The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty ...of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale production of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6 micrograms per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support the clinical development and testing of PiCoVacc for use in humans.
This study assessed the concentration characteristics of culturable airborne microbes in 60 family homes with children aged 1–15 years in Hangzhou, southeast China. The concentration of culturable ...airborne microbes ranged from 314 colony-forming units (CFU)/m
3
to 2903 CFU/m
3
, with a mean value of 873 CFU/m
3
. The mean fungal concentration (653 CFU/m
3
) was significantly higher than the mean bacterial concentration of the atmosphere (220 CFU/m
3
), and the proportion of airborne fungi (73.9%) was significantly higher than that of airborne bacteria (26.1%). Microbial concentrations in family homes with a male child were significantly higher than those in homes with a female child, and there was a negative correlation between microbial concentration and living area per capita in family homes. The mean microbial concentration was highest in summer, followed by spring and autumn, and lowest in winter. This study provides an exposure database of airborne microbes in family homes in southeast China, suggesting that child gender and human occupancy in family homes significantly influence the microbial concentration in the air.
There have been five waves of influenza A (H7N9) epidemics in Zhejiang Province between 2013 and 2017. Although the epidemiological characteristics of the five waves have been reported, the molecular ...genetics aspects, including the phylogeny, evolution, and mutation of hemagglutinin (HA), have not been systematically investigated. A total of 154 H7N9 samples from Zhejiang Province were collected between 2013 and 2017 and sequenced using an Ion Torrent Personal Genome Machine. The starting dates of the waves were 16 March 2013, 1 July 2013, 1 July 2014, 1 July 2015, and 1 July 2016. Single-nucleotide polymorphisms (SNPs) and amino acid mutations were counted after the HA sequences were aligned. The evolution of H7N9 matched the temporal order of the five waves, among which wave 3 played an important role. The 55 SNPs and 14 amino acid mutations with high frequency identified among the five waves revealed the dynamic occurrence of mutation in the process of viral dissemination. Wave 3 contributed greatly to the subsequent epidemic of waves 4 and 5 of H7N9. Compared with wave 1, wave 5 was characterized by more mutations, including A143V and R148K, two mutations that have been reported to weaken the immune response. In addition, some amino acid mutations were observed in wave 5 that led to more lineages. It is necessary to strengthen the surveillance of subsequent H7N9 influenza outbreaks.
Numerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) ...still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly.
In this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1-26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis.
The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.
Lactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; ...it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated.
One hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH (45 min, p = 0.020), hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment.
L. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height.
Ebola virus infection causes severe hemorrhagic fever, and its mortality rates varied from 25 to 90% in the previous outbreaks. The highly infectious and lethal nature of this virus highlights the ...need for reliable and sensitive diagnostic methods to distinguish it from other diseases present with similar clinical symptoms. Based on multiplex polymerase chain reaction (PCR) and oligonucleotide microarray technology, a cost-effective, multipathogen and high-throughput method was developed for simultaneous detection of Ebola virus and other pathogens associated with hemorrhagic fever, including Marburg virus, Lassa fever virus, Junin virus, Machupo virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, malaria parasite, hantavirus, severe fever with thrombocytopenia syndrome virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus, and influenza B virus. This assay had an excellent specificity for target pathogens, without overlap signal between the probes. The limit of detection was approximately 10
3
pathogen copies/μl. A total of 60 positive nucleic acid samples for different pathogens were detected, a concordance of 100% was observed between microarray assay and real-time PCR analysis. Consequently, the described oligonucleotide microarray may be specific and sensitive assay for diagnosis and surveillance of infections caused by Ebola virus and other species of hemorrhagic fever pathogens.
Dengue fever (DF) is a mosquito-borne viral disease caused by the dengue virus (DENV), which is considered one of the most important arboviruses in the world. This study aimed to determine the ...molecular, epidemiological, and phylogenetic characterization of 174 DENV-1 (132 indigenous cases and 42 imported cases) isolated from nine municipalities of Zhejiang province in 2019. The analyses of phylogenetics, haplotypes, and amino acid substitutions were conducted based on the full envelope (E) gene sequences. Sixty-four haplotypes were clustered into two main clades, with isolates from Wenzhou and Taizhou mainly clustered into clade I and Hangzhou and Ningbo cases clustered into clade II. Six sites of amino acid substitutions including A88T, F96L, M297V, T339S, I378L, and V436I were only observed in strains isolated from Ningbo and Hangzhou, while two sites of amino acid substitutions including V312L and V380I were observed in strains from Taizhou and Wenzhou. In our study, strains were in high homology with the strains from Southeast Asian countries, thus cases in Zhejiang were probably imported from Southeast Asian countries. The strains from different regions in Zhejiang were clustered in the same branch which may be caused by the continuous import of cases in the same country at different time periods. After the continuous outbreak in Zhejiang province, some sites of the dengue gene have mutated, and the effects need further study.
The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel ...technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (
= 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.
Dengue, a mosquito-borne disease caused by the dengue virus (DV), has been recognized as a global public health threat. In 2017, an unexpected dengue outbreak occurred in Zhejiang, China. To clarify ...and characterize the causative agent of this outbreak, data on dengue fever cases were collected from the China Information System for Disease Control and Prevention in Zhejiang province for subsequent epidemiological analysis. A total of 1,229 cases were reported, including 1,149 indigenous and 80 imported cases. Most indigenous cases (1,128 cases) were in Hangzhou. The epidemic peak occurred in late August and early September, and the incidence rate of elderly people (4.34 per 100,000) was relatively high. Imported cases were reported all year round, and most were from South-East Asia and Western Pacific regions. Young people and men accounted for a large fraction of the cases. Acute phase serums of patients were collected for virus isolation. And 35 isolates (including 25 DV-2, 8 DV-1, 1 DV-3, and 1 DV-4) were obtained after inoculation and culture in mosquito C6/36 cells. The E genes of the 35 new DV isolates and the complete genome of a DV-2 isolate (Zhejiang/HZ33/2017), and the E gene of a DV-2 isolate from
(Zhejiang/Aedes-1/2017) were determined. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, DVs of all four serotypes with multiple genotypes (mainly including 21 Cosmopolitan genotype DV-2, 4 Asian I genotype DV-2, 6 genotype I DV-1, and 2 genotype V DV-1) were present in the indigenous and imported cases in Zhejiang during the same period. Most of the isolates probably originated from South-East Asia and Western Pacific countries. The imported cases, high density of mosquito vector, and missed diagnosis might contribute to the 2017 outbreak in Zhejiang.
Viral respiratory infection (VRI) is a common contraindication to elective surgery. Asymptomatic shedding among pediatric surgery patients (PSPs) could potentially lead to progression of symptomatic ...diseases and cause outbreaks of respiratory diseases. The aim of this study is to investigate the incidence of infection among mild symptomatic PSP group and asymptomatic PSP group after surgical procedure.
We collected the induced sputum from enrolled 1629 children (under 18 years of age) with no respiratory symptom prior to pediatric surgery between March 2017 and February 2019. We tested 16 different respiratory virus infections in post-surgery mild symptomatic PSP group and asymptomatic PSP group using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay panel. We analyzed symptom data and quantitative viral load to investigate the association between viruses, symptoms and viral quantity in qRT-PCR-positive PSPs.
Out of 1629 children enrolled, a total of 204 respiratory viruses were present in 171 (10.50%) PSPs including 47 patients with mild symptoms and 124 with no symptoms after surgery. Commonly detected viruses were human rhino/enterovirus (HRV/EV, 42.19%), parainfluenza virus 3 (PIV3, 24.48%), coronavirus (CoV NL63, OC43, HKU1, 11.46%), and respiratory syncytial virus (RSV, 9.9%). PIV3 infection with a higher viral load was frequently found in PSPs presenting with mild symptoms, progressing to pneumonia with radiographic evidence after surgery. HRV/EV were the most commonly detected pathogens in both asymptomatic and mild symptomatic PSPs. CoV (OC43, HKU1) infections with a higher viral load were mostly observed in asymptomatic PSPs progressing to alveolar or interstitial infiltration.
Our study suggested that PIV3 is a new risk factor for VRI in PSPs. Employing a more comprehensive, sensitive and quantitative method should be considered for preoperative testing of respiratory viruses in order to guide optimal surgical timing.
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