Accurate virus identification with interpretable Raman signatures by machine learning.

Rapid identification of newly emerging or circulating viruses is an important first step toward managing the public health response to potential outbreaks. A portable virus capture device, coupled with label-free Raman spectroscopy, holds the promise of fast detection by rapidly obtaining the Raman signature of a virus followed by a machine learning (ML) approach applied to recognize the virus based on its Raman spectrum, which is used as a fingerprint. We present such an ML approach for analyzing Raman spectra of human and avian viruses. A convolutional neural network (CNN) classifier specifically designed for spectral data achieves very high accuracy for a variety of virus type or subtype identification tasks. In particular, it achieves 99% accuracy for classifying influenza virus type A versus type B, 96% accuracy for classifying four subtypes of influenza A, 95% accuracy for differentiating enveloped and nonenveloped viruses, and 99% accuracy for differentiating avian coronavirus (infectious bronchitis virus [IBV]) from other avian viruses. Furthermore, interpretation of neural net responses in the trained CNN model using a full-gradient algorithm highlights Raman spectral ranges that are most important to virus identification. By correlating ML-selected salient Raman ranges with the signature ranges of known biomolecules and chemical functional groups­for example, amide, amino acid, and carboxylic acid­we verify that our ML model effectively recognizes the Raman signatures of proteins, lipids, and other vital functional groups present in different viruses and uses a weighted combination of these signatures to identify viruses.

Diagnostic investigation of avian reovirus field variants circulating in broiler chickens in Pennsylvania of United States between 2017 and 2022.

Avian reovirus (ARV) has been continuously affecting the poultry industry in Pennsylvania (PA) in recent years. This report provides our diagnostic investigation on monitoring ARV field variants from broiler chickens in Pennsylvania. Genomic characterization findings of 72 ARV field isolates obtained from broiler cases during the last 6 years indicated that six distinct cluster variant strains (genotype I-VI), which were genetically diverse and distant from the vaccine and vaccine-related field strains, continuously circulated in PA poultry. Most of the variants clustered within genotype V (24/72, 33.3%), followed by genotype II (16/72, 22.2%), genotype IV (13/72, 18.1%), genotype III (13/72, 18.1%), genotype VI (05/72, 6.94%), and genotype I (1/72, 1.38%). The amino acid identity between 72 field variants and the vaccine strains (1133, 1733, 2408, 2177) varied from 45.3% to 99.7%, while the difference in amino acid counts ranged from 1-164. Among the field variants, the amino acid identity and count difference ranged from 43.3% to 100% and 0 to 170, respectively. Variants within genotype V had maximum amino acid identity (94.7-100%), whereas none of the variants within genotypes II and VI were alike. These findings indicate the continuing occurrence of multiple ARV genotypes in the environment.

A rapid and label-free platform for virus capture and identification from clinical samples.

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.

A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection.

Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 103 EID50 mL-1 (EID50 : 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme-linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off-chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low-cost and convenient biomarker detection.

Label-Free Virus Capture and Release by a Microfluidic Device Integrated with Porous Silicon Nanowire Forest.

Viral diseases are perpetual threats to human and animal health. Detection and characterization of viral pathogens require accurate, sensitive, and rapid diagnostic assays. For field and clinical samples, the sample preparation procedures limit the ultimate performance and utility of the overall virus diagnostic protocols. This study presents the development of a microfluidic device embedded with porous silicon nanowire (pSiNW) forest for label-free size-based point-of-care virus capture in a continuous curved flow design. The pSiNW forests with specific interwire spacing are synthesized in situ on both bottom and sidewalls of the microchannels in a batch process. With the enhancement effect of Dean flow, this study demonstrates that about 50% H5N2 avian influenza viruses are physically trapped without device clogging. A unique feature of the device is that captured viruses can be released by inducing self-degradation of the pSiNWs in physiological aqueous environment. About 60% of captured viruses can be released within 24 h for virus culture, subsequent molecular diagnosis, and other virus characterization and analyses. This device performs viable, unbiased, and label-free virus isolation and release. It has great potentials for virus discovery, virus isolation and culture, functional studies of virus pathogenicity, transmission, drug screening, and vaccine development.

Genomic characterization of a novel avian arthritis orthoreovirus variant by next-generation sequencing.

By using next-generation sequencing (NGS) technology, we have identified a divergent avian orthoreovirus (ARV) field variant (Reo/PA/Broiler/15511/13, or PA15511), isolated from broiler chickens with viral arthritis in Pennsylvania in 2013. The complete genome of the PA15511 field strain was 23,495 bp in length with 10 dsRNA segments encoding 12 viral proteins. The lengths of the genomic segments ranged from 1192 bp (S4) to 3958 bp (L1). Genomic analysis has revealed that this virus is distinct from reference ARV strains and meets criteria for a new or novel strain.

An Impedance Aptasensor with Microfluidic Chips for Specific Detection of H5N1 Avian Influenza Virus.

In this research a DNA aptamer, which was selected through SELEX (systematic evolution of ligands by exponential enrichment) to be specific against the H5N1 subtype of the avian influenza virus (AIV), was used as an alternative reagent to monoclonal antibodies in an impedance biosensor utilizing a microfluidics flow cell and an interdigitated microelectrode for the specific detection of H5N1 AIV. The gold surface of the interdigitated microelectrode embedded in a microfluidics flow cell was modified using streptavidin. The biotinylated aptamer against H5N1 was then immobilized on the electrode surface using biotin-streptavidin binding. The target virus was captured on the microelectrode surface, causing an increase in impedance magnitude. The aptasensor had a detection time of 30 min with a detection limit of 0.0128 hemagglutinin units (HAU). Scanning electron microscopy confirmed the binding of the target virus onto the electrode surface. The DNA aptamer was specific to H5N1 and had no cross-reaction to other subtypes of AIV (e.g., H1N1, H2N2, H7N2). The newly developed aptasensor offers a portable, rapid, low-cost alternative to current methods with the same sensitivity and specificity.

Identification, 16S rRNA-based characterization, and antimicrobial profile of Gallibacterium isolates from broiler and layer chickens.

Gallibacterium spp., particularly G. anatis, have received much attention as poultry pathogens in recent years. We report here the presence and antimicrobial resistance profile of 69 Gallibacterium isolates obtained from 2,204 diagnostic submissions of broiler and layer chickens in 2019-2021. Gallibacterium-positive chickens had lesions primarily in the respiratory tract, reproductive tract, and related serosal surfaces. Gallibacterium spp. were initially identified based on their typical cultural characteristics on blood agar. The isolates were confirmed by a genus-specific PCR spanning 16S-23S rRNA and MALDI-TOF mass spectrometry. Phylogenetic analysis based on 16S rRNA gene sequence revealed distinct clades. Of the 69 isolates, 68 clustered with the reference strains of G. anatis and 1 with Gallibacterium genomospecies 1 and 2. Antimicrobial susceptibility testing of 58 of the 69 isolates by a MIC method showed variable responses to antimicrobials. The isolates were all susceptible to enrofloxacin, ceftiofur, florfenicol, and gentamicin. There was a high level of susceptibility to trimethoprim-sulfamethoxazole (98.0%), streptomycin (98.0%), amoxicillin (84.0%), sulfadimethoxine (71.0%), and neomycin (71.0%). All of the isolates were resistant to tylosin. There was resistance to penicillin (98.0%), erythromycin (95.0%), clindamycin (94.0%), novobiocin (90.0%), tetracycline (88.0%), oxytetracycline (76.0%), and sulfathiazole (53.0%). A high rate of intermediate susceptibility was observed for spectinomycin (67.0%) and sulfathiazole (40.0%). Our findings indicate a potential role of G. anatis as an important poultry pathogen and cause of subsequent disease, alone or in combination with other pathogens. Continuous monitoring and an antimicrobial susceptibility assay are recommended for effective treatment and disease control.

New outbreaks of H5N1 highly pathogenic avian influenza in domestic poultry and wild birds in Cambodia in 2011.

Five outbreaks of H5N1 highly pathogenic avian influenza (HPAI) have been diagnosed in domestic poultry and wild birds in Cambodia from January to November of 2011. Of the five outbreaks, one occurred in a village backyard flock in Kandal province in January; two occurred in native Cambodian chickens and ducks in Banteay Meanchey province in July and August, respectively; one was seen in wild birds in Phnom Tamao Zoo in Kandal Province in July; and one outbreak occurred in commercial broilers at Opong Moan in Battambang province in northwestern Cambodia in early November. Clinically, HPAI-infected broilers and native chickens showed sudden death, severe depression, ruffled feathers, edema of heads and necks, swollen and cyanotic combs and wattles, and swollen and congested conjunctiva, with occasional hemorrhage, paralysis, and other neurologic signs. In ducks, significantly swollen sinuses and eyes, cloudy corneas, difficulty standing, or paralysis were commonly seen. Some affected ducks showed sudden death without obvious clinical symptoms. Necropsy lesions showed congestion and necrotic debris within sinuses and severe hemorrhages in gizzards, livers, and lungs in both affected native chickens and ducks during the new outbreaks in 2011. All five outbreaks were diagnosed as H5N1 HPAI by virus isolation and real-time reverse transcription-PCR tests. Once a backyard flock in a village or a poultry farm was diagnosed as positive for H5N1 HPAI; the whole village backyard poultry and all farm flocks were culled immediately by Cambodian provincial and central authorities as per the strategies adopted for the control of HPAI.

A SPR aptasensor for detection of avian influenza virus H5N1.

Rapid and specific detection of avian influenza virus (AIV) is urgently needed due to the concerns over the potential outbreaks of highly pathogenic H5N1 influenza in animals and humans. Aptamers are artificial oligonucleic acids that can bind specific target molecules, and show comparable affinity for target viruses and better thermal stability than monoclonal antibodies. The objective of this research was to use a DNA-aptamer as the specific recognition element in a portable Surface Plasmon Resonance (SPR) biosensor for rapid detection of AIV H5N1 in poultry swab samples. A SPR biosensor was fabricated using selected aptamers that were biotinylated and then immobilized on the sensor gold surface coated with streptavidin via streptavidin-biotin binding. The immobilized aptamers captured AIV H5N1 in a sample solution, which caused an increase in the refraction index (RI). After optimizing the streptavidin and aptamer parameters, the results showed that the RI value was linearly related (R(2) = 0.99) to the concentration of AIV in the range of 0.128 to 1.28 HAU. Negligible signal (<4% of H5N1) was observed from six non-target AIV subtypes. The AIV H5N1 in poultry swab samples with concentrations of 0.128 to 12.8 HAU could be detected using this aptasensor in 1.5 h.

Genetic characterization of a novel pheasant-origin orthoreovirus using Next-Generation Sequencing.

A field isolate (Reo/SDWF /Pheasant/17608/20) of avian orthoreovirus (ARV), isolated from a flock of game-pheasants in Weifang, Shandong Province, was genetically characterized being a field variant or novel strain in our recent research studies in conducting whole genome sequencing by using Next-Generation Sequencing (NGS) technique on Illumina MiSeq platform. Among a total of 870,197 35-151-mer sequencing reads, 297,711 reads (34.21%) were identified as ARV sequences. The de novo assembly of the ARV reads resulted in generation of 10 ARV-related contigs with the average sequencing coverage from 1390× to 1977× according to 10 ARV genome segments. The complete genomes of this pheasant-origin ARV (Reo/SDWF /Pheasant/17608/20) were 23,495 bp in length and consist of 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1) encoding 12 viral proteins. Sequence comparison between the SDWF17608 and classic ARV reference strains revealed that 58.1-100% nucleotide (nt) identities and 51.4-100% amino acid (aa) identities were in genome segment coding genes. The 10 RNA segments had conversed termini at 5' (5'-GCUUUU) and 3' (UCAUC-3') side, which were identical to the most published ARV strains. Phylogenetic analysis revealed that this pheasant ARV field variant was closely related with chicken ARV strains in 7 genome segment genes, but it possessed significant sequence divergence in M1, M3 and S2 segments. These findings suggested that this pheasant-origin field variant was a divergent ARV strain and was likely originated from reassortments between different chicken ARV strains.

Identification of 6 gene markers for survival prediction in osteosarcoma cases based on multi-omics analysis.

Multiple-omics sequencing information with high-throughput has laid a solid foundation to identify genes associated with cancer prognostic process. Multiomics information study is capable of revealing the cancer occurring and developing system according to several aspects. Currently, the prognosis of osteosarcoma is still poor, so a genetic marker is needed for predicting the clinically related overall survival result. First, Office of Cancer Genomics (OCG Target) provided RNASeq, copy amount variations information, and clinically related follow-up data. Genes associated with prognostic process and genes exhibiting copy amount difference were screened in the training group, and the mentioned genes were integrated for feature selection with least absolute shrinkage and selection operator (Lasso). Eventually, effective biomarkers received the screening process. Lastly, this study built and demonstrated one gene-associated prognosis mode according to the set of the test and gene expression omnibus validation set; 512 prognosis-related genes (P < 0.01), 336 copies of amplified genes (P < 0.05), and 36 copies of deleted genes (P < 0.05) were obtained, and those genes of the mentioned genomic variants display close associations with tumor occurring and developing mechanisms. This study generated 10 genes for candidates through the integration of genomic variant genes as well as prognosis-related genes. Six typical genes (i.e. MYC, CHIC2, CCDC152, LYL1, GPR142, and MMP27) were obtained by Lasso feature selection and stepwise multivariate regression study, many of which are reported to show a relationship to tumor progressing process. The authors conducted Cox regression study for building 6-gene sign, i.e. one single prognosis-related element, in terms of cases carrying osteosarcoma. In addition, the samples were able to be risk stratified in the training group, test set, and externally validating set. The AUC of five-year survival according to the training group and validation set reached over 0.85, with superior predictive performance as opposed to the existing researches. Here, 6-gene sign was built to be new prognosis-related marking elements for assessing osteosarcoma cases' surviving state.

Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 in peafowl (Pavo cristatus).

An outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first diagnosed in a "backyard" flock of peafowl (Pavo cristatus) raised on palace premises in the Kingdom of Saudi Arabia in December 3, 2007. The flock consisted of 40 peafowl, and their ages ranged from 3 to 5 years old. Affected birds suffered from depression, anorexia, and white diarrhea. Four dead birds were submitted for HPAI diagnosis at the Central Veterinary Diagnostic Laboratory in Riyadh. Brain and liver tissues and tracheal and cloacal swabs were taken from the dead birds and processed for a real-time reverse transcriptase (RT)-PCR test and virus isolation in specific-pathogen-free embryonating chicken eggs. The H5N1 subtype of avian influenza virus was isolated from the four dead birds and identified by a real-time RT-PCR before and after egg inoculation. The virus isolates were characterized as HPAI H5N1 virus by sequencing analysis. Phylogenetic comparisons revealed that the H5N1 viruses isolated from peafowl belong to the genetic clade 2.2 according to the World Health Organization nomenclature. The peafowl H5N1 virus falls into 2.2.2 sublineage II and clusters with the H5N1 viruses isolated from poultry in Saudi Arabia in 2007-08.

Epidemic outbreaks, diagnostics, and control measures of the H5N1 highly pathogenic avian influenza in the Kingdom of Saudi Arabia, 2007-08.

The first outbreak of H5N1 highly pathogenic avian influenza (HPAI) in the Kingdom of Saudi Arabia (KSA) occurred in two "backyard" flocks of Houbara bustards and falcons in February 2007. Subsequent outbreaks were seen through the end of 2007 in "backyard" birds including native chickens, ostriches, turkeys, ducks, and peacocks. From November 2007 through January 2008, H5N1 HPAI outbreaks occurred in 19 commercial poultry premises, including two broiler breeder farms, one layer breeder farm, one ostrich farm, and 15 commercial layer farms, with approximately 4.75 million birds affected. Laboratory diagnosis of all H5N1-positive cases was conducted at the Central Veterinary Diagnostic Laboratory (CVDL) in Riyadh, Saudi Arabia. A combination of diagnostic tests was used to confirm the laboratory diagnosis. A rapid antigen-capture test and real-time reverse transcriptase-PCR (rtRT-PCR) assay on clinical and field specimens were conducted initially. Meanwhile, virus isolation in specific-pathogen-free embryonating chicken eggs was performed and was followed by hemagglutinin (HA) and hemagglutination inhibition tests, then rapid antigen-capture and rtRT-PCR tests on HA-positive allantoic fluid samples. In most HPAI cases, a complete laboratory diagnosis was made within 24-48 hr at the CVDL. Saudi Arabian government officials made immediate decisions to depopulate all H5N1-affected and nonaffected flocks within a 5-km radius area and applied quarantine zones to prevent the virus from spreading to other areas. Other control measures, such as closure of live bird markets and intensive surveillance tests on all poultry species within quarantine zones, were in place during the outbreaks. As a result, the HPAI outbreaks were quickly controlled, and no positive cases were detected after January 29, 2008. The KSA was declared free of HPAI on April 30, 2008, by the World Animal Health Organization.

Rapid Size-Based Isolation of Extracellular Vesicles by Three-Dimensional Carbon Nanotube Arrays.

Recent discoveries reveal that extracellular vesicles (EVs) play an important role in transmitting signals. Although this emerging transcellular pathway enables a better understanding of neural communication, the lack of techniques for effectively isolating EVs impedes their studies. Herein, we report an emergent high-throughput platform consisting of three-dimensional carbon nanotube arrays that rapidly capture different EVs based on their sizes, without any labels. More importantly, this label-free capture maintains the integrity of the EVs when they are excreted from a host cell, thus allowing comprehensive downstream analyses using conventional approaches. To study neural communication, we developed a stamping technique to construct a gradient of nanotube herringbone arrays and integrated them into a microdevice that allowed us processing of a wide range of sample volumes, microliters to milliliters, in several minutes through a syringe via manual hand pushing and without any sample preparation. This microdevice successfully captured and separated EVs excreted from glial cells into subgroups according to their sizes. During capture, this technology preserved the structural integrity and originality of the EVs that enabled us to monitor and follow internalization of EVs of different sizes by neurons and cells. As a proof of concept, our results showed that smaller EVs (∼80 nm in diameter) have a higher uptake efficiency compared to larger EVs (∼300 nm in diameter). In addition, after being internalized, small EVs could enter endoplasmic reticulum and Golgi but not the largest ones. Our platform significantly shortens sample preparation, allows the profiling of the different EVs based on their size, and facilitates the understanding of extracellular communication. Thus, it leads to early diagnostics and the development of novel therapeutics for neurological diseases.

Smartphone-Based Point-of-Care Microfluidic Platform Fabricated with a ZnO Nanorod Template for Colorimetric Virus Detection.

Viruses pose serious infectious disease threats to humans and animals. To significantly decrease the mortality and morbidity caused by virus infections, there is an urgent need of sensitive and rapid point-of-care platforms for virus detection, especially in low-resource settings. Herein, we developed a smartphone-based point-of-care platform for highly sensitive and selective detection of the avian influenza virus based on nanomaterial-enabled colorimetric detection. The 3D nanostructures, which serve as a scaffold for antibody conjugation to capture the avian influenza virus, are made on PDMS herringbone structures with a ZnO nanorod template. After virus capture, the on-chip gold nanoparticle-based colorimetric reaction allows virus detection by naked eyes with a detection limit of 2.7 × 104 EID50/mL, which is one order of magnitude better than that of conventional fluorescence-based ELISA. Furthermore, a smartphone imaging system with data processing capability further improves the detection limit, reaching down to 8 × 103 EID50/mL. The entire virus capture and detection process can be completed in 1.5 h. We envision that this point-of-care microfluidic system integrated with smartphone imaging and colorimetric detection would provide a fast, cheap, sensitive, and user-friendly platform for virus detection in low-resource settings.

A Portable Device Integrated with Aligned Carbon Nanotubes for Sensitive Virus Capture and Detection.

Point-of-care virus diagnosis is highly desirable in worldwide infectious disease control. Here we report a hand-held device for capturing viruses by applying physical size based exclusion inside a point-of-care device integrated with vertically aligned carbon nanotube (VACNT) nanostructures to achieve label-free and high throughput virus capture. The microfluidic device is constructed from a VACNT channel wall synthesized bottom-up via chemical vapor deposition (CVD). The VACNT has ~117 nm average gap size and ~97& porosity. By bonding with a polydimethylsiloxane (PDMS) cover sealing the top, the aqueous sample containing virus particles filter through the VACNT channel wall under negative pressure applied at the outlet end. We have demonstrated that the device is capable of filtering 50 µL of PBS containing ~6.3 × 104 counts of lentivirus particles in 10 minutes with 97& of capture efficiency, quantified by the cell infectious titration technique.

Light-Emitting Transition Metal Dichalcogenide Monolayers under Cellular Digestion.

2D materials cover a wide spectrum of electronic properties. Their applications are extended from electronic, optical, and chemical to biological. In terms of biomedical uses of 2D materials, the interactions between living cells and 2D materials are of paramount importance. However, biointerfacial studies are still in their infancy. This work studies how living organisms interact with transition metal dichalcogenide monolayers. For the first time, cellular digestion of tungsten disulfide (WS2 ) monolayers is observed. After digestion, cells intake WS2 and become fluorescent. In addition, these light-emitting cells are not only viable, but also able to pass fluorescent signals to their progeny cells after cell division. By combining synthesis of 2D materials and a cell culturing technique, a procedure for monitoring the interactions between WS2 monolayers and cells is developed. These observations open up new avenues for developing novel cellular labeling and imaging approaches, thus triggering further studies on interactions between 2D materials and living organisms.

Emergency assistance in laboratory development for control of avian influenza in Laos and Cambodia.

Avian influenza (AI) diagnostic laboratories in Laos and Cambodia have been established recently to conduct AI surveillance and diagnostic tests to coordinate regional efforts for the control of highly pathogenic avian influenza (HPAI) in South East Asia. Two laboratories have been provisioned with equipment, supplies, and reagents for routine diagnostic testing. Laboratory staff has received training to conduct serologic and virologic tests for isolation and identification of AI virus. Development of a disease reporting system and an AI surveillance program is in progress in Laos and Cambodia. There are plans to further upgrade laboratory facilities and to provide more comprehensive and advanced molecular diagnostic tests for control of HPAI in Laos and Cambodia. These two countries are on the frontline in the battle to fight HPAI H5N1 virus and to prevent it from spreading to other regions and mutating to a major human pathogen.

Whole genome alignment based one-step real-time RT-PCR for universal detection of avian orthoreoviruses of chicken, pheasant and turkey origins.

Newly emerging avian orthoreovirus (ARV) variants have been continuously detected in Pennsylvania poultry since 2011. In this paper, we report our recent diagnostic assay development of one-step real-time RT-PCR (rRT-PCR) for the rapid and universal detection of all ARVs or reference strains of chicken, pheasant and turkey origins and six σC genotypes of the newly emerging field ARV variants in Pennsylvania (PA) poultry. Primers and probes for the rRT-PCR were designed from the conserved region of the M1 genome segment 5' end based on the whole-genome alignment of various ARV strains, including six field variants or novel strains obtained in PA poultry. The detection limit of the newly developed rRT-PCR for ARV was as low as 10 copies/reaction of viral RNA, and 10(0.50)-10(0.88) tissue culture infectious dose (TCID50)/100 µL of viruses. This new rRT-PCR detected all six σC genotypes from the 66 ARV field variant strains and reference strains tested in this study. There were no cross-reactions with other avian viruses. Reproducibility of the assay was confirmed by intra- and inter-assay tests with variability from 0.12% to 2.19%. Sensitivity and specificity of this new rRT-PCR for ARV were achieved at 100% and 88%, respectively, in comparison with virus isolation as the "gold standard" in testing poultry tissue specimen.