Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR

BackgroundCOVID-19 pandemic is underway. Some COVID-19 cases re-tested positive for SARS-CoV-2 RNA after discharge raising the public concern on their infectivity. Characterization of re-positive cases are urgently needed for designing intervention strategies. MethodsClinical data were obtained through Guangdong COVID-19 surveillance network. Neutralization antibody titre was determined using a microneutralization assay. Potential infectivity of clinical samples was evaluated after the cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. ResultsAmong 619 discharged COVID-19 cases, 87 were re-tested as SARS-CoV-2 positive in circumstance of social isolation. All re-positive cases had mild or moderate symptoms in initial diagnosis and a younger age distribution (mean, 30.4). Re-positive cases (n=59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n=150) parallel-tested in this study. No infective viral strain could be obtained by culture and none full-length viral genomes could be sequenced for all re-positive cases. ConclusionsRe-positive SARS-CoV-2 was not caused by the secondary infection and was identified in around 14% of discharged cases. A robust Nabs response and a potential virus genome degradation were detected from nearly all re-positive cases suggesting a lower transmission risk, especially through a respiratory route.

Genomic epidemiology of SARS-CoV-2 in Guangdong Province, China

COVID-19 is caused by the SARS-CoV-2 coronavirus and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, Chinas most populous province, during early 2020 resulted in 1,388 reported RNA positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain due to low virus genetic variation early in the pandemic. Our results illustrate how the timing, size and duration of putative local transmission chains were constrained by national travel restrictions and by the provinces large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required as the number of cases imported from other countries is increasing. HighlightsO_LI1.6 million molecular diagnostic tests identified 1,388 SARS-CoV-2 infections in Guangdong Province, China, by 19th March 2020 C_LIO_LIVirus genomes can be recovered using a variety of sequencing approaches from a range of patient samples. C_LIO_LIGenomic analyses reveal multiple virus importations into Guangdong Province, resulting in genetically distinct clusters that require careful interpretation. C_LIO_LILarge-scale epidemiological surveillance and intervention measures were effective in interrupting community transmission in Guangdong C_LI

Identification of a common deletion in the spike protein of SARS-CoV-2

Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

Generation of human monoclonal antibody Fab fragment to Zika Virus by Phage Display Technology / 中华实验和临床病毒学杂志

Objective@#In this study, phage display technology was used to construct the human anti-Zika virus(ZIKV), phage antibody library and to obtain and express the monoclonal antibody. The aim was to master the preparation and expression of human phage antibody library screening method for highly specific antibodies.@*Methods@#The whole blood samples of Zika patients were collected and the lymphocytes were isolated. The RT-PCR method was used to amplify the antibody light chain and heavy chain Fab gene from lymphocyte Ig mRNA. The pComb3H system was used to construct the gene with genetic diversity Preparation of human anti-ZIKV phage antibody library. The purified antibody library was screened by using the purified ZIKV and the obtained ZIKV E protein antigen.@*Results@#The monoclonal antibody Fab fragment gene was successfully obtained for the ZIKV E protein antigen. The gene can be efficiently expressed in Escherichia coli.@*Conclusions@#According to the sequence analysis, this study showed that the monoclonal antibody was a new human genetically engineered antibody against ZIKV, which laid the foundation for the early diagnosis of ZIKV, and obtain a specific monoclonal antibody to ZIKV for human treatment of ZIKV infection.

Serum antibodies against norovirus GⅠ.1 and GⅡ.4 in populations in central and eastern China / 中华微生物学和免疫学杂志

Objective@#To detect norovirus (NoV) GⅠ.1- and GⅡ.4-specific IgG, IgA and histo-blood group antigen (HBGA)-blocking antibodies in healthy populations of all age groups in China for better understanding the epidemiological features of norovirus in China from a serological point of view and providing basic data for vaccine development and clinical trial design.@*Methods@#Indirect ELISA and HBGA-blocking assay were used to detect NoV-specific IgG, IgA and HBGA-blocking antibodies in serum samples collected from healthy natural populations (n=839, aged from six months to 88 years old) in Guangzhou, Fuyang and Yantai. The results were statistically analyzed.@*Results@#The total positive rates of NoV GⅠ.1- and GⅡ.4-specific IgG antibodies were 91.9% and 93.0%. The positive rates of GⅠ.1- and GⅡ.4-specific IgA antibodies were 48.6% and 75.6%, and the titers of HBGA-blocking antibodies to GⅠ.1 and GⅡ.4 norovirus were 5.04 (95%CI: 4.63-5.49) and 18.15 (95%CI: 16.11-20.44). The positive rates of IgG and IgA antibodies generally showed an increasing trend with age. The positive rates of GⅠ.1- and GⅡ.4-specific IgG antibodies ranged from 79.2% to 100.0% and 76.7% to 100.0% in different age groups. They were 81.7% and 85.0% in the age group of 0.5-<1 year, 79.2% and 76.7% in the age group of 1-<2 years, and 98.1% and 96.3% in the age group of 12-<18 years, and maintained at 96% and 98% in the older age groups. The positive rates of GⅠ.1-specific IgA antibody ranged from 11.7% to 93.8% in different age groups and rapidly increased with age. It was 11.7% in the age group of 0.5-<1 year, and reached 93.3% in people aged 45-<60 years and 93.8% in people aged ≥60 years. The positive rates of GⅡ.4-specific IgA antibody ranged from 50.8% to 88.8% in different age groups with 50.8% in people aged 0.5-<1 year, and 86.7%-90.7% in people aged 12-<18 years and older. The titer of GⅠ.1 HBGA-blocking antibody generally increased with age. The antibody titer in populations aged 0.5-<12 years old was lower than that in those aged 18 years and above (GMT: 2.98-4.07 vs 8.21-11.62, P<0.001), and the titer in people of 12-<18 years old was lower than that in those of 45 years old and above (GMT: 5.21 vs 11.03-11.62, P<0.05). No obvious change with age was observed in the titer of GⅡ.4 HBGA-blocking antibody excepting the significant difference between populations of 2-<5 and 22-<45 years old (GMT: 26.73 vs 11.87, P<0.01).@*Conclusions@#This study revealed the characteristics of serum NoV GⅠ.1- and GⅡ.4-specific IgG, IgA and HBGA blocking antibodies in populations of different age groups in central and eastern China through analyzing their positive rates and titers and provided preliminary seroepidemiological data for the development of NoV vaccines in China.