratory tract infections were caused by the Victoria 2 than the Victoria 1 clade. Overall, our study reveals the complex epidemiological dynamics of different influenza B lineages within a single geographic locality and has implications for vaccination policy in southern China.

[Oseltamivir resistance among pandemic A(H1N1)2009 viruses between 2009 and 2011 in Guangzhou].

OBJECTIVE: To study the prevalence of oseltamivir-resistance among pandemic A (H1N1)2009 viruses isolated from patients in Guangzhou between 2009 and 2011, and to provide more information for clinical usage of oseltamivir. METHODS: Totally 192 pandemic A (H1N1)2009 viruses isolated from patients in Guangzhou between July 2009 and April 2011 were studies. The HA and NA genes of all strains were sequenced to reveal the evolution of viruses, and the susceptibility of viruses to oseltamivir was tested in vitro. RESULTS: One strain with a S247N mutation of the NA gene, which would make the virus resistant to oseltamivir, was found. The susceptibility (IC)50 of this viral strain to oseltamivir was 0.45 nmol/L, 2.5 times lower as compared to the wild-type strains. Phylogenetic analysis showed that this virus was not prevalent in Guangzhou from 2009-2011, and was not located in the same branch with the strains being epidemic in Australia and Singapore during the early seasons of 2011. CONCLUSION: The resistance rate of pandemic A(H1N1)2009 viruses isolated from Guangzhou to oseltamivir was low, but surveillance on resistant strains needs to be strengthened to control resistant viruses imported from abroad.

Preclinical evaluation of the SARS-CoV-2 Mpro inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir.

Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (Mpro), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from Mpro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 Mpro:RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named 'leritrelvir' for COVID-19 treatment in China.

Using discrete event simulation to optimize nucleic acid testing process for coronavirus disease 2019 (COVID-19).

Background: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus-2 (SARS-CoV-2) has placed enormous diagnostic burden on hospitals and testing laboratories. It is thus critical for such facilities to optimize the diagnostic process to enable maximum testing on minimum resources. The current standard of diagnosis is the detection of the viral nucleic acid in clinical specimens. Methods: In order to optimize the laboratory's nucleic acid testing system for COVID-19, we performed a Discrete-Event-Simulation using the Arena Simulation Software to model the detection process based on the data obtained from the First Affiliated Hospital of Guangzhou Medical University (FAHGMU). The maximum of total time that specimens spent and the equipment consumption was compared under different scenarios in the model. Results: Seven scenarios were performed to simulate actual situation and improved situations. We analyzed conditions that adding a new nucleic acid extraction system (NAES), shifting a member from night duty to morning duty, using specimen tubes containing guanidine isothiocyanate (GITC), then tested the maximum testing capacity in the current number of technicians. In addition, the costs including personal protective equipment (PPE) and testing kits was calculated. Conclusions: A work schedule based on specimen-load improves efficiency without incurring additional costs, while using the specimen tubes containing GITC could reduce testing time by 30 min. In contrast, adding new NAESs or polymerase chain reaction (PCR) instruments has minimal impact on testing efficiency.

SARS-CoV-2 environmental contamination associated with persistently infected COVID-19 patients.

BACKGROUND: Severe COVID-19 patients typically test positive for SARS-CoV-2 RNA for extended periods of time, even after recovery from severe disease. Due to the timeframe involved, these patients may have developed humoral immunity to SARS-CoV-2 while still testing positive for viral RNA in swabs. Data are lacking on exposure risks in these situations. Here, we studied SARS-CoV-2 environmental contamination in an ICU and an isolation ward caring for such COVID-19 patients. METHODS: We collected air and surface samples in a hospital caring for critical and severe COVID-19 cases from common areas and areas proximal to patients. RESULTS: Of the 218 ICU samples, an air sample contained SARS-CoV-2 RNA. Of the 182 isolation ward samples, nine contained SARS-CoV-2 RNA. These were collected from a facemask, the floor, mobile phones, and the air in the patient room and bathroom. Serum antibodies against SARS-CoV-2 were detected in these patients at the beginning of the study. CONCLUSIONS: While there is a perception of increased risk in the ICU, our study demonstrates that isolation wards may pose greater risks to healthcare workers and exposure risks remain with clinically improved patients, weeks after their initial diagnoses. As these patients had serum antibodies, further studies may be warranted to study the utility of serum antibodies as a surrogate of viral clearance in allowing people to return to work. We recommend continued vigilance even with patients who appear to have recovered from COVID-19.

Epidemiological and viral genome characteristics of the first human H7N9 influenza infection in Guangdong Province, China.

BACKGROUND: The first H7N9 human case in south of China was confirmed in Guangdong Province on August 2013, outside of the typical influenza season. For investigating the H7N9 virus source and transmission in the local community, we analyze the epidemiology and genome features of the virus isolated from the first human infection detected in Guangdong Province. METHODS: The data including medical records, exposure history and time line of events for the H7N9 patient and close contacts was collected. Variation and genetic signatures of H7N9 virus in Guangdong was analyzed using ClustalW algorithm and comparison with mutations associated with changes in biological characteristics of the virus. RESULTS: The female patient had a history of poultry exposure, and she was transferred from a local primary hospital to an intensive care unit (ICU) upon deterioration. No additional cases were reported. Similar to previous infections with avian influenza A (H7N9) virus, the patient presented with both upper and lower respiratory tract symptoms. Respiratory failure progressed quickly, and the patient recovered 4 weeks after the onset of symptoms. Genome analysis of the virus indicated that the predicted antigen city and internal genes of the virus are similar to previously reported H7N9 viruses. The isolated virus is susceptible to neuraminidase (NA) inhibitors but resistant to adamantine. Although this virus contains some unique mutations that were only detected in avian or environment-origin avian influenza A (H7N9) viruses, it is still quite similar to other human H7N9 isolates. CONCLUSIONS: The epidemiological features and genome of the first H7N9 virus in Guangdong Province are similar to other human H7N9 infections. This virus may have existed in the environment and live poultry locally; therefore, it is important to be alert of the risk of H7N9 re-emergence in China, including emergence outside the typical influenza season.

[Development and characterization of a stable cell line expressing respiratory syncytial virus non-structural protein NS1].

To develop a stable cell line that could express the RSV NS1, the full-length RSV NS1 gene was generated by RT-PCR amplification from respiratory syncytial virus. NS1 gene was ligated with pBABE-puro to construct the recombinant retroviral expression plasmid pBABE-NS1, which was cotransfected into 293FT packaging cells with PIK packaging plasmid by calcium phosphate co-precipitation. The supernatant of 293FT was collected to infect HEp-2 cells, the resulting cell clones stably expressing NS1 were screened by puromycin. Using QPCR, CPE staining method and indirect immunofluorescence assay, the expression of NS1 at both gene and protein levels was identified. The recombinant plasmid pBABE-NS1 was identified by EcoRI and BamHI endonuclease digestion and the sequence analysis. QPCR results showed that the NS1 gene amplification in HEp-2-NS1 cells was 8483 fold higher than that in HEp-2 cells. Although the exogenous interferon was added, all cells were destroyed after 48 hours post infection using CPE staining method, showing that HEp-2-NS1 cells remained sensitive to the VSV virus. The results of RT-PCR and indirect immunofluorescence assay showed that the NS1 gene in HEp-2 cells could not only transcribe mRNA, but also express NS1 protein steadily. We had successfully established HEp-2-NS1 cell lines with stable expression of respiratory syncytial virus non-structural protein NS1.