Desmoglein 2 (DSG2) Is A Receptor of Human Adenovirus Type 55 Causing Adult Severe Community-Acquired Pneumonia.

Human adenovirus type 55 (HAdV-B55) is a re-emergent acute respiratory disease pathogen that causes adult community-acquired pneumonia (CAP). Previous studies have shown that the receptor of HAdV-B14, which genome is highly similar with HAdV-B55, is human Desmoglein 2 (DSG2). However, whether the receptor of HAdV-B55 is DSG2 is undetermined because there are three amino acid mutations in the fiber gene between HAdV-B14 and HAdV-B55. Here, firstly we found the 3T3 cells, a mouse embryo fibroblast rodent cell line which does not express human DSG2, were able to be infected by HAdV-B55 after transfected with pcDNA3.1-DSG2, while normal 3T3 cells were still unsusceptible to HAdV-B55 infection. Next, A549 cells with hDSG2 knock-down by siRNA were hard to be infected by HAdV-B3/-B14/-B55, while the control siRNA group was still able to be infected by all these types of HAdVs. Finally, immunofluorescence confocal microscopy indicated visually that Cy3-conjugated HAdV-B55 viruses entered A549 cells by binding to DSG2 protein. Therefore, DSG2 is a major receptor of HAdV-B55 causing adult CAP. Our finding is important for better understanding of interactions between adenoviruses and host cells and may shed light on the development of new drugs that can interfere with these processes as well as for the development of potent prophylactic vaccines.

Development and Application of a Fast Method to Acquire the Accurate Whole-Genome Sequences of Human Adenoviruses.

The whole-genome sequencing (WGS) of human adenoviruses (HAdVs) plays an important role in identifying, typing, and mutation analysis of HAdVs. Nowadays, three generations of sequencing have been developed. The accuracy of first-generation sequencing is up to 99.99%, whereas this technology relies on PCR and is time consuming; the next-generation sequencing (NGS) is expensive and not cost effective for determining a few special samples; and the third-generation sequencing technology has a higher error rate. In this study, first, we developed an efficient HAdV genomic DNA extraction method. Using the complete genomic DNA instead of the PCR amplicons as the direct sequencing template and a set of walking primers, we developed the HAdV WGS method based on first-generation sequencing. The HAdV whole genomes were effectively sequenced by a set of one-way sequencing primers designed, which reduced the sequencing time and cost. More importantly, high sequence accuracy is guaranteed. Four HAdV strains (GZ01, GZ02, HK35, and HK91) were isolated from children with acute respiratory diseases (ARDs), and the complete genomes were sequenced using this method. The accurate sequences of the whole inverted terminal repeats (ITRs) at both ends of the HAdV genomes were also acquired. The genome sequence of human adenovirus type 14 (HAdV-B14) strain GZ01 acquired by this method is identical to the sequence released in GenBank, which indicates that this novel sequencing method has high accuracy. The comparative genomic analysis identified that strain GZ02 isolated in September 2010 had the identical genomic sequence with the HAdV-B14 strain GZ01 (October 2010). Therefore, strain GZ02 is the first HAdV-B14 isolate emergent in China (September 2010; GenBank acc no. MW692349). The WGS of HAdV-C2 strain HK91 and HAdV-E4 strain HK35 isolated from children with acute respiratory disease in Hong Kong were also determined by this sequencing method. In conclusion, this WGS method is fast, accurate, and universal for common human adenovirus species B, C, and E. The sequencing strategy may also be applied to the WGS of the other DNA viruses.

Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine: "Adenovirus Vaccine Within an Adenovirus Vector".

Human adenoviruses (HAdVs) are highly contagious and result in large number of acute respiratory disease (ARD) cases with severe morbidity and mortality. Human adenovirus type 3 (HAdV-3) is the most common type that causes ARD outbreaks in Asia, Europe, and the Americas. However, there is currently no vaccine approved for its general use. The hexon protein contains the main neutralizing epitopes, provoking strong and lasting immunogenicity. In this study, a novel recombinant and attenuated adenovirus vaccine candidate against HAdV-3 was constructed based on a commercially-available replication-defective HAdV-5 gene therapy and vaccine vector. The entire HAdV-3 hexon gene was integrated into the E1 region of the vector by homologous recombination using a bacterial system. The resultant recombinants expressing the HAdV-3 hexon protein were rescued in AD293 cells, identified and characterized by RT-PCR, Western blots, indirect immunofluorescence, and electron microscopy. This potential vaccine candidate had a similar replicative efficacy as the wild-type HAdV-3 strain. However, and importantly, the vaccine strain had been rendered replication-defective and was incapable of replication in A549 cells after more than twenty-generation passages in AD293 cells. This represents a significant safety feature. The mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against HAdV-3. Therefore, this recombinant, attenuated, and safe adenovirus vaccine is a promising HAdV-3 vaccine candidate. The strategy of using a clinically approved and replication-defective HAdV-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ARDs and perhaps other adenovirus-associated diseases.

High-Throughput Screening and Identification of Human Adenovirus Type 5 Inhibitors.

Human adenovirus infections can develop into diffuse multi-organ diseases in young children and immunocompromised patients, and severe cases can lead to death. However, there are no approved antiviral drugs available to treat adenovirus diseases. In this study, a chemiluminescence-based, high-throughput screening (HTS) assay was developed and applied to screen human adenovirus 5(HAdV5)inhibitors from 1,813 approved drug library and 556 traditional Chinese medicine-sourced small-molecule compounds. We identified three compounds with in vitro anti-HAdV5 activities in the low-micromolar range (EC50 values 0.3-4.5 µM, selectivity index values 20-300) that also showed inhibitory effects on HAdV3. Cardamomin (CDM) had good anti-HAdV5 activity in vitro. Furthermore, three dilutions of CDM (150, 75, and 37.5 mg/kg/d) administered to BALB/c mouse models inhibited HAdV5-fluc infection at 1 day post-infection by 80% (p < 0.05), 76% (p < 0.05), and 58% (p < 0.05), respectively. HE-staining of pathological tissue sections of mice infected with a wildtype adenoviral strain showed that CDM had a protective effect on tissues, especially in the liver, and greatly inhibited virus-induced necrosis of liver tissue. Thus, CDM inhibits adenovirus replication in vivo and in vitro. This study established a high-throughput screening method for anti-HAdV5 drugs and demonstrated CDM to be a candidate for HAdV5 therapy, potentially providing a new treatment for patients infected with adenoviruses.

COVID-19: Antiviral Agents, Antibody Development and Traditional Chinese Medicine.

The World Health Organization (WHO) has declared coronavirus disease 2019 (COVID-19) is the first pandemic caused by coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, there is no effective anti-SARS-CoV-2 drug approved worldwide for treatment of patients with COVID-19. Therapeutic options in response to the COVID-19 outbreak are urgently needed. To facilitate the better and faster development of therapeutic COVID-19 drugs, we present an overview of the global promising therapeutic drugs, including repurposing existing antiviral agents, network-based pharmacology research, antibody development and traditional Chinese medicine. Among all these drugs, we focus on the most promising drugs (such as favipiravir, tocilizumab, SARS-CoV-2 convalescent plasma, hydroxychloroquine, Lianhua Qingwen, interferon beta-1a, remdesivir, etc.) that have or will enter the final stage of human testing-phase III-IV clinical trials.

Characterization of Influenza A and B Viruses Circulating in Southern China During the 2017-2018 Season.

The trivalent seasonal influenza vaccine was the only approved and available vaccine during the 2016-2018 influenza seasons. It did not include the B/Yamagata strain. In this study, we report an acute respiratory disease outbreak associated with influenza B/Yamagata infections in Guangzhou, Southern China (January through March, 2018). Among the 9914 patients, 2241 (22.6%) were positive for the influenza B virus, with only 312 (3.1%) positive for the influenza A virus. The influenza B/Yamagata lineage dominated during this period in Southern China. The highest incidence of influenza A virus infection occurred in the children aged 5-14 years. In contrast, populations across all age groups were susceptible to the influenza B virus. Phylogenetic, mutations, and 3D structure analyses of hemagglutinin (HA) genes were performed to assess the vaccine-virus relatedness. The recommended A/H1N1 vaccine strain (A/Michigan/45/2015) during both 2017-2018 and 2018-2019 was antigen-specific for these circulating isolates (clade 6B.1) in Spring 2018. An outbreak of influenza B/Yamagata (clade 3) infections in 2018 occurred during the absence of the corresponding vaccine during 2016-2018. The recommended influenza B/Yamagata vaccine strain (B/Phuket/3073/2013) for the following season (2018-2019) was antigen-specific. Although there were only a few influenza B/Victoria infections in Spring 2018, five amino acid mutations were identified in the HA antigenic sites of the 19 B/Victoria isolates (clade 1A), when compared with the 2016-2018 B/Victoria vaccine strain. The number was larger than expected and suggested that the influenza B HA gene may be more variable than previously thought. One of the mutations (K180N) was noted to likely alter the epitope and to potentially affect the viral antigenicity. Seven mutations were also identified in the HA antigenic sites of 2018-2020 B/Victoria vaccine strain, of which some or all may reduce immunogenicity and the protective efficacy of the vaccine, perhaps leading to more outbreaks in subsequent seasons. The combined epidemiological, phylogenetic, mutations, and 3D structural analyses of the HA genes of influenza strains reported here contribute to the understanding and evaluation of how HA mutations affect vaccine efficacy, as well as to providing important data for screening and selecting more specific, appropriate, and effective influenza vaccine candidate strains.

COVID-19: Coronavirus Vaccine Development Updates.

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a newly emerged coronavirus, and has been pandemic since March 2020 and led to many fatalities. Vaccines represent the most efficient means to control and stop the pandemic of COVID-19. However, currently there is no effective COVID-19 vaccine approved to use worldwide except for two human adenovirus vector vaccines, three inactivated vaccines, and one peptide vaccine for early or limited use in China and Russia. Safe and effective vaccines against COVID-19 are in urgent need. Researchers around the world are developing 213 COVID-19 candidate vaccines, among which 44 are in human trials. In this review, we summarize and analyze vaccine progress against SARS-CoV, Middle-East respiratory syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, including inactivated vaccines, live attenuated vaccines, subunit vaccines, virus like particles, nucleic acid vaccines, and viral vector vaccines. As SARS-CoV-2, SARS-CoV, and MERS-CoV share the common genus, Betacoronavirus, this review of the major research progress will provide a reference and new insights into the COVID-19 vaccine design and development.

Regulation of Probiotics on Metabolism of Dietary Protein in Intestine.

Proteins are indispensable components of living organisms, which are derived mainly from diet through metabolism. Dietary proteins are degraded by endogenous digestive enzymes to di- or tripeptides and free amino acids (AAs) in the small intestine lumen and then absorbed into blood and lymph through intestinal epithelial cells via diverse transporters. Microorganisms are involved not only in the proteins' catabolism, but also the AAs, especially essential AAs, anabolism. Probiotics regulate these processes by providing exogenous proteases and AAs and peptide transporters, and reducing hazardous substances in the food and feed. But the core mechanism is modulating of the composition of intestinal microorganisms through their colonization and exclusion of pathogens. The other effects of probiotics are associated with normal intestinal morphology, which implies that the enterocytes secrete more enzymes to decompose dietary proteins and absorb more nutrients.

Prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows.

BACKGROUND: Overconditioned dairy cows are susceptible to excessive lipolysis and increased insulin resistance during the transition period. The associations among body fat reserve, insulin resistance, and lipolysis in adipose tissues (AT) remain to be elucidated. Therefore, this study aimed to investigate whether excessive fat reserves influence the insulin signaling pathway in AT postpartum. RESULTS: Twenty multiparous dairy cows were selected and assigned to one of two groups, according to prepartum body condition score (BCS): Control group (BCS = 3.0-3.5; n = 10) and Overconditioned group (BCS ≥ 4.0; n = 10). Blood samples were collected on days -14, -7, -4, -2, -1, 0, 1, 2, 4, 7, and 14 relative to parturition. Subcutaneous AT were collected on day 2 following parturition for quantitative real-time polymerase chain reaction and western blot analyses. No differences were observed between the two groups in serum glucose, non-esterified fatty acids, ß-hydroxybutyric acid, tumor necrosis factor (TNF) α, insulin, or leptin concentrations during the experimental period. Compared with the control cows, the overconditioned cows had lower serum triglyceride levels and higher adiponectin concentrations. In the AT postpartum, insulin receptor mRNA and protein levels were lower in the overconditioned cows than in the control cows, and no differences were found in glucose transporter 4 mRNA. Compared with the control cows, the overconditioned cows had lower mRNA levels of TNFα and higher mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) in AT postpartum. The phosphorylated protein kinase B (AKT) content and phosphorylation rate of AKT were increased in the overconditioned cows compared with the control cows, which suggested that the downstream insulin signaling in AT was affected. CONCLUSIONS: In the present study, transition dairy cows with higher BCS did not show more fat mobilization. The changes of insulin signaling pathway in AT postpartum of overconditioned cows may be partly related to the expression of PPARγ and TNFα, and the secretion of adiponectin.

Unraveling adsorption behavior and mechanism of perfluorooctane sulfonate (PFOS) on aging aquatic sediments contaminated with engineered nano-TiO2.

Engineered nano-TiO2 (Enano-TiO2) have inevitably discharged into aquatic sediments that resulted from their widespread use. The physicochemical characteristics of sediments might be changed because of remarkable properties of Enano-TiO2 and affected by the aging of sediments, thereby altering the environmental behavior and bioavailability of other pollutants such as perfluorooctane sulfonate (PFOS) in sediments. Here, adsorption behavior and mechanism of PFOS on aging aquatic sediments spiked with Enano-TiO2 at a weight ratio of 5.0% were investigated. The results showed that Enano-TiO2 significantly altered zero points of charge (pHzpc) and pore surface properties of sediments, manifested as pHzpc, the total surface area (SBET), the micro-pore surface area (Smicro), and the external surface area (Sext) of sediment particles contaminated with Enano-TiO2 clearly increased, instead average pore size decreased. Rapid intra-particle diffusion processes were well fitted by the pseudo-second-order rate model with the sorption rate (K2) following the order single (5.764 mg/(g·h)) > binary systems (3.393 mg/(g·h)). Freundlich model best described the sorption isotherm data with the larger sorption capacity (KF) and sorption affinity (1/n) of sediments spiked with Enano-TiO2 than that of sediments only. Additionally, Enano-TiO2 changed the adsorption thermodynamics of PFOS on the sediments with the absolute value of ∆G0, ∆H0, and ∆S0 increased. Fourier transform infrared (FT-IR) spectroscopy suggested possible formation of a negative charge-assisted H-bond between PFOS and the functionalities on sediment surfaces, including O-H of carboxyl, alcohol, phenols, and chemisorbed H2O as well as carbonyl groups (C=O) of ketone groups. Furthermore, the multilayer sorption of PFOS on sediments contaminated with Enano-TiO2 is plausible because of bridging effect of Cu2+ and Pb2+.

Dietary Clostridium butyricum Induces a Phased Shift in Fecal Microbiota Structure and Increases the Acetic Acid-Producing Bacteria in a Weaned Piglet Model.

Clostridium butyricum is known as a butyrate producer and a regulator of gut health, but whether it exerts a beneficial effect as a dietary supplement via modulating the intestinal microbiota remains elusive. This study investigated the impact of C. butyricum on the fecal microbiota composition and their metabolites 14 and 28 days after weaning with 10 g/kg dietary supplementation of C. butyricum. Dynamic changes of microbial compositions showed dramatically increasing Selenomonadales and decreasing Clostridiales on days 14 and 28. Within Selenomonadales, Megasphaera became the main responder by increasing from 3.79 to 11.31%. Following the prevalence of some acetate producers ( Magasphaera) and utilizers ( Eubacterium_hallii) at the genus level and even with a significant decrease in fecal acetate on day 28, the present data suggested that C. butyricum influenced microbial metabolism by optimizing the structure of microbiota and enhancing acetate production and utilization for butyrate production.

Combination of Clostridium butyricum and Corn Bran Optimized Intestinal Microbial Fermentation Using a Weaned Pig Model.

Experimental manipulation of the intestinal microbiota influences health of the host and is a common application for synbiotics. Here Clostridium butyricum (C. butyricum, C.B) combined with corn bran (C.B + Bran) was taken as the synbiotics application in a waned pig model to investigate its regulation of intestinal health over 28 days postweaning. Growth performance, fecal short chain fatty acids (SCFAs) and bacterial community were evaluated at day 14 and day 28 of the trial. Although the C.B + Bran treatment has no significant effects on growth performance (P > 0.05), it optimized the composition of intestinal bacteria, mainly represented by increased acetate-producing bacteria and decreased pathogens. Microbial fermentation in the intestine showed a shift from low acetate and isovalerate production on day 14 to enhanced acetate production on day 28 in the C.B + Bran treatment. Thus, C.B and corn bran promoted intestinal microbial fermentation and optimized the microbial community for pigs at an early age. These findings provide perspectives on the advantages of synbiotics as a new approach for effective utilization of corn barn.