Journal of virological methods最新文献

Introduction: Global outbreaks of mosquito-transmitted arbovirus infections, such as dengue (DENV) and chikungunya (CHIKV), are increasing. Differentiating these infections is challenging due to non-specific symptoms and serology limitations. PCR-based approaches offer higher sensitivity and specificity. This study evaluated the performance of TaqMan™ Arbovirus Triplex Kit (ZIKV/DENV/CHIKV) (TaqMan™ Kit) to detect DENV and CHIKV in clinical samples from patients in south India.

Methods: In total, 280 serum samples with 90 DENV-positive, 90 CHIKV-positive, and 100 negative samples were tested with TaqMan™ Kit and CDC Trioplex Real-Time RT-PCR assay. No Zika virus was detected. The sensitivity and specificity of viral RNA detection were determined, and discordant results were resolved using comparator PCRs, dengue NS1 antigen detection, virus-specific antibody results, or previously de-identified in-house PCR results.

Results: TaqMan™ Kit showed 100 % agreement with the comparator for DENV detection in 92 positive samples. Among 188 samples negative for DENV by the comparator, 30 showed positive results with the TaqMan™ kit, and 23 of those were confirmed as true positives. The resulting sensitivity and specificity for DENV detection were 100 % and 95.1 %, respectively. For CHIKV, 77 positive and 195 negative results were concordant. Eight samples showed discordant results, but upon resolution testing, sensitivity and specificity for CHIKV were 93.9 % and 100.0 %, respectively.

Conclusion: TaqMan™ Arbovirus Triplex Kit demonstrated high sensitivity and specificity (>93 %) for detecting circulating DENV and CHIKV strains. Multiplex PCR testing can improve case detection, surveillance, and public health responses while optimizing laboratory resources for outbreak control.

Monkeypox (mpox) is a rare viral disease that can cause severe illness in humans, with outbreaks occurring primarily in central and western Africa. Well-coordinated and synchronized efforts are necessary to understand the factors involved in disease transmission and develop effective health interventions. The aim of this study is to assess the relationship between climate factors and daily mpox cases, as well as to identify the most suitable predictive model for transmission. We analyzed confirmed mpox cases from May 5, 2022, to February 14, 2023, in the 33 most affected countries. We employed and compared the efficiency of four models: Poisson, negative binomial, zero-inflated Poisson, and zero-inflated negative binomial. We found a significant correlation between climate factors and daily mpox cases across most of the studied countries. Specifically, for each 1°C increase in the heat index (HI), daily cases increased by 7.7 % (IRR = 1.077, p < 0.05). Conversely, higher relative humidity (RH) decreased daily cases by 2.4 %, and increased wind speed (WS) reduced them by 7.3 %. The HI positively influences mpox spread, while RH and WS act as protective factors. Public health officials should consider these climate influences when developing targeted interventions.

Humanity faces an ongoing battle at the microscopic level to identify, contain, and treat outbreaks of numerous pathogens each year. Global genomic surveillance is the first step in monitoring outbreaks, but high-throughput methods are expensive and time-consuming. To solve this problem, we designed and manufactured a resequencing microarray capable of identifying 35 viral pathogens, 21 pathogenic bacteria, 16 antibiotic resistance genes, and 6 controls. We then developed an assay using these microarrays for the rapid detection of SARS-CoV-2, influenza A, and influenza B. Here, we present the clinical validation data for this test. The assay requires less than two hours to complete and has high-sensitivity, with limits of detection of 125 copies per mL for SARS-CoV-2, 0.01-0.05 TCID₅₀/mL for influenza A, and 0.005-0.01 TCID₅₀/mL for influenza B. The microarrays used for these assays are easily mass-produced using wafer-scale synthesis, making this an affordable option for pathogen screening with broad implications in public health.

Lumpy skin disease (LSD), caused by the LSD virus (LSDV) from the Capripoxvirus genus, affects cattle, water buffalo, and wild bovines, leading to significant economic losses. Characterised by fever, skin nodules, and mucosal lesions, LSD raises global concerns due to vector-borne transmission. The World Organisation for Animal Health (WOAH) classifies LSD as a notifiable disease, emphasising the need for rapid diagnostic methods for timely disease confirmation and control. This study evaluates the performance of two previously developed ELISA tests - competitive and indirect. The validation involved 450 field sera from infected and vaccinated herds in Albania (collected in 2016, during the LSD outbreak), 332 sera from vaccinated cattle in Serbia (collected in 2017 from farms with no prior history of LSD detection), 90 sera from experimental infections at Friedrich-Loeffler-Institut, and 412 field negative sera from a Capripox-free country. The comparison with the virus neutralisation test - the gold standard - demonstrated high specificity (≥0.95) and significant sensitivity (0.87-0.94), with 8-9 % of sera showing discordant results. The results diverged more in sera from animals with a single vaccination or sampled five months post-vaccination, indicating reduced antibody detectability over time. The study confirms the ELISAs' efficacy for large-scale LSDV serological surveillance, highlighting their potential to provide a cost-effective and rapid solution for monitoring and controlling LSD in endemic regions.

In this study, a novel Encephalomyocarditis virus (EMCV) reverse genetic operating system was developed utilizing CMV promoters, enabling EMCV genome expression under the transcriptional control of the CMV immediate early promoter and BGH polyA transcriptional-termination signal. The full-length cDNA of EMCV BJC3 was ligated to the pRK5 vector, incorporating the CMV eukaryotic promoter sequence, resulting in the construction of recombinant plasmid EMCV (pEMCV). Subsequently, the recombinant plasmid was transfected into BHK-21 cells to generate the rescue virus. Further investigation of the neuropathogenicity and histopathology of the rescue virus was conducted using a mouse infection model. The findings demonstrated that the proliferation dynamics of the virus verified that the rescue virus and the wild-type virus exhibited comparable proliferative traits in BHK-21 cells, and whole genome sequencing confirmed the genetic stability of the rescue virus. Although the neuropathogenicity of the rescue virus in mice was less than that of the wild-type parent virus, it retained robust proliferative capacity in vivo. The study indicates that CMV promoter-based EMCV infectious clones can efficiently and stably rescue the virus, preserving similar in vivo and in vitro biological characteristics as the wild-type parent viruses. The development of the infectious cDNA clone offers a genetic platform for studying gene function and facilitating the rational design of vaccines.

With the rapid development of the cell and gene therapy industry, there is an increasing demand for lentiviral vectors that can efficiently infect cells of different purposes. BaEV lentiviruses have been shown to efficiently infect hematopoietic stem cells, primary B cells, and NK cells, which traditional VSV-G lentiviruses cannot infect. However, there is a problem of low virus yield in the production of BaEV lentivirus. The formation of syncytium and apoptosis occur after plasmid transfection, and resulting in a reduction in virus production. In this study, the issue of low production of BaEV lentivirus was comprehensively improved. By increasing the cell density of inoculation, reducing the amount of BaEV plasmid, and adjusting the harvest time, the maximum titer of BaEV lentivirus reached 4.43E+ 06 IU/ml, representing an increase of 369 times compared to the unoptimized condition. Further, the purification method of lentivirus solution was optimized, and the infection titer of lentivirus reached 1.00E+ 08 IU/ml, which is 10300 times higher than pre-optimization levels.

Rapidly identifying and sequencing viral pathogens in poultry flocks can substantially reduce economic loss especially during disease outbreaks. Current next generation sequencing technologies require multi-step laboratory-intensive workflows to generate sequence data which precludes field adaptation. In this study, we hypothesized that direct RNA sequencing (DRS) using an Oxford Nanopore Technology (ONT) MinION device would enable sequencing of the full-length viral RNA genome of Orthoavulavirus javaense (OAVJ), the causative of Newcastle disease, a major poultry challenge. The data demonstrate that a custom OAVJ-specific adapter paired with the ONT DRS kits enables capture and sequencing of OAVJ viral RNAs. Further, the new ONT SQK-RNA004 chemistry and flow cells, paired with the associated super accurate base calling workflow improves on read quality and length compared to the previous SQK-RNA002 chemistry. This is the first report of a method to sequence near full-length viral RNA genome of a member of the Paramyxoviridae family. While additional improvements in DRS are needed before widespread adaptation of this method for rapid field sequencing, DRS of OAVJ has the potential to enable further studies into the viral epitranscriptome and its role in infection and pathogenesis.

Human norovirus (HuNoV) and human astrovirus (HAstV) are viral enteric pathogens and known causative agents of acute gastroenteritis. Identifying the presence of these viruses in environmental samples such as irrigation water, or foods exposed to virus contaminated water (e.g., shellfish, agricultural crops), remains an important goal in the field of food safety. Determining if a virus species present in a sample is infectious is complicated by the recalcitrance of many enteric virus species to grow in culture, and/or the lack of a common cell culture system(s). Human intestinal enteroids (HIEs) can support the replication of HuNoV and HAstV, and thus hold promise as a platform for demonstrating the replication of multiple enteric virus species within a single sample. The objective of this study was to determine if HIEs can support co-replication of two genetically distinct human enteric viruses, HAstV3 and HuNoV GII.4[P16]. In single virus infections, HuNoV GII.4[P16] RNA levels were highest at 48-72 hpi (6.3-9.1 x 10⁶ genome copy equivalents [gce]/well) and HAstV3 RNA levels were highest at 24 hpi (3.4 ×10⁸ gce/well). HAstV3-infected cells stained positive for viral capsid protein at 24 hpi and induced the synthesis of RNA and protein expression of interferon (IFN)-beta, -lambda 1 and 2/3, peaking at 24 hpi and 48 hpi respectively. HuNoV GII.4[P16] replication was negatively impacted by HAstV3 co-infection, but HAstV3 was unaffected by HuNoV. A reduction in HuNoV GII.4[P16] RNA during co-infections was observed at 72 hpi, with partial restoration achieved using neutralizing anti-IFN-antibodies. Human intestinal enteroids can support the co-infection and replication of HuNoVGII.4[P16] and HAstV3, even at more than 100-fold excess in one virus over the other, and compounds (e.g., anti-IFN antibodies) that interfere with HIE antiviral mechanism(s) can aid in maximizing HuNoV replication during co-infection.

The aim of this study was to compare the Sansure HIV-1 VL assay with the Roche Cobas HIV-1 assay in the quantitation of HIV-1 VL and evaluate its application in China. We collected plasma samples from patients infected with HIV-1 or interference patients infected with other viruses. The same samples were subsequently tested using the Sansure HIV-1 VL and Roche Cobas HIV-1 VL assays. Thirty plasma samples from patients not infected with HIV-1 were undetectable using two assays. Overall, agreement was observed for 289 of the 300 samples (96.33 %), with a κ value of 0.92. The Pearson correlation coefficient between the two assays was 0.96. A paired t test revealed no significant difference between the two assays (p = 0.64). Bland-Altman analysis revealed that 97.88 % (185/189) of the paired VLs fell within the 95 % confidence limits of agreement (-0.746-0.768 log10 copies/mL). In particular, the Pearson correlation coefficients for the samples of subtypes CRF01_AE, CRF07_BC, CRF08_BC, and CRF55_01B were 0.98, 0.97, 0.99, and 0.98, respectively. Compared with the Roche Cobas HIV-1 assay, the Sansure HIV-1 VL assay showed good accuracy and linearity and a high correlation, including with HIV subtypes common in China. In addition, the Sansure HIV-1 VL assay is more accessible because of its advantages of price, acquisition and suitability; thus, it can be readily used in China.