A detailed analysis of 16S rRNA gene sequencing and conventional PCR-based testing for the diagnosis of bacterial pathogens and discovery of novel bacteria.

This study represents the first analysis of the bacterial community in chickens affected by swollen head syndrome, utilizing 16S rRNA gene sequencing. Samples were obtained from clinical laying chickens and were examined for the presence of Avibacterium paragallinarum (APG) and Ornithobacterium rhinotracheale (ORT) using conventional polymerase chain reaction (PCR). From the samples, five APG-positive (APG) and APG-negative (N-APG) samples were chosen, along with five specific pathogen-free chickens, for 16S rRNA gene sequencing. Results showed that APG and ORT were widely detected in the chicken samples with swollen head syndrome (SHS, 9/10), while APG was detected in all five specific pathogen-free (SPF) samples. In contrast, conventional PCR sensitivity was found to be inadequate for diagnosis, with only 35.7% (5/14) and 11.1% (1/9) sensitivity for APG and ORT, respectively, based on 16S rRNA gene sequencing data. Furthermore, 16S rRNA gene sequencing was able to quantify the bacteria in the samples, revealing that the relative abundance of APG in the APG group ranged from 2.7 to 81.3%, while the relative abundance of APG in the N-APG group ranged from 0.1 to 21.0%. Notably, a low level of APG was also detected in all 5 SPF samples. The study also identified a significant number of animal and human common bacterial pathogens, including but not limited to Gallibacterium anatis, Riemerella columbina, Enterococcus cecorum, Mycoplasma synoviae, Helicobacter hepaticus, and Staphylococcus lentus. In conclusion, 16S rRNA gene sequencing is a valuable tool for bacterial pathogen diagnosis and the discovery of novel bacterial pathogens, while conventional PCR is not reliable for diagnosis.

Infectivity of pseudotyped SARS-CoV-2 variants of concern in different human cell types and inhibitory effects of recombinant spike protein and entry-related cellular factors.

Since the report of the first COVID-19 case in 2019, SARS-CoV-2 variants of concern (VOCs) have continued to emerge, manifesting diverse infectivity, evasion of host immunity and pathology. While ACE2 is the predominant receptor of SARS-CoV-2, TMPRSS2, Kim-1, NRP-1, CD147, furin, CD209L, and CD26 have also been implicated as viral entry-related cofactors. To understand the variations in infectivity and pathogenesis of VOCs, we conducted infection analysis in human cells from different organ systems using pseudoviruses of VOCs including Alpha, Beta, Gamma, and Delta. Recombinant spike S1, RBD, ACE2, Kim-1, and NRP-1 proteins were tested for their ability to block infection to dissect their roles in SARS-CoV-2 entry into cells. Compared with wild type SARS-CoV-2 (WT), numerous VOCs had significant increases of infectivity across a wide spectrum of cell types. Recombinant ACE2 protein more effectively inhibited the infection of VOCs including Delta and Omicron (BA.1 and BA.2) than that of WT. Interestingly, recombinant S1, RBD, Kim-1, and NRP-1 proteins inhibited the infection of all pseudoviruses in a manner dependent on the levels of ACE2 expression in different cell types. These results provide insights into the diverse infectivity of SARS-CoV-2 VOCs, which might be helpful for managing the emergence of new VOCs.

Ultrarapid and ultrasensitive detection of SARS-CoV-2 antibodies in COVID-19 patients via a 3D-printed nanomaterial-based biosensing platform.

Rapid detection of antibodies during infection and after vaccination is critical for the control of infectious outbreaks, understanding immune response, and evaluating vaccine efficacy. In this manuscript, we evaluate a simple ultrarapid test for SARS-CoV-2 antibodies in COVID-19 patients, which gives quantitative results (i.e., antibody concentration) in 10-12 s using a previously reported nanomaterial-based three-dimensional (3D)-printed biosensing platform. This platform consists of a micropillar array electrode fabricated via 3D printing of aerosolized gold nanoparticles and coated with nanoflakes of graphene and specific SARS-CoV-2 antigens, including spike S1, S1 receptor-binding domain (RBD) and nucleocapsid (N). The sensor works on the principle of electrochemical transduction, where the change of sensor impedance is realized by the interactions between the viral proteins attached to the sensor electrode surface and the antibodies. The three sensors were used to test samples from 17 COVID-19 patients and 3 patients without COVID-19. Unlike other serological tests, the 3D sensors quantitatively detected antibodies at a concentration as low as picomole within 10-12 s in human plasma samples. We found that the studied COVID-19 patients had higher concentrations of antibodies to spike proteins (RBD and S1) than to the N protein. These results demonstrate the enormous potential of the rapid antibody test platform for understanding patients' immunity, disease epidemiology and vaccine efficacy, and facilitating the control and prevention of infectious epidemics.

Neutralizing antibodies to SARS-CoV-2 variants of concern including Delta and Omicron in subjects receiving mRNA-1273, BNT162b2, and Ad26.COV2.S vaccines.

SARS-CoV-2 vaccines have contributed to the control of COVID-19 in some parts of the world. However, the constant emergence of variants of concern (VOCs) challenges the effectiveness of SARS-CoV-2 vaccines over time. In particular, Omicron contains a high number of mutations in the spike (S) protein gene, on which most vaccines were developed. In this study, we quantitated neutralizing antibodies in vaccine recipients at various times postvaccination using S protein-based pseudoviruses derived from wild type (WT) SARS-CoV-2 and five VOCs including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). We found that two-dose mRNA-1273 and BNT162b2 vaccines elicited robust neutralizing antibodies against WT, Alpha, Beta, Gamma, and Delta, but wanned after 6 months with a faster decline observed for BNT162b2. Both mRNA-1273 and BNT162b2 elicited weak neutralizing antibodies against Omicron. One dose of Ad26.COV2.S vaccine induced weaker neutralizing antibodies against WT and most VOCs than mRNA-1273 and BNT162b2 did but moderate neutralizing antibodies against Delta and Omicron, which lasted for 6 months. These results support current recommendations of the Centers for Disease Control and Prevention for a booster 5 months after full immunization with an mRNA-based vaccine and the use of an mRNA-based vaccine 2 months after Ad26.COV2.S vaccination.