Journal of virological methods最新文献

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.

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: FLOQSwabs® and Evalyn Brushes® 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™ 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 was 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 and storage conditions. 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 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.

The surface of HIV-1 is embedded with numerous host-derived proteins. Characterizing these proteins can enhance knowledge of virus biology and potentially identify novel therapeutic targets. As many of these proteins are present in low abundance on virion surfaces, their identification can be hindered by inherent variables in the methods employed to detect them, including their varying assay sensitivities, sample processing, quantitative capacity, and experimental reproducibility. Here, we have compared the quantification of virion-incorporated proteins using conventional virus immunocapture assays and western blotting, alongside an emerging technique called flow virometry (FV). Using four different pseudovirus models that each express a human protein of interest (CD14, CD38, CD59 and CD162), we compared four experimental techniques for their ability to reliably quantify the incorporation of those four proteins onto virion surfaces. Our results shed light on the advantages and caveats of each technique for detecting virion-incorporated proteins and highlight the breadth in quantification for each technique under different experimental conditions. Protein detection with (FV) provided distinct advantages as it enabled highly reproducible quantifications, had the lowest sample requirements and reagent costs, and minimal hands-on experimental time. We additionally highlight some important considerations in experimental design when studying virion-incorporated proteins, such as the effect of different antibody clones, assay incubation times, and contributions of extracellular vesicles. Most importantly, our data illustrate the importance of using a combination of orthogonal approaches to detect virus-associated proteins, to enable reliable and reproducible quantification that accounts for individual assay biases.