Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

A New Method for Ultra-sensitive P24 Antigen Assay Based on Near-infrared Fluorescent Microsphere Immunochromatography.

OBJECTIVE: To develop a rapid, highly sensitive quantitative method for detecting P24 antigen based on near-infrared fluorescent microsphere immunochromatography. METHODS: First, we prepared a lateral flow assay test strip, and labeled the detection antibody using a fluorescent microsphere. Second, we optimized the antibody labeling conditions. Third, we optimized the detection conditions. Fourth, we created a working curve. Fifth, we conducted a methodological assessment of the established fluorescent microsphere immunochromatography method. Sixty-six clinical samples were tested, and we compared the established fluorescent microsphere immunochromatography with the quantitative ELISA method. RESULTS: According to the working curve, the detection limit of the method is 3.4 pg/mL, and the detection range is 3.4 pg/mL to 10 ng/mL. The average intra-assay recovery was 99.6%, and the Coefficient of Variation (CV) was 5.4%-8.6%; the average inter-assay recovery was 97.3%, and the CV was 8.5%-11%. The detection rate of fluorescent microsphere immunochromatography was higher than ELISA method, and had a good correlation with ELISA. CONCLUSION: The P24 antigen quantitative detection method based on near-infrared fluorescent microsphere immunochromatography has the advantages of rapid detection, high sensitivity, and wide detection range; thus, it is suitable for early clinical diagnosis and continuous monitoring of AIDS.

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC.

OBJECTIVE: In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. METHODS: A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. RESULTS: Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. CONCLUSION: An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus.

OBJECTIVE: To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed. METHODS: By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay. RESULTS: With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%. CONCLUSION: A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

The Relationship between Japanese Encephalitis and Environmental Factors in China Explored Using National Surveillance Data.

Japanese encephalitis (JE) is a serious public health issue. This study was undertaken to better understand the relationship between JE distribution and environmental factors in China. JE data from 2005 to 2010 were retrieved from National Notifiable Disease Report System. ArcGIS, remote sensing techniques, and R software was used to exhibit and explore the relationship between JE distribution and environmental factors. Our results indicated that JE cases were mostly concentrated in warm-temperate, semitropical and tropical zones with annual precipitation > 400 mm; Broad-leaved evergreen forest, shrubs, paddy field, irrigated land, dryland, evergreen coniferous forest, and shrubland were risk factors for JE occurrence, and the former five were risk factors for counties with high JE incidence. These findings will inform the effective allocation of limited health resources such as intensive vaccination, surveillance and training in areas with high environmental risk factors.

A New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope.

OBJECTIVE: To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope. METHODS: The lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit. RESULTS: Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit. CONCLUSION: The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.

Molecular evolution of emerging Banna virus.

Banna virus (BAV) is an emerging pathogen that causes human viral encephalitis and has been isolated from types of blood-sucking insects and mammals in Asia. However, there are no reported systematic studies that describe the origin and evolution of BAV. Here, a phylogenetic analysis of BAVs isolated from a variety of potential vectors and vertebrate hosts worldwide revealed that BAVs emerged in the beginning of the 20th century and do not exhibit a species barrier. The mean substitution rate of BAVs was 2.467×10-2substitution/site/year (95% HPD, 1.093×10-3 to 5.628×10-2). The lineage is mainly composed of BAVs from high-latitude regions, which are the most recently emerged viruses with significantly higher substitution rates compared with the lineage comprised of the isolates from middle or low-latitude regions. The genetic differences between BAV strains are positively correlated with the geographic distribution. Strains from the same latitude regions are almost 100% identical, whereas the differences between strains from long distance regions with different latitudes could be >60%. Our results demonstrate that BAV is an emerging virus at a stage that involves rapid evolution and has great potential for introduction into non-endemic areas. Thus, enhanced surveillance of BAV is highly recommended worldwide.

Clinical and Virological Characteristics of Ebola Virus Disease Patients Treated With Favipiravir (T-705)-Sierra Leone, 2014.

BACKGROUND: During 2014-2015, an outbreak of Ebola virus disease (EVD) swept across parts of West Africa. No approved antiviral drugs are available for Ebola treatment currently. METHODS: A retrospective clinical case series was performed for EVD patients in Sierra Leone-China Friendship Hospital. Patients with confirmed EVD were sequentially enrolled and treated with either World Health Organization (WHO)-recommended supportive therapy (control group) from 10 to 30 October, or treated with WHO-recommended therapy plus favipiravir (T-705) from 1 to 10 November 2014. Survival and virological characteristics were observed for 85 patients in the control group and 39 in the T-705 treatment group. RESULTS: The overall survival rate in the T-705 treatment group was higher than that of the control group (56.4% [22/39] vs 35.3% [30/85]; P = .027). Among the 35 patients who finished all designed endpoint observations, the survival rate in the T-705 treatment group (64.8% [11/17]) was higher than that of the control group (27.8% [5/18]). Furthermore, the average survival time of the treatment group (46.9 ± 5.6 days) was longer than that of the control group (28.9 ± 4.7 days). Most symptoms of patients in the treatment group improved significantly. Additionally, 52.9% of patients who received T-705 had a >100-fold viral load reduction, compared with only 16.7% of patients in the control group. CONCLUSIONS: Treatment of EVD with T-705 was associated with prolonged survival and markedly reduced viral load, which makes a compelling case for further randomized controlled trials of T-705 for treating EVD.

A Centralized Report on Pediatric Japanese Encephalitis Cases from Beijing Children's Hospital, 2013.

Fifteen pediatric cases of suspected Japanese encephalitis (JE) were reported in Beijing Children's Hospital during the late summer of 2013. The clinical manifestations in most cases included high fever, seizures, and abnormal magnetic resonance imaging findings. Twelve of 15 cases were laboratory-confirmed as JE cases by pathogen identification. Epidemiological investigations showed that five of the 12 laboratory-confirmed patients had an incomplete JE vaccination history. Follow-up investigations after discharge indicated that seven laboratory-confirmed JE patients without JE vaccinations had relatively poor prognoses, with an average Modified Rankin Scale (MRS) score of 2.6 when compared with the other five laboratory-confirmed, JE-vaccinated patients with an average MRS score of 0.5. The observation of pediatric JE cases among those with a history of JE vaccination warrants further attention.

Ebola Virus Outbreak Investigation, Sierra Leone, September 28-November 11, 2014.

During 2014-2015, an outbreak of Ebola virus disease (EVD) swept across parts of West Africa. The China Mobile Laboratory Testing Team was dispatched to support response efforts; during September 28-November 11, 2014, they conducted PCR testing on samples from 1,635 suspected EVD patients. Of those patients, 50.4% were positive, of whom 84.6% lived within a 3-km zone along main roads connecting rural towns and densely populated cities. The median time from symptom onset to testing was 5 days. At testing, 75.7% of the confirmed patients had fever, and 94.1% reported at least 1 gastrointestinal symptom; all symptoms, except rash and hemorrhage, were more frequent in confirmed than nonconfirmed patients. Virus loads were significantly higher in EVD patients with fever, diarrhea, fatigue, or headache. The case-fatality rate was lower among patients 15-44 years of age and with virus loads of <100,000 RNA copies/mL. These findings are key for optimizing EVD control and treatment measures.

Real-time RT-PCR Assay for the detection of Tahyna Virus.

A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

Molecular characterization of full-length genome of Japanese encephalitis virus genotype V isolated from Tibet, China.

OBJECTIVE: To determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V. METHODS: The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. RESULTS: The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. CONCLUSION: The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

Isolation and full-length sequence analysis of Armigeres subalbatus totivirus, the first totivirus isolate from mosquitoes representing a proposed novel genus (Artivirus) of the family Totiviridae.

During an investigation of arboviruses in China, a novel dsRNA virus was isolated from adult female Armigeres subalbatus. Full genome sequence analysis showed the virus to be related to members of the family Totiviridae, and was therefore named 'Armigeres subalbatus totivirus' (AsTV). Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 40 nm. The AsTV genome is 7510 bp in length, with two ORFs. ORF1 (4443 nt) encodes the coat-protein and a dsRNA-binding domain (which may be involved in the evasion of 'gene silencing'), while ORF2 (2286 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). The AsTV coat protein shows a higher level of amino acid identity with Drosophila totivirus (DTV, 52 %) than with infectious myonecrosis virus (IMNV, 29 %). Similarly, the RdRp shows higher identity levels with DTV (51 %) than with IMNV (44 %). Identity levels to other members of the family Totiviridae, in either the coat protein or the RdRp, ranged from 6 to 11 %. Based on a recent reassessment of the coding strategy used by IMNV, we suggest that an AsTV coat-RdRp fusion protein could be synthesized via a -1 frameshift. Elements favouring -1 frameshift such as 'slippery heptamers' and pseudonkots, were identified in the AsTV, DTV and IMNV genomes. AsTV was shown to grow in both mosquito and mammalian cells, suggesting that it is an arbovirus that can infect mammals.

Banna virus, China, 1987-2007.

Banna viruses (BAVs) have been isolated from pigs, cattle, ticks, mosquitoes, and human encephalitis patients. We isolated and analyzed 20 BAVs newly isolated in China; this finding extends the distribution of BAVs from tropical zone to north temperate climates and demonstrate regional variations in BAV phylogeny and mosquito species possibly involved in BAV transmission.

[Isolation and identification of arboviruses from mosquito pools in some regions of Liaoning province, China].

OBJECTIVE: To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province. METHODS: Mosquitoes were collected from Shenyang, Yingkou, Panjin, Jinzhou and Dandong cities of Liaoning province in 2006. Viruses were isolated by inoculating the specimens onto C6/ 36 and BHK-21cells. The new isolates were identified using serological and molecular biological methods. RESULTS: 5410 mosquitoes were collected from the five cities in total. Three isolates produced CPE in C6/ 36 cell and five isolates produced CPE in both C6/36 and BHK-21 cell. Three isolates (LN0684, LN0688 and LN0689) were identified as Banna virus and one isolate (LN0636) was identified as Getah virus. Phylogenetic analysis showed that the three Banna virus strains were clustered into the same evolution branch as the other Chinese isolates. The identity of nucleotide sequence was between 91.2% and 94.7%, compared with other Banna virus strains. The new isolated Getah virus was clustered into the same branch with the strain of South Korea (swine). The identity of nucleotide sequence was 99.2%, when comparing with the strain of South Korea and was 95% to 99% with the strains from Russia, mainland of China and Taiwan region. Conclusion Eight virus isolates, including three Banna virus, one Getah virus and four unknown virus strains were isolated from mosquitoes in Liaoning province. Banna virus and Getah virus were reported for the first time in Liaoning province, while Getah virus showed the highest nucleotide homology with the South Korea strains.