#qRTPCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

 

Abstract

Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosphate-buffered saline and spiked with the virus to reach approximately 10 copies/mcL virus concentration, and 2) raw milk samples from two farms, each spiked with the inactivated AIV H5 followed by 5 series of dilution to reach AIV concentrations of 1000, 100, 10, 1 and 0.1 copies/mcL. Results show that despite the inhibitors in raw milk, AIV in all samples can be detected by both platforms. The MT platform showed higher sensitivity than the benchtop platform: the Ct values from the MT were ~2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for diagnosing AIV H5 in raw milk samples and support its use as an on-site diagnostic for rapid surveillance and response.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

DiaVac Biotech Co.

Schweitzer Biotech Co.

Source: 

Link: https://www.biorxiv.org/content/10.1101/2025.06.02.657307v3

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Longitudinal #serum #proteomics analyses reveal #biomarkers for porcine #influenza and #coronavirus infections

 

Abstract

Respiratory virus infections affect both humans and livestock, causing considerable mortality and morbidity. While respiratory pathogens such as swine influenza A virus (pH1N1) and porcine respiratory coronavirus (PRCV) often present with overlapping clinical symptoms, their pathological trajectories and outcomes differ. Given the propensity for pathogen spillover and the use of pigs as a physiologically relevant large-animal translational model, we aimed to characterise host serum protein signatures that detect and differentiate pH1N1 from PRCV, enabling improved disease monitoring and control. Using high-resolution mass spectrometry-based proteomics, we identified 162 serum proteins that were significantly dysregulated across 3 infection timepoints (1, 5, and 12 days post-infection (DPI)), with signatures correlating with viral shedding and lung pathology as early as 1 DPI. Notably, multiplexed targeted analysis of a subset of proteins in an independent cohort from a different breed and geographic location demonstrated detection, femtomole-level targeted quantitation, and validation of SRGN as a diagnostic marker for pH1N1 and PRCV (AUC=0.85). Further, SOD1 was validated as an early marker for PRCV, increasing as early as 1 DPI (AUC= 0.9). Finally, a multi-peptide signature composed of SRGN, SOD1, and RAN demonstrated reasonable predictive power for pH1N1 (AUC=0.75) and PRCV (AUC=0.65) at 1 DPI. Our data validate the proteomic screening, provide insights into the role of early protein markers in distinguishing respiratory viral infections, and pave the way for the development of point-of-care diagnostics and targeted prevention strategies, enhancing preparedness against emerging zoonotic threats.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

UKRI Biotechnology and Biological Sciences Research Council (BBSRC), BB/X019780/1, BBS/E/PI/230002A, BBS/E/PI/230002B, BBS/E/PI/230002C, BS/E/PI/23NB0004

University of Surrey, https://ror.org/00ks66431

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.04.21.719833v1

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Next-Generation #Sequencing #Strategies During the 2024–2025 Avian #Influenza #H5N1 #Emergency Response in the #US

 

Abstract

The first influenza A(H5N1) human case associated with the A(H5N1) dairy cattle outbreak in the United States was identified in April 2024. The U.S. CDC response to this outbreak was activated days later and remained active until July 2025. During this time, 70 human cases of influenza A(H5N1) were detected with a range of epidemiological links to sources of exposure. Next-generation sequencing (NGS) of human samples was an effectual mechanism for tracking and analyzing the outbreak evolution throughout the response. Due to the specimens’ importance and their variable physical quality, an assortment of laboratory methods was utilized including influenza segment-specific amplification, enrichment capture, short-read, and long-read sequencing. Combining these methods allowed for high-quality genomic data production with rapid turnaround times—typically 2 days from sample receipt to public database submission. By leveraging replicate sequencing, enrichment capture, and sequencing of diagnostic amplicons, valuable genomic data could be produced directly from human clinical specimens that would have normally been considered too weak for routine virologic surveillance sequencing. The resulting assemblies were characterized and analyzed by CDC and shared with local and state public health authorities to facilitate case investigations and risk assessment. These data were further used for phylogenetic analyses of viruses from human cases to investigate likely animal-to-human transmission events and identify clusters within the outbreak that might indicate trends in the types of exposures. Through the adaptable laboratory workflow and the rapid release of viral genomic data, the public health risk mitigation strategies could be evaluated and adjusted in real time.

Source: 

Link: https://www.mdpi.com/1999-4915/18/4/482

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Confirming #ERVEBO #Vaccination to Support #Ebola Virus #Surveillance

 

Abstract

Accurate confirmation of Ebola vaccination (ERVEBO) is essential for interpreting serologic data and assessing vaccine coverage during Ebola virus (EBOV) outbreaks. Current GP1,2-based assays cannot reliably distinguish vaccine-induced immunity from responses generated by natural infection. We developed a multiplex Luminex assay incorporating EBOV GP1,2, secreted glycoprotein (sGP), and a modified vesicular stomatitis virus nucleoprotein (VSV-P-N), a vector antigen encoded by ERVEBO but absent from wild-type EBOV. By using samples from US vaccinees and controls and a small comparison set from the Democratic Republic of the Congo, we found sGP and VSV-P-N demonstrated 100% sensitivity and >97.6% specificity for identifying vaccinees. In samples collected after a ring vaccination campaign in Guinea, combined sGP and VSV-P-N positivity confirmed vaccination in 94.8% of persons with written and 90.8% of persons with verbal confirmation of vaccination history. Our findings show that sGP and VSV-P-N provide a reliable signature of ERVEBO vaccination and support improved Ebola surveillance.

Source: 

Link: https://wwwnc.cdc.gov/eid/article/32/4/25-1906_article

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Rapid GeneXpert #surveillance of #influenza A virus in #seabirds and the #environment provides early warning for #wildlife health in #Aotearoa New Zealand

 

Abstract

The global expansion of highly pathogenic avian influenza (HPAI) virus A(H5N1) underscores the need for rapid surveillance at high-risk wildlife interfaces. Taiaroa Head (45.7828 S, 170.7333 E) in the South Island of Aotearoa New Zealand hosts a plethora of aquatic wildlife including a large red-billed gull (Chroicocephalus novaehollandiae scopulinus) colony as well as the only mainland breeding colony of northern royal albatross (Diomedea sanfordi). The Royal Albatross Centre is also a major nature tourism destination, attracting tens of thousands of visitors annually, thereby creating a dense ecological and human-wildlife interface vulnerable to viral incursion. We evaluated the GeneXpert II platform using the Xpert Xpress Flu/RSV cartridge as a field-deployable tool for avian influenza virus detection in environmental and wildlife-associated samples. The assay detected synthetic influenza A viral RNA and multiple endemic low pathogenic avian influenza virus subtypes (A(H3N8), A(H1N9), A(H5N2) and A(H7N7)) circulating in New Zealand birds. Influenza A virus was reliably identified in spiked environmental water samples with no consistent PCR inhibition as well as naturally occurring avian influenza virus in duck pond water. Field deployment demonstrated that the system could be operated by non-laboratory personnel with minimal training in a non-clinical setting. This study establishes the feasibility of near-real-time environmental monitoring. Repurposing clinical cartridge-based point-of-care diagnostics offers a practical early warning approach for avian influenza virus surveillance at ecologically and economically significant locations.

Competing Interest Statement

Steven G Badman is employed by Cepheid who supplied the Xpert Flu/RSV kits for free. Jo-Ann Stanton is the owner of JStanton Consulting Ltd who contributed in kind resources to the project. JLS has received support from Cepheid and Roche to attend scientific meetings.

Funder Information Declared

Te Niwha, TN/SWC/24/UoOJG

Rutherford Discovery Fellowship, RDF-20-UOO-007

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.03.23.713605v1

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Developing and #Benchmarking #OneHealth Genomic #Surveillance #Tools for #Influenza A Virus in #Wastewater

 

Abstract

Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool. However, IAV is at low abundance in wastewater, making it difficult to sequence. We benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods. Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes. However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes, but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches, potentially enabling broader surveillance and detection of emerging strains. However, probes were costly, labor-intensive, and less sensitive than tiled-amplicon. When testing compatibility of sequencing methods with upstream virus concentration and extraction methods, ultrafiltration-based virus concentration outperformed large-volume direct extraction with all four sequencing methods. This set of benchmarking comparisons and custom panels provides needed information for the translation of IAV genomic sequencing into a routine component of wastewater surveillance.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

University of California, Berkeley, L22CR4507

NIH Common Fund, 4R00GM144747-03

Source: 

Link: https://www.biorxiv.org/content/10.1101/2025.09.19.676942v2

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#Nipah virus molecular #detection from whole #blood and respiratory #swabs in a rapid field-ready protocol

 

Highlights

• A Nipah virus real-time RT-PCR was developed for this study and display dynamic amplification, with sensitive (limit of detection 3.7-4.2 copies/µL) and specific detection.

• The assay was adapted for use on a portable, battery-powered real-time thermocycler.

• When paired with instrument-free RNA extraction, Nipah virus RNA was rapidly detected from contrived whole blood and nasopharyngeal swabs without electricity.

• The combined of Extract & Store and the Palm PCR S1e device offers a viable solution for field-based molecular detection of Nipah virus.

Abstract

Background

Nipah virus (NiV) is a highly pathogenic, zoonotic paramyxovirus with significant public health implications due to high associated mortality and potential for human-to-human transmission. Current diagnostic testing options for NiV are limited and require extensive laboratory infrastructure.

Objective

Develop a field-deployable testing workflow for timely NiV detection.

Study design

A NiV real-time RT-PCR (rRT-PCR) was designed for a highly conserved region of the nucleocapsid gene and tested with RNA from Bangladesh and Malaysia NiV strains. The NiV rRT-PCR was evaluated on Rotor-Gene Q and Palm PCR S1e thermocyclers following instrument free RNA extraction (Extract & Store).

Results

Initial analytical evaluation, on a Rotor-Gene Q, demonstrated dynamic amplification and a limit of detection (LoD) of 3.7-4.2 copies/µL without amplification of related paramyxoviruses. The assay was adapted for the portable, battery-powered, self-contained Palm PCR S1e thermocycler, and exhibited linear detection with a LoD of 30.7 copies/µL. RNA extraction from contrived whole blood and pharyngeal swabs using the Extract & Store workflow yielded comparable results to automated extraction on a KingFisher Apex instrument. The entire assay, including extracted and stabilized RNA controls from BSL-1 strains, was successfully transferred to Aga Khan University with ambient temperature shipping and yielded similar performance.

Conclusions

The combination of Extract & Store and the Palm PCR S1e device offers a viable solution for field-based molecular detection of NiV. While limitations were noted for reaction setup on the Palm PCR, this presents a flexible and accessible workflow for rapid, portable detection of high-consequence pathogens in resource-constrained settings.

Source: 

Link: https://www.sciencedirect.com/science/article/abs/pii/S138665322600020X?dgcid=rss_sd_all

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Evaluating #primer and #probe #mismatch tolerance in an #Influenza A #matrix gene RT #qPCR using contemporary human and zoonotic strains

 

Abstract

Background: 

Genetic drift and host-associated adaptation in influenza A viruses threaten the long-term reliability of RT-qPCR-based diagnostics, particularly when nucleotide mismatches arise within primer and probe binding regions. Conventional assay evaluations often emphasize sequence conservation but rarely assess functional mismatch tolerance across divergent subtypes and hosts. 

Methods: 

We performed an in silico evaluation of a matrix (M) gene–targeted RT-qPCR assay by aligning primer and probe binding regions against 22 H1N1 isolates and representative H3N2 and H5N1 reference strains, including recent zoonotic isolates from avian and bovine hosts. Nucleotide mismatches were identified, quantified, and mapped relative to assay components and oligonucleotide termini. Mismatch burden was summarized by subtype and assay region. 

Results: 

H1N1 isolates exhibited complete conservation across primer and probe regions. In contrast, H3N2 and H5N1 strains demonstrated subtype-specific sequence variability, with a total of eleven mismatches identified across seven non-H1N1 isolates (mean mismatch per isolate = 2.43). Probe mismatches predominated (63.6%), occurring primarily at internal positions, while primer mismatches were infrequent and largely avoided 3′ terminal nucleotides. Recent H5N1 isolates (2023–2024) shared conserved internal mismatches in the probe and forward primer, whereas a historical H5N1 isolate (2016) exhibited a distinct profile including a terminal probe mismatch. Despite this variability, mismatch patterns were consistent with preserved amplification potential. 

Conclusion: 

This study demonstrates that the evaluated influenza A M gene RT-qPCR assay exhibits inherent mismatch tolerance across human and zoonotic subtypes. By shifting diagnostic evaluation from strict sequence identity to functional resilience, our findings provide a framework for designing and maintaining robust molecular assays suitable for surveillance and pandemic preparedness amid ongoing viral evolution.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.02.23.707407v1

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Zoonotic #Influenza #Preparedness: Dutch Medical #Labs Efficiently Detect Animal Influenza A Viruses - External #Quality #Assessment, 2023

 

Highlights

• Concern over H5N1 bird flu testing and detection in the Netherlands is increasing.

• 50 human laboratories in the Netherlands, Aruba, Bonaire, and Curacao were assessed.

• The laboratories detected animal influenza viruses with high performance.

• Few laboratories identified the animal subtype of detected influenza A viruses.

• National reference laboratory capacity to identify the animal subtype is critical.

Abstract

Background

Since 2022, highly pathogenic H5N1 influenza A virus clade 2.3.4.4b has caused global outbreaks among wild birds and poultry, with increasing mammalian and sporadic human infections. This elevates concerns about zoonotic transmission and pandemic risk, highlighting the need for accurate detection and identification of animal influenza A viruses by human clinical diagnostic laboratories (hCDL).

Methods

To evaluate routine diagnostic performance, an External Quality Assessment (EQA) panel containing inactivated influenza A viruses of avian (three subtype H5, one H7), swine (two H1, one H3), and human (one H1pdm09, one H3) origin was distributed to 50 hCDL in the Netherlands, Aruba, Bonaire, and Curaçao. Laboratories conducted their routine molecular influenza virus detection and, if available, subtyping workflows.

Results

A total of 118 detection workflows were reported. Of these, 109 (91%) successfully detected influenza A virus in all positive specimens. At least one workflow in 49/50 (98%) laboratories reliably detected all animal influenza viruses as type A influenza virus. Most false negatives occurred with swine H1N1v. Only 24 workflows from 20 laboratories attempted subtyping for one or multiple panel specimens (total 109 subtype-specific results reported): for human viruses, 37/39 results were correct; for avian viruses, 13/14 were correct (including 12/12 for H5); for swine viruses, only 2/56 were correct (both swine H3N2 using broad-reactive H3 assays).

Conclusions

hCDL in the Netherlands demonstrate high performance for detecting animal influenza A viruses. However, subtyping capacity is limited, necessitating referral of specimens of suspected zoonotic influenza cases to the National Influenza Centre for further characterization.

Source: 

Link: https://www.sciencedirect.com/science/article/abs/pii/S1386653226000168?dgcid=rss_sd_all

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Detection of #LaCrosse Virus #RNA in Clinical #Specimens Obtained from #Children with La Crosse Infection

 

Highlights

• Viremia in children with La Crosse Virus infection is transient; viral RNA was detected in only 3.2% of sera

• Detection of La Crosse Virus RNA in respiratory samples is slightly higher at 21.7% and may reflect the temporal distribution of the virus after infection

• NAAT has limited utility in routine diagnosis of La Crosse Virus encephalitis in children but may still be useful in cases with delayed seroconversion

Abstract

Background

La Crosse virus (LACV), a member of family Peribunyaviridae, genus Orthobunyavirus, is the leading cause of neuroinvasive arboviral infection in children in the United States. Diagnosis relies on detecting specific antibodies (IgG or IgM), a 4-fold titer rise or seroconversion, in patients with compatible presentations. NAAT used for LACV detection has largely been limited to mosquito, animal models or postmortem brain tissue. There is a lack of data on the performance of NAATs in clinical specimens from living patients.

Methods

Children who had positive arbovirus serology tests and a diagnosis of LACV encephalitis were identified. Remnant specimens including plasma, serum, CSF, throat swab (THT) or nasopharyngeal sample (NP) submitted to the laboratory for other diagnostic testing were retrieved and tested with LACV-PCR. Medical records were reviewed for demographics, presenting symptoms and test results.

Results

From June 2015 to October 2021, 61 patients had remnant specimens available for LACV-PCR and were included in this study. A total of 179 clinical specimens from these patients were tested, including 64 sera, 31 plasma, 33 CSF, 23 THT and 28 NP. Ten (5.3%) samples collected from 8 (13.1%) unique patients were positive for LACV RNA. The positive rates were 3.2%, 0, 6.5%, 3.5% and 21.7% for sera, plasma, CSF, NP and THT respectively.

Conclusion

There is limited utility of NAATs for diagnosis of LACV infection. NAATs may be useful in cases with delayed seroconversion or in immunocompromised individuals.

Source: 

Link: https://www.sciencedirect.com/science/article/abs/pii/S1386653226000120?dgcid=rss_sd_all

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Detection of Avian #Influenza #H5–Specific #Antibodies by Chemiluminescent Assays

 

Abstract

We evaluated 2 electrochemiluminescence serologic assays to detect avian influenza H5 antibodies. Both assays identified H5 antibodies from both serum and dried blood spots and had strong specificity and minimal cross-reactivity in human and avian samples. Such assays can support populationwide serologic surveys aimed at assessing population-level immunity.

Source: 

Link: https://wwwnc.cdc.gov/eid/article/32/1/25-1117_article

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Development of a multi-species #luciferase-based double #antigen #ELISA for the detection of #antibodies against #Influenza A virus #H5 clade 2.3.4.4b

 

Abstract

The highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 represent a major threat to animal and public health. The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses and a high frequency of spillover events to unexpected novel mammalian hosts such as dairy cows. The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission. Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface. For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screenings are critically needed. The newly developed double antigen ELISA format employing a luminescence-based detection technology has demonstrated to comply with such prerequisites. The assay allowed the detection of H5-specific antibodies even early after infection or vaccination in a wide range of birds and mammals including humans. It further demonstrated superior analytical sensitivity and high specificity for antibodies directed against H5 hemagglutinin of clade 2.3.4.4b as no cross-reactivity with other hemagglutinin subtypes was observed. Thus, the assay represents a valuable contribution to existing serological diagnostic tests for a clade-optimized detection of influenza A virus antibodies in a broad range of species.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.01.05.697617v1

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#Molnupiravir clinical trial simulation suggests that #PCR underestimates #antiviral #potency against #SARS-CoV-2

 

Abstract

Molnupiravir is an antiviral medicine that induces lethal copying errors during SARS-CoV-2 RNA replication. Molnupiravir reduced hospitalization in one pivotal trial by 50% and had variable effects on reducing viral RNA levels in three separate trials. We used mathematical models to simulate these trials and closely recapitulated their virologic outcomes. Model simulations suggested lower antiviral potency against pre-Omicron SARS-CoV-2 variants than against Omicron. We estimated that in vitro assays underestimated in vivo potency by 6- to 7-fold against Omicron variants. Our model suggested that because polymerase chain reaction detects molnupiravir mutated variants, the true reduction in non-mutated viral RNA was underestimated by approximately 0.4 log10 in the two trials conducted while Omicron variants dominated. Viral area under the curve estimates differed significantly between non-mutated and mutated viral RNA. Our results reinforce past work suggesting that in vitro assays are unreliable for estimating in vivo antiviral drug potency and suggest that virologic endpoints for respiratory virus clinical trials should be catered to the drug mechanism of action.

Source: Journal of Clinical Investigation, https://www.jci.org/articles/view/192052

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Developing and #Benchmarking #OneHealth #Genomic #Surveillance Tools for Influenza A Virus in #Wastewater

 

Abstract

Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool. However, IAV is at low abundance in wastewater, making it difficult to sequence. We benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods. Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes. However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes, but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches, potentially enabling broader surveillance and detection of emerging strains. However, probes were costly, labor-intensive, and less sensitive than tiled-amplicon. When testing compatibility of sequencing methods with upstream virus concentration and extraction methods, ultrafiltration-based virus concentration outperformed large-volume direct extraction with all four sequencing methods. This set of benchmarking comparisons and custom panels provides needed information for the translation of IAV genomic sequencing into a routine component of wastewater surveillance.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

University of California, Berkeley, L22CR4507

NIH Common Fund, 4R00GM144747-03

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.09.19.676942v1

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#Syndromic approach for rapid #detection and differentiation of #human pathogenic #alphaviruses

 

Highlights

• Most vector-borne viruses like alphaviruses are not included in routine diagnostics

• Lack of testing results in misdiagnoses and underdetection

• A new multiplexed real-time PCR assay detects all human pathogenic alphaviruses

• The new multiplex assay is more sensitive than available tests and highly specific

• The multiplex test can be applied broadly for diagnostics and molecular surveillance

Abstract

Background

Knowledge of epidemiology, pathogenesis, and public health burden is scarce for many arthropod-borne viruses (arboviruses). Insufficient knowledge is partly due to lack of exhaustive laboratory diagnostics due to resource limitations. Among arboviruses, arthritogenic and encephalitogenic alphaviruses are globally widespread, can cause severe disease, and can co-occur regionally.

Objectives

We developed and validated a multiplexed real-time reverse transcription-PCR assay for the detection of all alphaviruses commonly causing human disease except Barmah Forest virus.

Study design

The assay combines five antigenic complex-specific assays and one Chikungunya virus-specific assay in a single parallelized reaction.

Results

Comparisons with previously published PCR-based protocols for broad alphavirus detection using 20 different human-pathogenic alphaviruses revealed a significantly higher sensitivity of the new multiplexed assay (Fisher’s exact test, p<0.0001). Detection limits with the new assay ranged from 0.83 cps/μl of extracted O’nyong-nyong virus to 33.05 cps/μl of extracted Western equine encephalitis virus. Antigenic complexes could be clearly differentiated by reactivity, Ct values (T-test, p<0.0025) and signal intensities (T-test, p<0.0001), even when testing high alphavirus concentrations potentially capable of causing false-positive PCR results. Testing of high-titred cell culture supernatants of eight important non-alphaviral arboviruses, of 4,308 serum samples collected from febrile patients in Benin and Peru, of seven CHIKV positive diagnostic samples from Brazil, and of non-targeted alphaviruses confirmed excellent diagnostic performance by the new assay, including improved detection of Mayaro and Venezuelan equine encephalitis virus in clinical specimens.

Conclusions

Short turn-around time, applicability in resource-limited settings, antigenic complex determination, and higher sensitivity compared to previously available tests make the new assay a useful tool for alphavirus surveillance and routine patient diagnostics.

Source: Journal of Clinical Virology, https://www.sciencedirect.com/science/article/pii/S1386653225001143?dgcid=rss_sd_all

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Laboratory #Diagnosis of #Hendra and #Nipah: Two Emerging Zoonotic Diseases with One Health Significance

Abstract

Hendra virus (HeV) and Nipah virus (NiV) are two highly pathogenic RNA viruses with zoonotic potential, which can cause severe diseases with high mortality rates (50–100%) in humans and animals. Given this context, these viruses are classified as Biosafety Level 4 (BSL-4) pathogens, thus limiting research studies. Despite the high case fatalities, there are currently no human vaccines available for either virus, owing in part to the limitations in research and hesitancy in funding. In the absence of widespread vaccination, diagnostic tests are crucial for the rapid identification of cases and disease surveillance. This review synthesizes current knowledge on the epidemiology, transmission dynamics, and pathogenesis of NiV and HeV to contextualize a detailed assessment of the available diagnostic tools. We examined molecular and serological assays, including RT-PCR, ELISA, and LAMP, highlighting sample sources, detection windows, and performance. Diagnostic considerations across human and animal hosts are discussed, with emphasis on outbreak applicability and field-readiness, given the need for diagnostic assays that are suitable for use in low-income areas. Further development of diagnostic assays, including isothermal amplification tests and other next-generation approaches, is recommended to fill the gap in rapid, point-of-care diagnostics.

Source: Viruses, https://www.mdpi.com/1999-4915/17/7/1003

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qRTPCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

Abstract

Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosphate-buffered saline and spiked with the virus to reach approximately 10 copies/mcL virus concentration, and 2) raw milk samples from two farms, each spiked with the inactivated AIV H5 followed by 5 series of dilution to reach AIV concentrations of 1000, 100, 10, 1 and 0.1 copies/mcL. Results show that despite the inhibitors in raw milk, AIV in all samples can be detected by both platforms. The MT platform showed higher sensitivity than the benchtop platform: the Ct values from the MT were ~2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for diagnosing AIV H5 in raw milk samples and support its use as an on-site diagnostic for rapid surveillance and response.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.06.02.657307v2

____

qRT-PCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

Abstract

Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosphate-buffered saline and spiked with the virus to reach approximately 10 copies/mcL virus concentration, and 2) raw milk samples from two farms, each spiked with the inactivated AIV H5 followed by 5 series of dilution to reach AIV concentrations of 1000, 100, 10, 1 and 0.1 copies/mcL. Results show that despite the inhibitors in raw milk, AIV in all samples can be detected by both platforms. The MT platform showed higher sensitivity than the benchtop platform: the Ct values from the MT were ~2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for diagnosing AIV H5 in raw milk samples and support its use as an on-site diagnostic for rapid surveillance and response.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.06.02.657307v1

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