Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort: Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which has been characterized by fever, respiratory, and gastrointestinal symptoms as well as shedding of virus RNA into feces. We performed a systematic review and meta-analysis of published gastrointestinal symptoms and detection of virus in stool and also summarized data from a cohort of patients with COVID-19 in Hong Kong. METHODS: We collected data from the cohort of patients with COVID-19 in Hong Kong (N = 59; diagnosis from February 2 through February 29, 2020),and searched PubMed, Embase, Cochrane, and 3 Chinese databases through March 11, 2020, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We analyzed pooled data on the prevalence of overall and individual gastrointestinal symptoms (loss of appetite, nausea, vomiting, diarrhea, and abdominal pain or discomfort) using a random effects model. RESULTS: Among the 59 patients with COVID-19 in Hong Kong, 15 patients (25.4%) had gastrointestinal symptoms, and 9 patients (15.3%) had stool that tested positive for virus RNA. Stool viral RNA was detected in 38.5% and 8.7% among those with and without diarrhea, respectively (P = .02). The median fecal viral load was 5.1 log10 copies per milliliter in patients with diarrhea vs 3.9 log10 copies per milliliter in patients without diarrhea (P = .06). In a meta-analysis of 60 studies comprising 4243 patients, the pooled prevalence of all gastrointestinal symptoms was 17.6% (95% confidence interval [CI], 12.3-24.5); 11.8% of patients with nonsevere COVID-19 had gastrointestinal symptoms (95% CI, 4.1-29.1), and 17.1% of patients with severe COVID-19 had gastrointestinal symptoms (95% CI, 6.9-36.7). In the meta-analysis, the pooled prevalence of stool samples that were positive for virus RNA was 48.1% (95% CI, 38.3-57.9); of these samples, 70.3% of those collected after loss of virus from respiratory specimens tested positive for the virus (95% CI, 49.6-85.1). CONCLUSIONS: In an analysis of data from the Hong Kong cohort of patients with COVID-19 and a meta-analysis of findings from publications, we found that 17.6% of patients with COVID-19 had gastrointestinal symptoms. Virus RNA was detected in stool samples from 48.1% patients, even in stool collected after respiratory samples had negative test results. Health care workers should therefore exercise caution in collecting fecal samples or performing endoscopic procedures in patients with COVID-19, even during patient recovery.

SMRT sequencing revealed the diversity and characteristics of defective interfering RNAs in influenza A (H7N9) virus infection.

Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.

Triple combination of FDA-approved drugs including flufenamic acid, clarithromycin and zanamivir improves survival of severe influenza in mice.

Seasonal influenza virus remains a common cause of mortality despite the use of neuraminidase inhibitors. This study evaluated the efficacy of a triple combination of zanamivir, clarithromycin and flufenamic acid (FFA) in the treatment of influenza virus A(H1N1) infection. An in vitro cell protection assay and a multiple-cycle growth assay showed that the antiviral activity of zanamivir was enhanced when combined with clarithromycin or FFA. A mouse challenge model was used here for the evaluation of the in vivo efficacy of the triple combination treatment. We found that mice receiving the triple combination of FFA, zanamivir, and clarithromycin had a significantly better survival rate than those receiving the double combination of zanamivir and clarithromycin (88% versus 44%, P = 0.0083) or zanamivir monotherapy (88% versus 26%, P = 0.0002). Mice in the FFA-zanamivir-clarithromycin triple combination group also exhibited significantly less body weight loss than those in the zanamivir-clarithromycin double combination group. There was no significant difference in the lung viral titers among the different groups from day 2 to day 6 postinfection. However, the levels of IL-1ß, TNF-α and RANTES in the FFA-zanamivir-clarithromycin triple combination group were significantly lower than those in the zanamivir-clarithromycin double combination group, zanamivir monotherapy group, or solvent group on day 2 postinfection. Our findings showed that the FFA-zanamivir-clarithromycin triple combination improved the inflammatory markers and survival of severe influenza A(H1N1) infection in mice.

Copper transporter 1 in human colorectal cancer cell lines: Effects of endogenous and modified expression on oxaliplatin cytotoxicity.

Oxaliplatin-based chemotherapy is the mainstay for the treatment of advanced colorectal cancer. Copper transporter proteins have been implicated in the transport of platinum-based anticancer drugs, but their expression in human colorectal cancer cell lines and roles in controlling their sensitivity to oxaliplatin are not well studied or understood. The endogenous and modified expression of copper uptake transporter 1 (hCTR1) was studied in a panel of human colorectal cancer cell lines (DLD-1, SW620, HCT-15 and COLO205) with ~20-fold variation in oxaliplatin sensitivity. hCTR1 protein was expressed more abundantly than ATP7A and ATP7B proteins, but with broadly similar levels and patterns of expression across four colorectal cancer cell lines. In a colorectal cancer cell-line background (DLD-1), stable transfection of the hCtr1 gene enhanced hCTR1 protein expression and increased the sensitivity of the cells to the cytotoxicity of copper and oxaliplatin. Treatment with copper chelators (ammonium tetrathiomolybdate, bathocuproinedisulfonic acid and D-penicillamine) increased expression of hCTR1 protein in DLD-1 and SW620 cells, and potentiated the cytotoxicity of oxaliplatin in DLD-1 but not SW620 cells. Treatment with copper chloride altered neither the expression of copper transporters nor cytotoxicity of oxaliplatin in colorectal cancer lines. In conclusion, human colorectal cancer cell lines consistently express hCTR1 protein despite their variable sensitivity to oxaliplatin. Genetic or pharmacological modification of hCTR1 protein expression may potentiate oxaliplatin sensitivity in some but not all colorectal cancer cell lines.

Human H7N9 virus induces a more pronounced pro-inflammatory cytokine but an attenuated interferon response in human bronchial epithelial cells when compared with an epidemiologically-linked chicken H7N9 virus.

BACKGROUND: Avian influenza virus H7N9 has jumped species barrier, causing sporadic human infections since 2013. We have previously isolated an H7N9 virus from a patient, and an H7N9 virus from a chicken in a live poultry market where the patient visited during the incubation period. These two viruses were genetically highly similar. This study sought to use a human bronchial epithelial cell line model to infer the virulence of these H7N9 viruses in humans. METHODS: Human bronchial epithelial cell line Calu-3 was infected with two H7N9 viruses (human H7N9-HU and chicken H7N9-CK), a human H5N1 virus and a human 2009 pandemic H1N1 virus. The infected cell lysate was collected at different time points post-infection for the determination of the levels of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α] and interleukin 6 [IL-6]), anti-inflammatory cytokines (interleukin 10 [IL-10] and transforming growth factor beta [TGF-ß]), chemokines (interleukin 8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]), and interferons (interferon ß [IFN-ß] and interferon lambda 1 [IFNL1]). The viral load in the cell lysate was also measured. RESULTS: Comparison of the human and chicken H7N9 viruses showed that H7N9-HU induced significantly higher levels of TNF-α at 12 h post-infection, and significantly higher levels of IL-8 from 12 to 48 h post-infection than those of H7N9-CK. However, the level of IFNL1 was lower for H7N9-HU than that of H7N9-CK at 48 h post-infection (P < 0.001). H7N9-HU had significantly higher viral loads than H7N9-CK at 3 and 6 h post-infection. H5N1 induced significantly higher levels of TNF-α, IL-6, IL-8, IL-10 and MCP-1 than those of H7N9 viruses at 48 h post-infection. Conversely, H1N1 induced lower levels of TNF-α, IL-10, MCP-1, IFNL1 and IFN-ß when compared with H7N9 viruses at the same time point. CONCLUSIONS: H7N9-HU induced higher levels of pro-inflammatory IL-6 and IL-8 and exhibited a more rapid viral replication than H7N9-CK. However, the level of antiviral IFNL1 was lower for H7N9-HU than H7N9-CK. Our results suggest that the gained properties in modulating human innate immunity by H7N9-HU transformed it to be a more virulent virus in humans than H7N9-CK.

ABC Transporter Inhibitors in Reversing Multidrug Resistance to Chemotherapy.

The superfamily of human ATP-binding cassette (ABC) transporters comprises seven subfamilies (ABCA to G) with 48 members. In addition to their profound physiological and pharmacological functions, ABC transporters play important roles in instigating multidrug resistance (MDR) in cancer by mediating the efflux of many anticancer drugs, particularly, ABCB1, ABCG2 and ABCC subfamily members. Previous development of ABCB1 transporter inhibitors has provided insights into seeking novel strategies in developing new classes of compound that inhibit ABCB1 and other MDRrelated ABC transporters. We herein review and evaluate current evidence in this area, with an emphasis on experimental and investigational agents that are under preclinical and clinical tests, including tyrosine kinase inhibitors, natural products, microRNAs and novel chemical entities. New strategies targeting ABC transporters in cancer stem cells and future perspectives in this field are also discussed.

A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be emphasized in the investigation of disease outbreaks in human and animals.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.

BACKGROUND: The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). METHODOLOGY/PRINCIPAL FINDINGS: The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.

Leptin mediates the pathogenesis of severe 2009 pandemic influenza A(H1N1) infection associated with cytokine dysregulation in mice with diet-induced obesity.

BACKGROUND: Obesity is associated with a high circulating leptin level and severe 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) infection. The mechanism for severe lung injury in obese patients and the specific treatment strategy remain elusive. METHOD: We studied the pathogenesis of A(H1N1)pdm09 infection in a mouse model of diet-induced obesity. RESULTS: Obese mice had significantly higher initial pulmonary viral titer and mortality after challenge with A(H1N1)pdm09, compared with age-matched lean mice. Compared with lean mice, obese mice had heightened proinflammatory cytokine and chemokine levels and more severe pulmonary inflammatory damage. Furthermore, obese mice had a higher preexisting serum leptin level but a lower preexisting adiponectin level. Recombinant mouse leptin increased the interleukin 6 (IL-6) messenger RNA expression in mouse single-lung-cell preparations, mouse macrophages, and mouse lung epithelial cell lines infected with A(H1N1)pdm09. Administration of anti-leptin antibody improved the survival of infected obese mice, with associated reductions in pulmonary levels of the proinflammatory cytokines IL-6 and interleukin 1ß but not the pulmonary viral titer. CONCLUSIONS: Our findings suggest that preexisting high levels of circulating leptin contribute to the development of severe lung injury by A(H1N1)pdm09 in mice with diet-induced obesity. The therapeutic strategy of leptin neutralization for the reduction of proinflammatory responses and pulmonary damage in obese patients warrants further investigations.