Impact of Wheat Streak Mosaic Virus on Peroxisome Proliferation, Redox Reactions, and Resistance Responses in Wheat

Although peroxisomes play an essential role in viral pathogenesis, and viruses are known to change peroxisome morphology, the role of genotype in the peroxisomal response to viruses remains poorly understood. Here, we analyzed the impact of wheat streak mosaic virus (WSMV) on the peroxisome proliferation in the context of pathogen response, redox homeostasis, and yield in two wheat cultivars, Patras and Pamir, in the field trials. We observed greater virus content and yield losses in Pamir than in Patras. Leaf chlorophyll and protein content measured at the beginning of flowering were also more sensitive to WSMV infection in Pamir. Patras responded to the WSMV infection by transcriptional up-regulation of the peroxisome fission genes PEROXIN 11C (PEX11C), DYNAMIN RELATED PROTEIN 5B (DRP5B), and FISSION1A (FIS1A), greater peroxisome abundance, and activation of pathogenesis-related proteins chitinase, and β-1,3-glucanase. Oppositely, in Pamir, WMSV infection suppressed transcription of peroxisome biogenesis genes and activity of chitinase and β-1,3-glucanase, and did not affect peroxisome abundance. Activity of ROS scavenging enzymes was higher in Patras than in Pamir. Thus, the impact of WMSV on peroxisome proliferation is genotype-specific and peroxisome abundance can be used as a proxy for the magnitude of plant immune response.

Precise localization and dynamic distribution of Japanese encephalitis virus in the rain nuclei of infected mice

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.

SARS-CoV-2 Infects Syncytiotrophoblast and Activates Inflammatory Responses in the Placenta

SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 55 placentas (41%). Three placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise, stillbirth, and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and, to a lesser extent, endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.

Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.

Establishment and Application of a Taqman Reverse Transcriptase Quantitative Real Time Pcr Assay for Feline Calicivirus

Feline calicivirus (FCV) is an infectious pathogen that causes disease in cats. With the current emergence of FCV-associated virulent systemic disease (FCV VSD) worldwide, the establishment of a rapid, sensitive, and reproducible diagnostic assay for its detection is important to inform prevention and control strategies. In this study, specific primers and TaqMan-FAM probes were designed based on the conserved regions of the FCV genome sequence, and a TaqMan reverse transcriptase quantitative real time PCR assay was established. This assay could specifically detected the FCV genome. The assay had a wide dynamic range, with linear detection in the range of 9.6×109 copies/μL to 9.6×100 copies/μL, with a limit of detection of 9.6×100 copies/μL, showing high sensitivity and repeatability. In addition, we used this assay to evaluated clinical samples (n=100) taken from cats from across China for the presence/absence of FCV genetic material For samples with low virus content, the positive detection rate of TaqMan reverse transcriptase quantitative real time PCR assay (RT-qPCR) was much higher than that of conventional reverse transcriptase PCR assay (cRT-PCR). And The qRT-PCR assay was used to detect the viral load of cat swabs within 17 days after FCV infection. From days 1-9, the oral and nasal swabs generally had higher viral loads than the anal swabs. While from days 10-17, the levels in the oral and nasal swabs being generally lower than those in the anal swabs. Overall, this FCV TaqMan RT-qPCR assay assay represents a rapid and accurate.

Detection of Southern Rice Black-Streaked Dwarf Virus Using Western Blotting With P6

The southern rice black-streaked dwarf virus (SRBSDV) is a severe threat to the yield and quality of rice products worldwide. Traditional detection methods for diagnosing SRBSDV infection show several false positives and thus provide inaccurate findings. However, Western blotting (WB) can precisely solve this problem. In this study, P6—a viral RNA-silencing suppressor—was expressed and purified in vitro. Two polyclonal P6 antibodies were obtained and quantified by enzyme-linked immunosorbent assay and WB. Subsequently, WB was performed using the P6 antibodies to identify SRBSDV antigens derived from the suspected rice samples collected from nine districts in Guizhou, China. The assay results showed that Libo, Pingtang, Huishui, Dushan, and Anshun districts had experienced an SRBSDV outbreak. The virus content in the sampled rice tissues was quantified by WB. Our results revealed that SRBSDV mainly accumulated in rice stems rather than rice leaves. Thus, the findings of our study show that the SRBSDV P6 antibody can be used in WB for detecting and monitoring SRBSDV infection in infected rice plants.

ISOLATION AND CHARACTERIZATION OF AVIAN INFLUENZA VIRUS H5N1 SUBTYPE FIELD ISOLATES FROM BALI FOR VACCINE CANDIDATES

A research on the isolation and characterization of the Avian Influenza H5N1 subtype field isolate has been carried out at the BSL-3 Laboratory of PT Sanbio Laboratories, Bogor. The aim of the study was to prepare a candidate for the H5N1 subtype Avian Influenza virus vaccine. Virus isolates were taken from field isolates from Bali. A total of seven field H5N1 AI subtypes from Bali were characterized in Bogor. The isolates were: isolate 3A, isolate 4A, isolate 9C, isolate 10 A, isolate 10 C, isolate P65, isolate P67. The passage of isolates was carried out on 9-day-old embryonic Specific Pathogenic (SPF) chicken eggs by injecting 0.1 mL of SPF isolates/eggs through the allantoic cavity. Each isolate was placed in five SPF eggs and then incubated in an incubator at 37 C and candled every day. Since day 2-4 post inoculation, embryo death has occurred. The eggs are harvested by their allantoic fluid and tested for haemagglutination test(HA/HI). The HI test results were confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using the front primer FPHA232_13 (ATTGGTTAYCATGCAAAYAACTCG) and the back primer BPHA232_597 (GGAAAYATAGGTRGTTGGRTTYTGATAG) The results were five of the seven isolates were positive AI subtype B5 585 - 581 The five isolates of AI subtype H5N1 were subsequently sequenced, the results were all positive for AI virus subtype H5N1 clade 2.3.2. Each field isolate was given the name A / Chicken / Bali3A / GAY / 2019; A / Chicken / Bali9C / GAY / 2019; A / Chicken / BaliA4 / GAY / 2019; A / Chicken / Bali10A / GAY / 2016 and A / Chicken / Bali10C / GAY / 2019. One A / Chicken / Bali 9C / GAY / 2016 isolate was subsequently repeated 7 times until a stable H5N1 subtype AI virus titer was obtained. The results of matching with bioinformatics turned out that A / Chicken / Bali 9C / GAY / 2016 isolates had a kinship of 98.62% with AI subtype H5N1 Banyuwangi, amounting to 98.45% with AI subtype H5N1 Lamongan, amounting to 98.10% with AI-H5N1 Lumajang, 97.58% with AI-H5N1 Kediri, 97.07% with AIH5N1 Blitar, 96.72% with AI-H5N1 Denpasar, 96.72% with AI-H5N1 Buleleng and 96.72% with AI-H5N1 Sukoharjo. The conclusion is one of isolate namely A / Chicken / Bali 9C / GAY / 2019 including AI subtype H5N1 clade 2.3.2, is’t stable at passage on SPF eggs, has a kinship of 96.72% with A / duck / Sukoharjo / BBVW-1428- 9/2012, the virus content is 106.9 ELD50 so it is potential for vaccine candidates.

Application of the real-time PCR in veterinary practice

To date the molecular genetic methods of analysis are widely used for laboratory diagnostic tests in various infectious diseases of animals. This discourse reflects information about the history of the invention of real-time polymerase chain reaction (PCR-RV), the nature of the processes that occur during this reaction, the main stages of the reaction, the preparation of biological material for research in PCR-RV. The spectrum of possibilities of using the PCR-RV method for a qualitative study of biological material in cases of suspected infection of animals with certain viral and bacterial agents, as well as a quantitative assessment of the virus content in tissues, organs or in the body by analogy with conventional methods for titrating infectiousness without direct manipulation with pathogenic agents, is presented. . A quantitative PCR-RV option allows veterinarians to evaluate the pathogenetic dynamics of the development of the disease, monitor the effect of antiviral and antibacterial therapy, and monitor the emergence of pathogen variants with high resistance to the drugs used. Thanks to the development of ARRIAH, the qualitative and quantitative PCR-RV method can now be used in domestic veterinary science and laboratory practice for the diagnosis of a wide range of animal infectious diseases.

Multi-route respiratory infection: when a transmission route may dominate

The exact transmission route of many respiratory infectious diseases remains a subject for debate to date. The relative contribution ratio of each transmission route is largely undetermined, which is affected by environmental conditions, human behavior, the host and the microorganism. In this study, a detailed mathematical model is developed to investigate the relative contributions of different transmission routes to a multi-route transmitted respiratory infection. It is illustrated that all transmission routes can dominate the total transmission risk under different scenarios. Influential parameters considered include dose-response rate of different routes, droplet governing size that determines virus content in droplets, exposure distance, and virus dose transported to the hand of infector. Our multi-route transmission model provides a comprehensive but straightforward method to evaluate the transmission efficiency of different transmission routes of respiratory diseases and provides a basis for predicting the impact of individual level intervention methods such as increasing close-contact distance and wearing protective masks. (Word count: 153)HighlightsA multi-route transmission model is developed by considering evaporation and motion of respiratory droplets with the respiratory jet and consequent exposure of the susceptible.We have illustrated that each transmission route may dominate during the influenza transmission, and the influential factors are revealed.The short-range airborne route and infection caused by direct inhalation of medium droplets are highlighted.