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.

Background: Self-sampling is now a key component within HPV-based cervical screening programmes to engage individuals and enhance participation. As self-sampling is relatively new, information on the influence of pre-analytical parameters such as transit-temperature and time between sampling and testing on HPV test results requires detailed investigation.

Methods: FLOQSwab® and Evalyn Brush® were used to assess HPV and cellular stability over a 30-week period (0w,4w,12w,30w) at 4°C, ambient, and 37°C. Vaginal self-samples were simulated by inoculating the devices with an HPV16-positive cell-line suspension. Devices were tested using two DNA-based (Anyplex^TM II HPV28, Papilloplex® HR-HPV), one mRNA-based (APTIMA HR-HPV,) and one in-house beta-globin qPCR assay.

Results: No loss of qualitative HPV detection was observed after 12-weeks storage at ambient or 4°C irrespective of device or assay. For DNA-based assays, no loss of qualitative HPV detection was observed over time (30w) irrespective of temperature/device. Loss of qualitative mRNA signal was observed when devices were stored at 37°C for 12-weeks or longer; however, no loss of detection was observed at 30-weeks when either device stored at 4°C.

Conclusion: HPV nucleic acid is stable on proxies of self-taken samples, however, the duration of stability was affected by the device-assay combination Such differences should be considered when optimising self-sampling exercises.

Background: Hepatitis C virus (HCV) resistance-associated substitutions (RASs) have a significant impact on the treatment of HCV with direct-acting antivirals (DAAs). However, limited research has been conducted, and no standardized methods for detecting RASs in mainland China.

Objectives: To develop and apply a novel method for detecting HCV RASs in HCV RNA-positive patients in Linzhou, China.

Study design: In total, 103 HCV RNA-positive serum specimens and epidemiological questionnaires were collected. A PCR method for detecting HCV RASs encompassing the NS3 to NS5B region was developed.

Results: Demographic analysis revealed a predominance of females (66/103, 64.1%), with an average age of 70 years. Genotype 1b (GT1b) (17/103, 16.5%) and GT2a (86/103, 83.5%) were identified. The prevalence of RASs was higher (17/17, 100%) in GT1b than in GT2a (7/86, 8%). In GT1b, a higher frequency of RASs was observed in the NS5B region (17/17, 100%) than in the NS3 (14/17, 82%) and NS5A (10/17, 59%) regions. C316N (100%) was the most prevalent, followed by S122G (71%) and R30Q (35%).

Conclusions: We introduced an innovative approach for the detection of HCV RASs and provided a wealth of information on HCV RASs in Linzhou, China. The findings support the cautious selection of treatment regimens, potentially improving patient outcomes.

Hundreds of millions of the domestic dogs worldwide are routinely inoculated with the modified live vaccines for canine distemper virus (CDV) every year. However, the corresponding serological diagnostic and detections are always lacking, thus, there is an urgent demand to establish its unique diagnostic technologies to produce high-quality antigenic biomolecules. In the present study, the ectodomain (et) of CDV hemagglutinin (H) protein was firstly expressed in a soluble and secreted forms by an Expi293F transient transfection system based on its antigenic secondary structure analysis. The yields of CDV H(et) protein was 2.6 g/L with purity over 95 % in supernatant of Expi293F cells. The CDV H(et) protein elicited comparative antibodies levels to the CDV virions in rabbit by ELISA and neutralization test. The purified polyclonal antibodies of immunized with CDV H(et) protein recognized both wild-type and vaccine CDV strains. More importantly, the purified polyclonal antibodies of CDV H(et) protein revealed significantly higher viral neutralizing activity than those from CDV-3 virions, which highlighted that the critical role of CDV H protein to elicit viral specific and protective neutralizing antibodies. Taken together, the CDV H(et) protein produced in mammalian expression systems was high-quality and good immunogenicity, and would be with great potential to serve as a serological diagnostic antigen or a novel CDV subunit vaccine in future.

The emergence of SARS-CoV-2 raised concerns about the potential for interspecies transmission, particularly among domestic animals. We evaluated the seroprevalence of SARS-CoV-2 antibodies in domestic cats from various sites in North America. A total of 216 serum samples collected between December 2019 and February 2022, were analyzed using four different enzyme-linked immunosorbent assays (ELISAs), including a commercial surrogate virus neutralization test (sVNT), a commercial double antigen test (dN ELISA), and two in-house developed indirect ELISAS based on receptor-binding domain (RBD iELISA) and the nucleocapsid (N iELISA) proteins, respectively. Seropositive samples in the commercial ELISAs were subject to virus neutralization test (cVNT) employing the Wuhan-like USA-WA1/2020 SARS-CoV-2 isolate. Our findings revealed that, 6 % (12/216) of the cat serum samples tested positive by the sVNT, while 4 % (9/216) tested positive for the dN-ELISA. Interestingly, the N iELISA showed a higher seroprevalence, with 31 % of the samples testing positive, possibly due to cross-reactive antibodies against the N protein of other coronavirus commonly found in cats. There was a high concordance between sVNT, cVNT, and RBD iELISA. Among positive sVNT cat serum samples, 75 % (9/12) exhibited neutralizing titers with all samples also being positive by RBD iELISA. Notably, the RBD iELISA and sVNT demonstrated high sensitivity and specificity in detecting SARS-CoV-2 antibodies (100 and 79 %; 100 and 90 %, respectively). In conclusion, our study provides important insights into the seroprevalence of SARS-CoV-2 antibodies in domestic cats, highlighting the potential for interspecies transmission and the need for continued monitoring of SARS-CoV-2 in animal populations.

Rabies virus (RABV), remains a significant public health concern, with bat-maintained lineages accounting for all currently documented cases in Brazil. Despite the availability of pharmacological prophylaxis for humans and animals, the high genetic diversity of RABV in diverse natural bat hosts and continued circulation in multiple animals pose challenges for effective surveillance. Here, we developed and validated a novel, rapidly deployable amplicon-based sequencing approach for RABV genomic surveillance. This "all-in-one" protocol integrates whole RABV genome sequencing with host species identification through COI gene amplification and sequencing, addressing the challenges posed by RABV's high genetic diversity and complex transmission dynamics. We assessed the protocol's effectiveness by sequencing 25 near-complete RABV genomes from host species across four distinct families (Bovidae, Equidae, Felidae, and Microchiroptera) obtained from the Rabies Control and Surveillance Program from the Rio Grande do Sul State, Southern Brazil. The method achieved an average genome coverage of 91.4 % at a minimum 5x read depth, with a mean depth coverage of 816x across sequenced genomes. The results demonstrated significant Bat-Clade sublineage diversity, which was classified using the MADDOG RABV lineage system. The protocol successfully identified three bat species (Tadarida brasiliensis, Desmodus rotundus, and Myotis nigricans) among the samples, highlighting its capability for precise host identification. This study presents a powerful tool for high-resolution evaluation of RABV genomic features and host identification, enabling more targeted animal and human health interventions. This new approach has the potential to enhance RABV surveillance capabilities, contributing to more effective rabies control strategies within a One Health framework.