TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Deciphering the antiviral nature of endophytic Bacillus spp. against groundnut bud necrosis virus in cowpea and tomato.

Tomato, the important vegetable crop, is severely affected by Orthotospovirus arachinecrosis which impacts heavy economic losses. The application of insecticide to manage viral diseases is not an environmentally safe approach. In view of these issues, we investigated the antiviral efficacy of 21 bacterial endophytes against GBNV in local lesion host (Cowpea-VBN3). Based on the reduction in lesion number and virus titer as estimated through both DAC ELISA and qPCR in cowpea, the bacterial endophytes viz., Bacillus licheniformis Soya1, Bacillus tequilensis NBL6, and Bacillus velezensis VB7 were selected and further tested in tomato. The study revealed the well-defined antiviral efficacy of these endophytes against GBNV. The percentage of disease incidence ranged from 16 to 24% in endophyte-treated tomato plants compared with untreated plants (88%). In addition, symptom severity was reduced, and the application of endophytes also in promotion of the growth compared with untreated control. DAC ELISA revealed that the tomato plants treated with bacterial endophytes challenged with GBNV showed reduction in the virus titer (0.26-0.39 @ OD 405 nm) at different days of interval after inoculation (0, 5, and 10 days) compared with untreated control (3.475 @ OD 405 nm). Additionally, reduction in the viral copy number in bacterial endophyte-treated plants was evident by real-time PCR. Furthermore, tomato plants bacterized with endophytes depicted significant correlation and reduction in viral load and disease incidence as revealed by the principal-component biplot analysis. Thus, the application of bacterial endophytes has a potential role in reducing the disease incidence, severity, and titer value of GBNV, which will be the promising management approach in future to mitigate the virus infection in tomato plants.

Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line.

Antiviral vaccines for pig diseases are essential to prevent epidemic outbreaks. However, their production is often hindered by inefficient manufacturing processes that yield lower quantities of the vaccine. To accelerate the progress of various areas of bioproduction, we have considered the necessity of enhancing viral replication efficiency by optimizing ST (swine testicular) cell lines that are commonly utilized in virus manufacturing. CRISPR/Cas9 gene-editing technology were utilized to create IRF3 or IRF7 knockout cell lines that facilitate high-titer viral stock production. Compared to the parental cell lines, the ST IRF3/7 KO cell line displayed a compromised antiviral response to a panel of viruses (Porcine epidemic diarrhea virus, Senecavirus A, Parainfluenza virus 5, and Getah virus), as evidenced by decreased expression of interferon and certain antiviral factors. The inhibition of these responses led to heightened viral replication and increased cytopathic effects, ultimately promoting apoptosis. As a result, the development of these cell lines offers a more efficient approach for biopharmaceutical companies to boost their virus production and reduce associated costs.

Impact of an autophagy-inducing peptide on immunogenicity and protection efficacy of an adenovirus-vectored SARS-CoV-2 vaccine.

Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.

Characterization of the camel pox virus strain used in producing camel pox virus vaccine.

Background: The camel pox virus (CMLV) is a widespread infectious viral disease of camels. It is necessary to conduct research on new strains for the development of vaccines. Aim: The research aims to characterize a novel strain isolated from the CMLV used to produce a CMLV vaccine. Methods: The objects of the study were the "M-0001" strain isolated from a sample of animals infected with the CMLV during the epidemic. The cultural and reproductive properties of the virus isolate were studied using primary cell lines from primary trypsinized lamb kidney and testicular cell cultures (LK and LT). Other samples included kidney cell lines from transplanted sheep as well as a kidney cell line from transplanted cattle, Vero (transplanted green monkey kidney cell line), and calf trachea. The strain was polymerase chain reaction (PCR)-tested and sequenced for characterization purposes. Results: The PCR results show that the study sample is species specific and corresponds to the CMLV by the size of the cumulative amplifications, which is 241 bp. Given the maximum percentage of a sequence match analyzed by the BLAST algorithm based on the international database and the results of phylogenetic analysis, the M0001 sample was determined to belong to the CMLV (gene bank inventory number KP768318.1). Conclusion: The sample "M0001" is located on the same branch with a representative from CMLV. Among the cell cultures tested, the LK and LT cell lines were the most sensitive to the isolated CMLV isolate. Reproducing the virus in these cell cultures remains stable even after 15 consecutive passes. The cytopathic effect of the virus was less pronounced and low in transplanted cell lines, and the cytopathic effect was no longer apparent in the third passage. A genome alignment of the virus has identified potentially conserved sites, and analysis of loci in different virus types revealed one maximally conserved locus. An epizootic strain of the camelina virus "M-0001" candidate to produce vaccines for the camels was obtained. An experimental vaccine sample based on an isolated and charred camellia virus will be created in the future.

Differential Seasonal Prevalence of Yellowing Viruses Infecting Melon Crops in Southern California and Arizona Determined by Multiplex RT-PCR and RT-qPCR.

Viruses transmitted by the whitefly (Bemisia tabaci) are an increasing threat to cucurbit production in the southwestern United States and many other cucurbit production regions of the world. The crinivirus cucurbit yellow stunting disorder virus (CYSDV) has severely impacted melon production in California and Arizona since its 2006 introduction to the region. Within the past few years, another crinivirus, cucurbit chlorotic yellows virus (CCYV), and the whitefly-transmitted ipomovirus squash vein yellowing virus (SqVYV) were found infecting melon plants in California's Imperial Valley. CYSDV, CCYV, and an aphid-transmitted polerovirus, cucurbit aphid-borne yellows virus (CABYV), occur together in the region and produce identical yellowing symptoms on cucurbit plants. Mixed infections of these four viruses in the Sonoran Desert and other regions pose challenges for disease management and efforts to develop resistant varieties. A multiplex single-step RT-PCR method was developed that differentiates among these viruses, and this was used to determine the prevalence and distribution of the viruses in melon samples from fields in the Sonoran Desert melon production region of California and Arizona during the spring and fall melon seasons from 2019 through 2021. TaqMan probes were developed, optimized, and applied in a single-step multiplex RT-qPCR to quantify titers of these four viruses in plant samples, which frequently carry mixed infections. Results of the multiplex RT-PCR analysis demonstrated that CYSDV is the predominant virus during the fall, whereas CCYV was by far the most prevalent virus during the spring each year. Multiplex RT-qPCR was used to evaluate differential accumulation and spatiotemporal distribution of viruses within plants and suggested differences in competitive accumulation of CCYV and CYSDV within melon. This study provides the first official report of SqVYV in Arizona and offers an efficient method for virus detection and quantification for breeding and disease management in areas impacted by cucurbit yellowing viruses.

Machine-learning-based automated quantification machine for virus plaque assay counting.

The plaque assay is a standard quantification system in virology for verifying infectious particles. One of the complex steps of plaque assay is the counting of the number of viral plaques in multiwell plates to study and evaluate viruses. Manual counting plaques are time-consuming and subjective. There is a need to reduce the workload in plaque counting and for a machine to read virus plaque assay; thus, herein, we developed a machine-learning (ML)-based automated quantification machine for viral plaque counting. The machine consists of two major systems: hardware for image acquisition and ML-based software for image viral plaque counting. The hardware is relatively simple to set up, affordable, portable, and automatically acquires a single image or multiple images from a multiwell plate for users. For a 96-well plate, the machine could capture and display all images in less than 1 min. The software is implemented by K-mean clustering using ML and unsupervised learning algorithms to help users and reduce the number of setup parameters for counting and is evaluated using 96-well plates of dengue virus. Bland-Altman analysis indicates that more than 95% of the measurement error is in the upper and lower boundaries [±2 standard deviation]. Also, gage repeatability and reproducibility analysis showed that the machine is capable of applications. Moreover, the average correct measurements by the machine are 85.8%. The ML-based automated quantification machine effectively quantifies the number of viral plaques.

Comparison among plaque assay, tissue culture infectious dose (TCID50) and real-time RT-PCR for SARS-CoV-2 variants quantification.

Background and Objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissibility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine development. Materials and Methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID50) and real-time RT-PCR. Results: Plaque assay showed viral titers between 0.15 ± 0.01×107 and 1.95 ± 0.09×107 PFU/mL while viral titer by TCID50 assay was between 0.71 ± 0.01×106 to 4.94 ± 0.80×106 TCID50/mL for the five SARS-CoV-2 isolates. The PFU/mL titer obtained by plaque and the calculated from TCID50 assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×108 to 3.38 ± 0.04×108 RNA copies/µL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu. Conclusion: TCID50 assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data demonstrated a correlation among PFU/mL and E gene RNA copies/µL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes depending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.

Automated, label-free TCID50 assay to determine the infectious titer of virus-based therapeutics.

A robust and precise infectivity assay is a prerequisite for the development and market supply of virus-based biologics. Like other cell-based assays, traditional infectivity assays suffer from high variability and require extensive hands-on time. Therefore, a faster and more robust method to measure infectivity is needed to fulfill the requirements of a higher sample throughput and speed in drug development. We developed a label-free tissue culture infectious dose 50 (TCID50) assay using automated image analysis that determines the cell confluence to discriminate between cytopathic effect-positive and -negative wells. In addition, we implemented semi-automated bench-top pipetting robots for the required pipetting steps to further shorten the hands-on time of the assay. The automated image analysis categorized >99 % of the wells similar as operators did via visual evaluation and there was a close correlation between the titers that were determined by using either the automated image analysis or visual evaluation (r² = 0.99). Thus, here we present a label-free TCID50 method with a stable automated image analysis that is ∼3.6x faster and more standardized compared to the classical TCID50 assay.

Construction of Genomic and Sub-Genomic Dengue Infectious Replicons.

Dengue replicons are powerful tools used in studying virus biology as well as in high-throughput screening of drug candidates. Replicon constructs are developed as genomic (consists of all the viral protein genes) or sub-genomic (consists of only nonstructural protein genes) and are used to study various aspects of the virus life cycle such as genome replication and virus assembly. In addition, a replicon usually includes a reporter gene used in monitoring virus replication. In this chapter, we provide methods to develop both genomic and sub-genomic dengue replicons with a luciferase reporter and describe different assays to utilize these systems.

Effective Inhibition of Newly Emerged A/H7N9 Virus with Oligonucleotides Targeted to Conserved Regions of the Virus Genome.

Newly emerged highly pathogenic A/H7N9 viruses with pandemic potential are effectively transmitted from birds to humans and require the development of novel antiviral drugs. For the first time, we studied the in vitro and in vivo antiviral activity against A/H7N9 of oligodeoxyribonucleotides (ODNs), which were delivered into the cells in the proposed TiO2-based nanocomposites (TiO2∼ODN). The highest inhibition of A/H7N9 in vitro (∼400-fold) and efficient, sequence-specific, and dose-dependent protection (up to 100%) of A/H7N9-infected mice was revealed when ODN was targeted to the conserved terminal 3'-noncoding region of viral (-)RNA. After the treatment with ODN, the virus titer values in the lungs of mice decreased by several orders of magnitude. The TiO2∼ODN nanocomposite did not show toxicity in mice under the treatment conditions. The proposed approach for effective inhibition of the A/H7N9 can be tested against other viruses, for example, new emerging influenza viruses and coronaviruses with pandemic potential.

Infectivity assay for detection of SARS-CoV-2 in samples from patients with COVID-19.

Since the coronavirus disease 2019 (COVID-19) outbreak, laboratory diagnosis has mainly been conducted using reverse-transcription polymerase chain reaction (RT-PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there have been no quantitative investigations conducted for infectious SARS-CoV-2 in clinical samples. Therefore, in the present study, a rapid and simple focus-forming assay using the peroxidase-antiperoxidase technique was developed to quantify infectious SARS-CoV-2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID-19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real-time RT-PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = -0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID-19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others.

MSCs loaded with oncolytic reovirus: migration and in vivo virus delivery potential for evaluating anti-cancer effect in tumor-bearing C57BL/6 mice.

BACKGROUND AND AIMS: Several oncolytic viruses applications have been approved in the clinic or in different phases of clinical trials. However, these methods have some rudimentary problems. Therefore, to enhance the delivery and quality of treatment, considering the advantage of cell carrier-based methods such as Mesenchymal Stem Cells (MSC) have been proposed. This study was designed to evaluate the performance and quality of cancer treatment based on MSCs loaded by oncolytic reovirus in the cancerous C57BL/6 mouse model. Also, we evaluated MSCs migration potency in vitro and in vivo following the oncolytic reovirus infection. METHODS: C57BL/6 mice were inoculated with TC-1 cell lines and tumors were established in the right flank. Mice were systemically treated with reovirus, MSCs-loaded with reovirus, MSCs, and PBS as a control in separated groups. Effects of infected AD-MSCs with reovirus on tumor growth and penetration in the tumor site were monitored. All groups of mice were monitored for two months in order to therapeutic and anticancer potential. After treatments, tumor size alteration and apoptosis rate, as well as cytokine release pattern was assessed. RESULTS: The results of the current study indicated that the effect of reovirus infection on AD-MSCs is not devastating the migration capacity especially in MOI 1 and 5 while intact cells remain. On the other hand, MSCs play an efficient role as a carrier to deliver oncolytic virus into the tumor site in comparison with systemic administration of reovirus alone. Apoptosis intensity relies on viral titration and passing time. Followed by systemic administration, treatment with oncolytic reovirus-infected AD-MSCs and MSCs alone had shown significant inhibition in tumor growth. Also, treatment by reovirus causes an increase in IFN-γ secretion. CONCLUSION: The results of in vitro and in vivo study confirmed the tumor-homing properties of infected AD-MSCs and the significant antitumor activity of this platform. Hence, our results showed that the cell carrier strategy using oncolytic reovirus-loaded AD-MSCs enhanced virus delivery, infiltration, and antitumor activity can be effectively applied in most cancers.

Pronounced therapeutic potential of oligonucleotides fixed on inorganic nanoparticles against highly pathogenic H5N1 influenza A virus in vivo.

This study describes the effective attack of oligonucleotides on the viral genome of highly pathogenic H5N1 influenza A virus (IAV) in vivo using for the first time the new delivery system consisting of biocompatible low-toxic titanium dioxide nanoparticles and immobilized polylysine-containing oligonucleotides with the native (ODN) and partially modified (ODNm) internucleotide bonds. Intraperitoneal injection of the TiO2•PL-ODN nanocomposite provided 65-70% survival of mice, while intraperitoneal or oral administration of TiO2•PL-ODNm was somewhat more efficient (~80% survival). The virus titer in the lung was reduced by two-three orders of magnitude. The nanocomposites are nontoxic to mice under the used conditions. TiO2 nanoparticles, unbound ODN, and the nanocomposite bearing the random oligonucleotide showed an insignificant protective effect, which indicates the ability of targeted oligonucleotides delivered in mice in the nanocomposites to site-specifically interact with complementary RNAs. The protection of oligonucleotides in nanocomposites by TiO2 nanoparticles and partial modification of the internucleotide bonds provides a continued presence of oligonucleotides in the body for the effective and specific action on the viral RNA. The proposed oligonucleotide delivery system can claim not only to effectively inhibit IAV genes but also to turn off other genes responsible for diseases caused by nucleic acids.

Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry.

Rapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus-based products. Here, we report the development of a high-throughput single-particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory-built nano-flow cytometer (nFCM) was employed to simultaneously detect the side-scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production.

Novel Sources of Turnip Yellows Virus Resistance in Brassica and Impacts of Temperature on Their Durability.

Turnip yellows virus (TuYV; family Solemoviridae, genus Polerovirus) is the most widespread and economically damaging virus of canola (Brassica napus L.) production in Australia. However, no Australian commercial seed companies market TuYV-resistant canola cultivars, and little information is available on the susceptibility of those available. To identify potential sources of TuYV resistance, 100 B. napus accessions from the ERANET ASSYST diversity set were screened in the field and five of these were selected for further phenotyping via aphid inoculation. Furthermore, 43 Australian canola cultivars, six B. napus genotypes with previously reported resistance, and 33 B. oleracea and B. rapa cultivars were phenotyped. All Australian cultivars were susceptible except for 'ATR Stingray'. Stronger resistance to systemic TuYV infection (IR) was identified in diversity set accessions 'Liraspa-A', 'SWU Chinese 3', and 'SWU Chinese 5'. As indicated by lower relative enzyme-linked immunosorbent assay absorbance values (R-E405) in infected plants, resistance to TuYV accumulation (AR) often accompanied IR. Moderate IR was identified in four B. oleracea cultivars and one B. rapa cultivar. Very strong AR was identified in four B. oleracea cultivars and AR of some degree was common across many cultivars of this species tested. The impact of temperature during the inoculation access period or post-inoculation incubation on the resistance identified was examined. Infection rates were significantly higher in resistant B. napus genotypes when inoculated at 16°C than at 26°C, suggesting an increase in aphid transmission efficiency. IR in B. napus genotypes was strong when incubated at 16°C, but weakened at elevated temperatures with almost total breakdown in most genotypes at 30°C. However, infected plants of B. napus and B. oleracea genotypes with AR maintained lower R-E405 values than susceptible controls at all temperatures tested. Novel sources of resistance identified in this study offer potential as breeding material in Australia and abroad.

Grassy Weeds and Corn as Potential Sources of Barley yellow dwarf virus Spread Into Winter Wheat.

Barley yellow dwarf virus (BYDV) is an important vector-borne pathogen of cereals. Although many species of grasses are known to host BYDV, knowledge of their role in virus spread in regional agroecosystems remains limited. Between 2012 and 2016, Idaho winter wheat production was affected by BYDV. BYDV-PAV and the bird cherry-oat aphid (BCOA) (Rhopalosiphum padi L.) vector were commonly present in the affected areas. A series of greenhouse bioassays were performed to examine whether two types of corn (Zea mays L.), dent and sweet, and three commonly found grassy weeds, downy brome (Bromus tectorum L.), green foxtail (Setaria viridis L.), and foxtail barley (Hordeum jubatum L.), can be inoculated with BYDV (species BYDV-PAV) by BCOA and also act as sources of the virus in winter wheat. BCOA successfully transmitted BYDV-PAV to both corn types and all weed species. Virus titers differed between the weed species (P = 0.032) and between corn types (P = 0.001). In transmission bioassays, aphids were able to survive on these host plants during the 5-day acquisition access period and later successfully transmitted BYDV-PAV to winter wheat (var. SY Ovation). Transmission success was positively correlated with the virus titer of the source plant (P < 0.001) and influenced by weed species (P = 0.028) but not corn type. Overall, the results of our inoculation and transmission assays showed that the examined weed species and corn types can be inoculated with BYDV-PAV by BCOA and subsequently act as sources of infections in winter wheat.

Magnetic bead purification of enveloped alphavirus and flavivirus.

Virus production is an essential part of virology. The quality of virus preparation, in terms of purification and yield, can affect the outcome of experiments. Purified virus is critical for assays, as contamination from cell culture supernatants may affect results. Purified virus is also needed for vaccine preparations. The traditional process of gradient purification of virus is time consuming, with multiple steps spanning over days, and produces significant amounts of biohazard waste. Developing a more efficient alternative that provides a purified virus product is highly desirable. In this study, we performed magnetic bead purification of Zika virus (ZIKV) and Mayaro virus (MAYV) with the Mag4C LV kit from OZ Biosciences. Currently, this kit is only indicated for use with lentiviruses and retroviruses. We found that we were able to successfully concentrate and purify both ZIKV and MAYV using the kit. Therefore, we concluded that the Mag4C LV kit provides a quick and simple alternative to traditional virus purification methods.•Our protocol is customized by using an alphavirus (MAYV) and flavivirus (ZIKV). This method has not been previously used for these viruses.

A Comparative Analysis of Methods for Titering Reovirus.

BACKGROUND: A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers. OBJECTIVE: Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT50) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID50) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology. METHODS: Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI50) values of reovirus infected L929 cells with TCID50 titers were analyzed by RTCA, the CI50-TCID50 standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT50-TCID50 standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID50 method. RESULTS: L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL. CONCLUSION: The CIT50-TCID50 standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID50 method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID50 method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.

A Well-Defined H9N2 Avian Influenza Virus Genotype with High Adaption in Mammals was Prevalent in Chinese Poultry Between 2016 to 2019.

H9N2 subtype avian influenza virus (AIV) is widely prevalent in poultry, and the virus is becoming adaptive to mammals, which poses pandemic importance. Here, BALB/c mice were employed as a model to evaluate the adaption in mammals of 21 field H9N2 viruses isolated from avian species between 2016 to 2019 in China. The replication capacity of the viruses was evaluated in the lungs of mice. The pathogenicity of the viruses were compared by weight loss and lung lesions from infected mice. The whole genomic sequences of the viruses were further characterized to define the associated phenotypes of the H9N2 viruses in vitro and in vivo. The results showed that most viruses could replicate well and cause lesions in the mouse lungs. The propagation capacity in MDCK cells and damage to respiratory tissues of the infected mice corresponded to relative viral titers in the mouse lungs. Further genome analysis showed that all of the H9N2 viruses belonged to the same genotype, G57, and contained a couple of amino acid substitutions or deletions that have been demonstrated as avian-human markers. Additionally, nine amino acids residues in seven viral proteins were found to be correlated with the replication phenotypes of the H9N2 viruses in mammals. The study demonstrated that a well-defined H9N2 AIV genotype with high adaption in mammals was prevalent in China in recent years. Further investigations on the role of the identified residues and continuous surveillance of newly identified mutations associated with host adaption should be strengthened to prevent any devastating human influenza pandemics.