Differences In Viral Titers Research Articles

Nicotinic Acetylcholine Receptor Alpha6 Contributes to Antiviral Immunity via IMD Pathway in Drosophila melanogaster.

Currently, insecticides that target nicotinic acetylcholine receptors (nAChR) are widely used. Studies on the sublethal effects of insecticides have found that they can affect the amount of virus in insects. The mechanism by which insecticides affect insect virus load remain unclear. Here, we show that nAChR targeting insecticide can affect viral replication through the immune deficiency (IMD) pathway. We demonstrate that a low dose of spinosad (6.8 ng/mL), acting as an antagonist to Drosophila melanogaster nicotinic acetylcholine receptor α6 (Dα6), significantly elevates Drosophila melanogaster sigmavirus (DMelSV) virus titers in adults of Drosophila melanogaster. Conversely, a high dose of spinosad (50 ng/mL), acting as an agonist to Dα6, substantially decreases viral load. This bidirectional regulation of virus levels is absent in Dα6-knockout flies, signifying the specificity of spinosad's action through Dα6. Furthermore, the knockdown of Dα6 results in decreased expression of genes in the IMD pathway, including dredd, imd, relish, and downstream antimicrobial peptide genes AttA and AttB, indicating a reduced innate immune response. Subsequent investigations reveal no significant difference in viral titers between relish mutant flies and Dα6-relish double mutants, suggesting that the IMD pathway's role in antiviral defense is dependent on Dα6. Collectively, our findings shed light on the intricate interplay between nAChR signaling and the IMD pathway in mediating antiviral immunity, highlighting the potential for nAChR-targeting compounds to inadvertently influence viral dynamics in insect hosts. This knowledge may inform the development of integrated pest management strategies that consider the broader ecological impact of insecticide use.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.

A preliminary survey reveals that common viruses are found at low titers in a wild population of honey bees (Apis mellifera).

A major threat to honey bee (Apis mellifera Linnaeus, Hymenoptera: Apidae) health continues to be parasitism by the mite Varroa destructor, which has been linked to high colony losses worldwide. Besides feeding on developing and adult bees, Varroa is also a prolific vector of honey bee-associated viruses. Because they live in unmanaged conditions, wild honey bee colonies are not treated against Varroa, which has enabled the natural selection of more mite-tolerant bees. To date, few studies have explored the prevalence of viruses in unmanaged colonies. The Welder Wildlife Refuge (WWR) in Texas is a unique site to study the viral landscape of unmanaged honey bees in the United States. The goals of this study were to identify and quantify viruses in wild colonies at the WWR, to examine changes in the prevalence of viruses in these colonies over time, and to compare the presence and titers of viruses between wild colonies at the WWR and those from the nearest managed apiary. We collected bees from colonies at the WWR in 2013, 2016, and 2021, and analyzed selected viruses for their presence and titers via quantitative polymerase chain reaction. In 2021, we also sampled bees from the nearest managed apiary for comparison. We found low average virus titers in all wild colonies sampled, and no difference in virus titers between colonies at the WWR and those from the managed apiary. Our study indicates that virus titers in wild colonies at the WWR are similar to those found in nearby colonies, and that these titers fluctuate over time.

Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example

After entry of a quarantine/regulated pathogen, infected plants shall be destroyed, and the cultivated area (e.g., greenhouse) shall be disinfected. Therefore, the selection of an effective disinfectant plays an important role. With the availability of different methods for virus quantification, we investigated the application of quantitative ELISA (qELISA), RT-qPCR (reverse transcription-quantitative polymerase chain reaction), and bioassays for the quantification of disinfectant efficacy. Therefore, we estimated the titer reduction in tomato brown rugose fruit virus (ToBRFV), a regulated pathogen, in plant sap and on germ carriers after treatment with MENNO Florades 4% for 16 h. The virus load before and after the treatment was measured with the mentioned methods. The RT-qPCR and qELISA methods showed very low efficacy in the presence of the disinfectant. Although bioassays are time-consuming, need purified particles for establishing the quantification models, and are less sensitive than RT-qPCR, they were able to quantify the differences in virus titer in the presence/absence of disinfectant. Interestingly, the bioassays reached at least the lower limit sensitivity of a qELISA. By being less sensitive to the presence of the disinfectant, bioassays proved to be the only technique for the determination of the disinfectant efficacy against ToBRFV on different germ carriers as well as on virus-infected plant sap.

Dynamic changes in virus-induced volatiles in cotton modulate the orientation and oviposition behavior of the whitefly Bemisia tabaci.

Manipulation of insect vector behavior by virus-induced plant volatiles is well known. But how the viral disease progression alters the plant volatiles and its effect on vector behavior remains less explored. Our studies tracked changes in volatile profile in progressive infection stages of cotton leaf curl virus (CLCuV) infected plants and their effect on B. tabaci behavior. Significant differences in virus titers were noticed between progressive infection stages showing distinct symptoms. Whiteflies initially settled on CLCuV infected plants, but their preference was shifted to healthy plants over time. GC-MS analysis revealed subtle quantitative/qualitative changes in volatile organic compounds (VOCs) between the healthy and selected CLCuV infection stages. VOCs such as hexanal, (E)-2-hexen-1-ol, (+)-α-pinene, (−)-β-pinene, (Z)-3-hexen-1-ol, (+)-sylvestrene, and (1S,2E,6E, 10R)-3,7,11,11-tetramethylbicycloundeca-2,6-diene (Bicyclogermacrene) were associated with the infection stage showing upward curling of leaves; (E)-2-hexen-1-ol, β-myrcene, β-ocimene, and copaene were associated with the infection stage showing downward curling. Validation studies with eight synthetic VOCs indicated that γ-terpinene elicited attraction to B. tabaci (Olfactometric Preference Index (OPI) = 1.65), while β-ocimene exhibited strong repellence (OPI = 0.64) and oviposition reduction (66.01%–92.55%). Our studies have demonstrated that progression of CLCuV disease in cotton was associated with dynamic changes in volatile profile which influences the behavioural responses of whitefly, B.tabaci. Results have shown that VOCs such as (+)-α-pinene, (−)-β-pinene γ-Terpinene, α-guaiene; 4- hydroxy- 4 methyl-2- pentanone and β-ocimene emitted from Begomovirus infected plants could be the driving force for early attraction and later repellence/oviposition deterrence of B. tabaci on virus-infected plants. The findings of this study offer scope for the management of whitefly, B. tabaci through semiochemicals.

Patient-derived SARS-CoV-2 mutations impact viral replication dynamics and infectivity in vitro and with clinical implications in vivo

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally with more than 33 million patients diagnosed, taking more than a million lives. Abundant mutations were observed but the functional consequences of these mutations are largely unknown. We report the mutation spectrum, replication dynamics, and infectivity of 11 patient-derived viral isolates in diverse cell lines, including the human lung cancer cell line Calu-3. We observed 46 mutations, including 9 different mutations in the spike gene. Importantly, these viral isolates show significant and consistent variations in replication dynamics and infectivity in tested cell lines, up to a 1500-fold difference in viral titers at 24 h after infecting Calu-3 cells. Moreover, we show that the variations in viral titers among viral isolates are positively correlated with blood clotting function but inversely correlated with the amount of red blood cell and hemoglobin in patients. Therefore, we provide direct evidence that naturally occurring mutations in SARS-CoV-2 can substantially change its replication dynamics and infectivity in diverse human cell lines, with clinical implications in vivo.

Induction of cross-reactive antibodies in mice immunized with conserved influenza A virus neuraminidase-derived linear B-cell epitopes

Introduction. Influenza is a socially significant infection that causes profound damage to populational health and national economy annually. Preventive vaccination is the most effective means to manage influenza and its complications. Diverse influenza vaccines exist, but their common drawback is the narrow specificity, the need for annual renewal of the strain composition, not always satisfactory immunogenicity and effectiveness. In this regard, close attention is paid to developing universal influenza vaccines aimed to induce cross-reactive immune-related cues against most conserved parts of viral proteins. Antibodies against neuraminidase (NA) are able to provide heterosubtypic protection, which is important due to potential threat posed by influenza viruses differed in hemagglutinin and neuraminidase sequence in comparison to currently circulating viruses. The present study is aimed to search for new and analysis of earlier predicted NA linear B-cell epitopes conserved among all influenza A virus subtypes. Results. Eight conserved linear B-cell epitopes were identified around the active site of neuraminidase, three of which (MNPNQKIITIGS, ILRTQESEC, and DNWKGSNRP) were synthesized de novo, conjugated with bovine serum albumin to be next used for mouse immunization. IgG antibodies were detected by ELISA in the sera of immunized mice. Antibodies were found to specifically bind to various influenza A viruses containing NA subtypes N1, N2, N3, and N9. Immunization with NA peptides did not protect mice from substantial body weight loss after infection with lethal H1N1 influenza virus. However, all immunized mice survived during the observation period, whereas in the control group the survival rate was as low as 28.6%. Analyzing lung viral load in the mice infected with the H1N1 virus revealed no differences in virus titers on either day 4 or 8 post-infection. Nevertheless, the protective effect lacked after the mice were challenged with lethal H7N9 influenza virus: Mortality rate, body weight loss, and lung virus titers were comparable in immunized and control mice. Conclusions. The data obtained evidenced about serum cross-reactive anti-NA antibodies induced by immunization with NA peptides, as well as protective efficacy against infection caused by H1N1 virus, but not H7N9 virus. Such results hold promise and indicate that NA linear B-cell epitopes can be used for designing epitope-directed influenza vaccines, but a deeper and more comprehensive study on the specificity of conserved NA epitopes, as well as optimization of immunization schemes for achieving higher protective efficacy are required.

Relationships of virus titers and transmission rates among sympatric and allopatric virus isolates and thrips vectors support local adaptation

Plant viruses rely on insect vectors for transmission among plant hosts, but many of the specifics of virus-vector interactions are not fully understood. Thrips tabaci, which transmits Tomato spotted wilt virus (TSWV) in a persistent and propagative manner, varies greatly in its ability to transmit different isolates of TSWV. Similarly, TSWV isolates are transmitted at different efficiencies by different populations of T. tabaci. This study characterizes differences in virus titers in the vector among TSWV isolate-T. tabaci isoline pairings in relation to differences in transmission rates, and demonstrates that although transmission rates were higher for sympatric than allopatric TSWV isolate-T. tabaci isoline pairings, virus titers in the thrips vector were significantly lower in the sympatric pairings. Results further demonstrate that TSWV titers in the vector were unrelated to virus titers in the leaf tissue from which they acquired the virus and provide evidence for the importance of specific vector-virus interactions and local adaptation in determining transmission efficiency of TSWV by T. tabaci.

EPA and DHA can modulate cell death via inhibition of the Fas/tBid-mediated signaling pathway with ISKNV infection in grouper fin cell line (GF-1) cells

Polyunsaturated fatty acids (PUFAs) play important roles in organisms, including the structure and liquidity of cell membranes, anti-oxidation and anti-inflammation. Very little has been done in terms of the effect of PUFAs on cell death, especially on DNA virus. In this study, we demonstrated that the infectious spleen and kidney necrosis virus (ISKNV) can induce host cell death via the apoptotic cell death pathway, which correlated to modulation by PUFAs in grouper fin cell line (GF-1) cells. We screened the PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for the ability of different dosages to prevent cell death in GF-1 cells with ISKNV infection. In the results, each 10 μM of DHA and EPA treatment enhanced host cell viability up to 80% at day 5 post-infection. Then, in Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay, DHA- and EPA-treated groups reduced TUNEL positive signals 50% in GF-1 cells with ISKNV infection. Then, through studies of the mechanism of cell death, we found that ISKNV can induce both the Bax/caspase-3 and Fas/caspase-8/tBid death signaling pathways in GF-1 cells, especially at day 5 post-infection. Furthermore, we found that DHA and EPA treatment can either prevent caspase-3 activation on 17-kDa form cleavage or Bid cleaved (15-kDa form) for activation by caspase-8, apparently. On the other hand, the anti-apoptotic gene Bcl-2 was upregulated 0.3-fold and 0.15-fold at day 3 and day 5, respectively, compared to ISKNV-infected and DHA-treated cells; that this did not happen in the EPA-treated group showed that different PUFAs trigger different signals. Finally, ISKNV-infected GF-1 cells treated with either DHA or EPA showed a 5-fold difference in viral titer at day 5. Taken together, these results suggest that optimal PUFA treatment can affect cell death signaling through both the intrinsic and extrinsic death pathways, reducing viral expression and viral titer in GF-1 cells. This finding may provide insight in DNA virus infection and control.

Lack of influence by endosymbiont Wolbachia on virus titer in the common bed bug, Cimex lectularius

BackgroundThe common bed bug, Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius.MethodsWe used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia.ResultsThere was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia-positive and Wolbachia-cured groups.ConclusionsThese findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of Wolbachia, and our conclusions underscore the need for future research.

Accurate Quantification and Characterization of Adeno-Associated Viral Vectors.

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.

Characterization of Sex Differences in Ocular Herpes Simplex Virus 1 Infection and Herpes Stromal Keratitis Pathogenesis of Wild-Type and Herpesvirus Entry Mediator Knockout Mice.

Sex differences related to immune response and inflammation play a role in the susceptibility and pathogenesis of a variety of viral infections and disease (S. L. Klein, Bioessays 34:1050-1059, 2012, https://doi.org/10.1002/bies.201200099). Herpes simplex virus 1 (HSV-1) causes chronic inflammatory disease in the cornea, an immune-privileged tissue, resulting in irreversible damage and blindness in affected individuals (A. Rowe, A. St Leger, S. Jeon, D. K. Dhaliwal, et al., Prog Retin Eye Res 32:88-101, 2013, https://doi.org/10.1016/j.preteyeres.2012.08.002). Our research focuses on the role of herpesvirus entry mediator (HVEM) as an immune regulator during ocular HSV-1 infection. Mice lacking HVEM (HVEM knockout [KO] mice) exhibit lower levels of immune cell infiltrates and less severe ocular disease in the cornea than wild-type (WT) mice. As sex differences contribute to pathogenesis in many inflammatory diseases, we tested whether sex acts as a biological variable in the immune response to HSV-1 infection and herpes stromal keratitis (HSK) pathogenesis. Adult male and female WT and HVEM KO mice were inoculated with HSV-1 via corneal scarification and monitored daily for disease course. Viral titers were determined, and immune cell infiltrates were collected and analyzed. Our results indicated no significant differences in viral titers in tear film or affected tissues, in immune cell infiltration, or in clinical symptoms between males and females of either genotype. These results suggest that sex is not a significant biological variable in this experimental model and that male and female mice of the C57BL/6 background can be used similarly in studies of ocular HSV-1 pathogenesis.IMPORTANCE Sex hormones have come to be considered an important factor for the development of certain diseases only recently and as such should continue to be considered a biological variable. Ocular HSV-1, and the resulting HSK, is the leading cause of infectious blindness worldwide. We compared levels of ocular HSV-1 infection and pathogenesis in the two sexes and found no significance differences between male and female WT mice or HVEM KO mice.

Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality

BackgroundSuspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease.MethodsChanges to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation.ResultsCapsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A24-2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media.ConclusionThe selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.

Absolute Quantification of Tomato leaf curl New Delhi virus Spain strain, ToLCNDV-ES: Virus Accumulation in a Host-Specific Manner.

Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) has recently been introduced in western Mediterranean countries. Isolates in Spain constitute a new strain, denominated ToLCNDV-ES, that is causing losses in commercial zucchini and melon crops; however, it is also, although less often, detected in commercial tomato crops. We developed a tissue-print hybridization test to detect the two genomic components of the virus and a TaqMan quantitative polymerase chain reaction (qPCR) test to estimate the number of genome copies in plants. qPCR was approximately 104 to 106 times more sensitive than tissue-print hybridization to detect viral genomic DNA-A and DNA-B, respectively. It also detected the virus in more experimentally and naturally ToLCNDV-ES-infected zucchini squash and tomato plants. ToLCNDV-ES DNA-A titers were significantly lower in tomato than in zucchini plants, often falling below the detection limits in the hybridization test. In addition, the DNA-B accumulation was impaired in tomato when compared with zucchini. According to the data obtained in this study, the differences in viral titers of both plant species contribute to explain the dissimilarities in symptom expression, capability of detection, and transmission of the virus.

Gene expression profiling of immune responsive and fibrosis genes in hepatitis C virus infected patients.

Hepatitis C virus (HCV) is a dreadful viral disease, responsible for 170 million cases worldwide, of which most are from Asia and Africa and approximately 10 million people are from Pakistan. Currently, the pegylated interferon alpha (PEG-INF-α) has been approved as the standard of care in combination with ribavirin and Boceprevir/Telaprevir. Many studies regarding gene expression analysis of liver biopsy samples of patients with chronic HCV infection have been carried out previously. However, there are very few reports of expression analysis carried out using blood samples of HCV patients. Therefore, in this study, gene expression of human immune responsive genes (MMP-9, OAS1) and fibrogenic responsive gene (KRT19) was done in the peripheral blood mononuclear cells (PBMCs) of chronic HCV infected patients having differences in viral titers. Blood samples were collected from different hospitals in Pakistan. RNA was extracted and cDNA was synthesized according to the protocol prescribed by the Enzynomics™ M-MLV Reverse Transcriptase(®) Kit. The synthesized cDNA was amplified through polymerase chain reaction (PCR) using specific primers of immune responsive genes. The results were further evaluated using real-time PCR. There was a significant increase in the expression of the immune responsive genes (MMP-9, OAS1, CXCL6, CXCR3, ApoA1, and MYC) of HCV genotype 3a patients compared to controls. Similarly, the expression of the fibrosis genes was upregulated in HCV genotype 3a patients compared to controls. The information gained through this study is helpful to identify a noninvasive marker to determine liver fibrosis, and may also give useful information to understand HCV pathogenesis and develop better therapeutic regimens.

Early Host Responses of Seasonal and Pandemic Influenza A Viruses in Primary Well-Differentiated Human Lung Epithelial Cells

Replication, cell tropism and the magnitude of the host's antiviral immune response each contribute to the resulting pathogenicity of influenza A viruses (IAV) in humans. In contrast to seasonal IAV in human cases, the 2009 H1N1 pandemic IAV (H1N1pdm) shows a greater tropism for infection of the lung similar to H5N1. We hypothesized that host responses during infection of well-differentiated, primary human bronchial epithelial cells (wd-NHBE) may differ between seasonal (H1N1 A/BN/59/07) and H1N1pdm isolates from a fatal (A/KY/180/10) and nonfatal (A/KY/136/09) case. For each virus, the level of infectious virus and host response to infection (gene expression and apical/basal cytokine/chemokine profiles) were measured in wd-NHBE at 8, 24, 36, 48 and 72 hours post-infection (hpi). At 24 and 36 hpi, KY/180 showed a significant, ten-fold higher titer as compared to the other two isolates. Apical cytokine/chemokine levels of IL-6, IL-8 and GRO were similar in wd-NHBE cells infected by each of these viruses. At 24 and 36 hpi, NHBE cells had greater levels of pro-inflammatory cytokines including IFN-α, CCL2, TNF-α, and CCL5, when infected by pandemic viruses as compared with seasonal. Polarization of IL-6 in wd-NHBE cells was greatest at 36 hpi for all isolates. Differential polarized secretion was suggested for CCL5 across isolates. Despite differences in viral titer across isolates, no significant differences were observed in KY/180 and KY/136 gene expression intensity profiles. Microarray profiles of wd-NHBE cells diverged at 36 hpi with 1647 genes commonly shared by wd-NHBE cells infected by pandemic, but not seasonal isolates. Significant differences were observed in cytokine signaling, apoptosis, and cytoskeletal arrangement pathways. Our studies revealed differences in temporal dynamics and basal levels of cytokine/chemokine responses of wd-NHBE cells infected with each isolate; however, wd-NHBE cell gene intensity profiles were not significantly different between the two pandemic isolates suggesting post-transcriptional or later differences in viral-host interactions.

Ian Urquhart Fraser

<h3>Abstract</h3> Rapid testing methods can identify outbreaks and trigger preventive strategies for slowing the spread of SARS-CoV-2, the virus that causes COVID-19. The “gold-standard” detection method for SARS-CoV-2 is reverse...

Identification of swine influenza A virus and Stenotrophomonas maltophilia co-infection in Chinese pigs

BackgroundInfluenza virus virulence can be exacerbated by bacterial co-infections. Swine influenza virus (SIV) infection together with some bacteria is found to enhance pathogenicity.MethodsSIV-positive samples suspected of containing bacteria were used for bacterial isolation and identification. Antimicrobial susceptibility testing was performed by disc diffusion methods. To investigate the interaction of SIV and the bacteria in vitro, guinea pigs were used as mammalian hosts to determine the effect on viral susceptibility and transmissibility. Differences in viral titers between groups were compared using Student’s t-test.ResultsDuring surveillance for SIV in China from 2006 to 2009, seven isolates (24.14%) of 29 influenza A viruses were co-isolated with Stenotrophomonas maltophilia from nasal and tracheal swab samples of pigs. Antimicrobial susceptibility testing showed that the bacteria possessed a high level of resistance towards clinically used antibiotics. To investigate the interaction between these two microorganisms in influencing viral susceptibility and transmission in humans, guinea pigs were used as an infection model. Animals were inoculated with SIV or S. maltophilia alone or co-infected with SIV and S. maltophilia. The results showed that although no transmission among guinea pigs was observed, virus–bacteria co-infections resulted in higher virus titers in nasal washes and trachea and a longer virus shedding period.ConclusionsThis is the first report of influenza virus co-infection with S. maltophilia in the Chinese swine population. Increased replication of virus by co-infection with multidrug resistant bacteria might increase the infection rate of SIV in humans. The control of S. maltophilia in clinics will contribute to reducing the spread of SIV in pigs and humans.

Application of the pseudo-plaque assay for detection and titration of Crimean-Congo hemorrhagic fever virus

A pseudo-plaque assay was developed for detection and quantitation of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06. Enzyme-catalyzed color development of infected cells probed with anti-Crimean-Congo hemorrhagic fever virus antibodies was used for determining the titer of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 and for its detection in samples from persons infected with the Crimean-Congo hemorrhagic fever virus. The pseudo-plaque assay accuracy was confirmed by comparing pseudo-plaque assay titers with fluorescent immunofocus assay and focus formation assay titers using three stocks of virus. No significant difference in virus titers of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 among the three methods was observed. The pseudo-plaque assay is more sensitive than the fluorescent immunofocus assay for detecting the virus in primary isolates of Crimean-Congo hemorrhagic fever virus collected from humans, but no difference in sensitivity between the two methods was observed in the cell-adapted strain of Crimean-Congo hemorrhagic fever Turkey-Kelkit06. The pseudo-plaque assay is suitable for titration of Crimean-Congo hemorrhagic fever Turkey-Kelkit06, which does not develop plaques, suggesting it may also be suitable for the detection of other viruses.

Isolation, preliminary characterization, and full-genome analyses of tick-borne encephalitis virus from Mongolia

Tick-borne encephalitis virus (TBEV) causes one of the most important inflammatory diseases of the central nervous system, namely severe encephalitis in Europe and Asia. Since the 1980s tick-borne encephalitis is known in Mongolia with increasing numbers of human cases reported during the last years. So far, however, data on TBEV strains are still sparse. We herein report the isolation of a TBEV strain from Ixodes persulcatus ticks collected in Mongolia in 2010. Phylogenetic analysis of the E-gene classified this isolate as Siberian subtype of TBEV. The Mongolian TBEV strain showed differences in virus titers, plaque sizes, and growth properties in two human neuronal cell-lines. In addition, the 10,242 nucleotide long open-reading frame and the corresponding polyprotein sequence were revealed. The isolate grouped in the genetic subclade of the Siberian subtype. The strain Zausaev (AF527415) and Vasilchenko (AF069066) had 97 and 94% identity on the nucleotide level. In summary, we herein describe first detailed data regarding TBEV from Mongolia. Further investigations of TBEV in Mongolia and adjacent areas are needed to understand the intricate dispersal of this virus.