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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (SARS-CoV) pose a great threat to humanity. Every pandemic involving these coronaviruses has seriously affected human health and economic development. Currently, there are no approved therapeutic drugs against their infections. Therefore, the development of vaccines is particularly important to combat these coronaviruses. In this review, we summarized and analyzed the progress of vaccines against SARS-CoV, MERS-CoV, and SARS-CoV-2, including inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, and viral vector vaccines. In addition, we compared the levels of neutralizing antibodies in the serum of patients with these three kinds of coronaviruses at different stages, and their ability and effects against SARS-CoV-2, MERS-CoV, and SARS-CoV. This review provides useful information for vaccine evaluation and analysis.

The chikungunya virus (CHIKV) is widespread. In Zhejiang province, China, CHIKV infection is often associated with travelers from tropical and subtropical countries. In the present study, three CHIKV isolates from serum samples of travelers in Zhejiang province in 2019 were sequenced, and phylogenetically analyzed to study their molecular characteristics. Sequence analysis showed that the non-structural protein and the structural protein had 37 and 28 amino acid mutations, respectively; no mutation site was found at the E1-A226 residue, which could increase the adaptability of CHIKV to Aedes albopictus. All three samples carried two mutations, namely, E1-K211E and E2-V264A, which were introduced to Bangladesh around late 2015 and Thailand in early 2017. Phylogenetic analysis revealed that these three CHIKVs were Indian Ocean lineage of the East Africa/Central/South Africa genotype (ECSA) and that the MF773566 strain from Bangladesh (Australia/Bangladesh 2017) had the closest evolutionary relationship. The three CHICKs imported into Zhejiang province in 2019 belonged to the ECSA genotype and had multiple amino acid variation sites. The variation in the three samples provides a certain reference for the subsequent research on CHIKV evolution.

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 (p = 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.

Background: Dengue virus (DENV) is the most widespread mosquito-borne virus, which can cause dengue fever with mild symptoms, or progress to fatal dengue hemorrhagic fever and dengue shock syndrome. As the main target cells of DENV, macrophages are responsible for the innate immune response against the virus. Methods: In this study, we investigated the role of pyroptosis in the pathogenic mechanism of dengue fever by examining the level of pyroptosis in DENV-1-infected macrophages and further screened differentially expressed microRNAs by high-throughput sequencing to predict microRNAs that could affect the pyroptosis of the macrophage. Results: Macrophages infected with DENV-1 were induced with decreased cell viability, decreased release of lactate dehydrogenase and IL-1β, activation of NLRP3 inflammasome and caspase-1, cleavage of GSDMD to produce an N-terminal fragment bound to cell membrane, and finally induced macrophage pyroptosis. MicroRNA expression profiles were obtained by sequencing macrophages from all periods of DENV-1 infection and comparing with the negative control. Sixty-three microRNAs differentially expressed in both the early and later stages of infection were also identified. In particular, miR-223-3p, miR-148a-3p, miR-125a-5p, miR-146a-5p and miR-34a-5p were recognized as small molecules that may be involved in the regulation of inflammation. Conclusions: In summary, this study aimed to understand the pathogenic mechanism of DENV through relevant molecular mechanisms and provide new targets for dengue-specific therapy.