Clinical and molecular epidemiology of enterovirus D68 from 2013 to 2020 in Shanghai.

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

Real-time reverse transcription-polymerase chain reaction assay panel for the detection of severe acute respiratory syndrome coronavirus 2 and its variants.

BACKGROUND: With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time. METHODS: We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7). RESULTS: Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively. CONCLUSIONS: Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.

Viral and Bacterial Etiology of Acute Febrile Respiratory Syndrome among Patients in Qinghai, China.

OBJECTIVE: This study was conducted to investigate the viral and bacterial etiology and epidemiology of patients with acute febrile respiratory syndrome (AFRS) in Qinghai using a commercial routine multiplex-ligation-nucleic acid amplification test (NAT)-based assay. METHODS: A total of 445 nasopharyngeal swabs specimens from patients with AFRS were analyzed using the RespiFinderSmart22kit (PathoFinder BV, Netherlands) and the LightCycler 480 real-time PCR system. RESULTS: Among the 225 (225/445, 51%) positive specimens, 329 positive pathogens were detected, including 298 (90.58%) viruses and 31 (9%) bacteria. The most commonly detected pathogens were influenza virus (IFV; 37.39%; 123/329), adenovirus (AdV; 17.02%; 56/329), human coronaviruses (HCoVs; 10.94%; 36/329), rhinovirus/enterovirus (RV/EV; 10.03%; 33/329), parainfluenza viruses (PIVs; 8.51%; 28/329), and Mycoplasma pneumoniae (M. pneu; 8.51%; 28/329), respectively. Among the co-infected cases (17.53%; 78/445), IFV/AdV and IFV/M. pneu were the most common co-infections. Most of the respiratory viruses were detected in summer and fall. CONCLUSION: In our study, IFV-A was the most common respiratory pathogen among 22 detected pathogens, followed by AdV, HCoV, RV/EV, PIV, and M. pneu. Bacteria appeared less frequently than viruses, and co-infection was the most common phenomenon among viral pathogens. Pathogens were distributed among different age groups and respiratory viruses were generally active in July, September, and November. Enhanced surveillance and early detection can be useful in the diagnosis, treatment, and prevention of AFRS, as well as for guiding the development of appropriate public health strategies.

Detection of six common human paramyxoviruses in patients with acute febrile respiratory symptoms using a novel multiplex real-time RT-PCR assay.

Human metapneumovirus (hMPV), respiratory syncytial virus type A (RSV-A), RSV-B, and human parainfluenza viruses 1, 2, and 3 (HPIV-1, HPIV-2, and HPIV-3) are common respiratory paramyxoviruses. Here, we developed a two-tube triplex one-step real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) and evaluated its performance using clinical samples. The data showed that this novel assay was 100% consistent with the monoplex real-time RT-PCR assay (in-house), which was superior to the commercial routine multiplex-ligation-NAT-based assay. Meanwhile, the clinical nasopharyngeal swabs of 471 patients with the acute febrile respiratory syndrome (AFRS) were analyzed using the established method. The results showed that 52 (11.7%) cases were positive for paramyxovirus. Among them, HPIVs and RSV-A had the highest detection rate. The age and seasonal distribution of human paramyxovirus infection were analyzed. In conclusion, we developed a novel multiplex real-time RT-PCR assay for the rapid detection of six common human paramyxoviruses, which were dominant in patients with AFRS in Qinghai.

[Molecular detection assays for 2012 identified novel human coronavirus (HCoV) and probe modification with locked nucleic acid (LNA)].

OBJECTIVE: To develop and optimize the molecular detection assays for recently identified human coronavirus (HCoV) infection. METHODS: Based on the 208 base pair(bp) sequence of novel HCoV reported by HPA of UK, we designed and obtained several pairs of primer (F-1, R-1; F-2, R-2) and Taqman probes (TZ1,TZ2) for detection of novel HCoV. Two of probes were modified with LNA (LNA-TZ1, LNA-TZ2). Then, RT-PCR and various real time RT-PCR assays were developed and optimized in this study. We also compared our assays with the real time RT-PCR assays reported recently by Europe team based on upE or ORF1b target. RESULTS: The RT-PCR or real time RT-PCR assays for novel HCoV were developed without cross-reactivity with other HCoV and several common respiratory viruses using clinical specimen panel. The analytical sensitivity of assays were less than 50-500 copies per reaction and the detection was improved when Taqman probe modified with LNA-tagged, compared to no LNA-tagged in real time RT-PCR assays. The upE and LNA-TZ1 based assays were better than others. CONCLUSION: The molecular detection sensitivity and specificity of TaqMan-based real time PCR assay could be improved when probe tagged with LNA. The upE or LNA-TZ1 based real time RT-PCR assay was recommend for detection of novel HCoV. This study laid a foundation for improving the performance of novel HCoV detection.

[Characterization and development of recombinant vaccinia viruses expressing different segments of spike protein derived from human coronavirus NL-63].

The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.

[The non-replicating recombinant vaccinia virus expressing six genes of HIV-1 can be passaged stably in CEF].

To investigate the genetic stability (including the vector of vaccinia virus and six foreign genes: gp160, gag, pol, rev, tat and nef) of the HIV-1 non-replicating recombinant vaccinia virus (rNTV-C). rNTV-C was serially passaged to passage 25 (P25) in primary chicken embryo fibroblast (CEF). P9, P12, P15 and P25 were selected to study the genetic stability in four aspects, including the genetic stability of viral vector, the genetic stability of six foreign genes, the expressing stability of foreign genes and the genetic loss of foreign genes. The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably; foreign gene sequences matched with designed sequences, the insert sites were right, and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C; the target proteins could be expressed effectively, and the expression level was stable within different passages of rNTV-C; the genetic loss of gag and nef was less than 5% within different passages of rNTV-C. The above results provided important data for the vaccine production.

[Peptide mapping of H-2d restricted T-cell epitope against six antigens of HIV-1 subtype B'/C by ELISPOT assay].

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.

Human Coronaviruses HCoV-NL63 and HCoV-HKU1 in Hospitalized Children with Acute Respiratory Infections in Beijing, China.

The human coronaviruses (HCoVs) HCoV-NL63 and HCoV-HKU1 are two recently discovered coronaviruses that circulate widely and are associated with acute respiratory infections (ARI). We detected HCoV-NL63 and HCoV-HKU1 in specimens collected from May 2008 to March 2010 from patients with ARI aged <7.75 years of age attending the Beijing Children's Hospital. Thirty-two (8.4%) and 57 (14.9%) of 382 specimens tested positive for HCoV-NL63 and HCoV-HKU1, respectively, by real-time RT-PCR. Use of a Luminex xTAG RVP Fast kit showed that coinfection with respiratory syncytial virus and parainfluenza 3 virus was common among patients infected with either virus type. In HCoV-HKU1-infected patients, the predominant clinical symptoms were cough, fever, and expectoration. In HCoV-NL63-infected patients they were cough, fever, and rhinorrhea. Phylogenetic studies showed that the HCoV-HKU1 nucleoprotein gene was relatively conserved compared to NCBI reference sequences, while the 1ab gene of HCoV-NL63 showed more variation.

[Development of serological detection assays for human coronavirus HKU1 infection and its application].

OBJECTIVE: To express the nuclear capsid protein (N protein) and the spike protein (S protein) of HCoV-HKU1, and to develop the corresponding serum assay for antibody detection. METHODS: The N protein of HCoV-HKU1 was expressed in E. Coli, anti-N antibody assay was established using Western Blotting with turn-based membrane. HCoV-HKU1 S protein was constructed in the eukaryotic expression plasmids, and confirmed by Western Blotting, S antibody assay was established using indirect immunofluorescence assay (IFA). We analyzed anti-S and anti-N antibody among 100 normal adult serum. RESULTS: Expression of S and N protein were confirmed; 100 normal adult serum were analyzed using the established serological detection assay, in which HCoV-HKU1 S antibody positive rate was 47%, N antibody positive rate was 48%, Both S and N antibodies positive were 21%, Both S and N antibodies negative were 22%. Co-detection S and N antibody was achieved 74% positive rate. CONCLUSION: The methods we established here could be used for serological analysis of HCoV-HKU1. Either detection of HCoV-HKU1 S or N antibodies achieved good results. Higher positive detection rate of anti-S or anti-N antibody was found in the normal adults.

[Characterization of human coronavirus 229E infection among patients with respiratory symptom in Beijing, Oct-Dec, 2007].

OBJECTIVE: To know the etiology, prevalence, clinical symptoms associated with the infection of the HCoV-229E in the respiratory specimens sampled from adult patients in Beijing. METHODS: 158 nasopharyngeal swab specimens were collected from adult patients with fever in Beijing between October and December, 2007. We performed the screening of HCoV-229E by real-time RT-PCR and sequencing of HCoV-229E gene fragments derived from conventional PCR. At meantime, we also screened the HCoV-229E positive samples for the co-infection with HCoV-NL63, HCoV-HKU1 and HMPV by real-time RT-PCR. Finally, demographic and clinical data associated with HCoV-229E infection were examined retrospectively. RESULTS: We detected 103 (62.5%) of 158 specimens were positive for HCoV-229E by real-time RT-PCR. When tested for other respiratory viruses, 26 HCoV-229E positive patients were found to be co-infected with other viruses. Of which HCoV-NL63 was observed in 3 specimens (11.5%), HCoV-HKU1 in 3 (11.5%) and HMPV in 20 (76.9%). The main clinical manifestations were noted as: headache (in 70.9%), sore throat (69%), chills (68%), cough (33%), sputum (21.3%), rhinorrhea (21.4%), nasal obstruction (16.5%), and a few of patients were visible as vomiting (6.8%), dyspnea (3.9%), diarrhea (in 1.9%). The rate of HCoV-229E infection in adult patients was found no relative with age and gender. CONCLUSION: Our data showed that HCoV-229E is a common and important pathogen in adult patients with acute respiratory symptoms but usually resulted in milder influenza-like illnesses. There might have a local outbreak of HCoV-229E infection in Beijing, Oct-Dec, 2007.

[Development and comparison of real-time and conventional RT-PCR assay for detection of human coronavirus NL63 and HKU1].

We designed specific primers and fluorescence-labeled probes to develop real-time and conventional RT-PCR assays for detection of human coronavirus NL63 or HKU1. Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The detection limit of the real-time RT-PCR assays was 10 RNA copies per reaction mixture. No cross-reactivity was observed between RNA samples derived from designed HCoV and other HCoV or human metapneumovirus. A total of 158 nasopharyngeal swab specimens collected from adult patients with acute respiratory tract infection in Beijing were screened for the presence of human coronavirus NL63 and HKU1 by using real-time RT-PCR and conventional RT-PCR method. The fluorescence quantitative RT-PCR method detected six specimens positive for human coronavirus NL63, five specimens positive for human coronavirus HKU1; and conventional RT-PCR method detected three HCoV-NL63 positive and three HCoV-HKU1 positive, respectively. The convention RT-PCR products of positive samples were obtained and sequence analysis confirmed the reliability of the above methods. In summary, the real-time RT-PCR assay for HCoV- NL63 or HKU1 was more sensitive than conventional RT-PCR and with less time (less than 4 hours) for completion. It may be suitable for molecular epidemiological surveillance and clinical diagnosis for human coronavirus NL63 and HKU1.