Indirect Immunofluorescence Assay Research Articles

Development and biological characterization of an infectious cDNA clone of NADC34-like PRRSV

IntroductionPorcine Reproductive and Respiratory Syndrome virus (PRRSV) causes high abortion rates in gestating sows and stillbirths, as well as high piglet mortality, seriously jeopardizing the pig industry in China and worldwide.MethodsIn this study, an infectious clone containing the full-length genome of NADC34-like PRRSV was constructed for the first time using reverse genetic techniques. The gene was amplified segmentally onto a plasmid, transfected into BHK-21 cells, and the transfected supernatant was harvested and transfected into PAM cells, which showed classical cytopathic effects (CPE).ResultsThe virus rJS-KS/2021 was successfully rescued which could be demonstrated by Western Blot and indirect immunofluorescence assays. Its growth curve was similar to the original strain. Replace the 5’UTR and 3’UTR of rJS-KS/2021 with 5’UTR and 3’UTR of HP-PRRSV (strain SH1) also failed to propagate on MARC-145.DiscussionIn this study, an infectious clone of NADC34-like was constructed by reverse genetics, replacing the UTR and changing the cellular tropism of the virus. These findings provide a solid foundation for studying the recombination of different PRRSVs and the adaption of PRRSVs on MARC-145 in the future.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

Seroprevalence of the novel swine acute diarrhea syndrome coronavirus in China assessed by enzyme-linked immunosorbent assay.

The endemic outbreak of SADS-CoV has resulted in economic losses and potentially threatened the safety of China's pig industry. The molecular epidemiology of SADS-CoV in pig herds has been investigated in many provinces in China. However, there are no data over a long-time span, and there is a lack of extensive serological surveys to assess the prevalence of SADS-CoV in Chinese swine herds since the discovery of SADS-CoV. In this study, an indirect anti-SADS-CoV IgG enzyme-linked immunosorbent assay (ELISA) based on the SADS-CoV S1 protein was established to investigate the seroprevalence of SADS-CoV in Chinese swine herds. Cross-reactivity assays, indirect immunofluorescence, and western blotting assays showed that the developed ELISA had excellent SADS-CoV specificity. In total, 12,978 pig serum samples from 29 provinces/municipalities/autonomous regions in China were tested from 2022 to 2023. The results showed that the general seroprevalence of SADS-CoV in China was 59.97%, with seroprevalence ranging from 16.7% to 77.12% in different provinces and from 42.61% to 68.45% in different months. SADS-CoV is widely prevalent in China, and its seroprevalence was higher in Northeast China, North China, and Central China than in other regions. Among the four seasons, the prevalence of SADS-CoV was the highest in spring and the lowest in autumn. The results of this study provide the general seroprevalence profile of SADS-CoV in China, facilitating the understanding of the prevalence of SADS-CoV in pigs. More importantly, this study is beneficial in formulating preventive and control measures for SADS-CoV and may provide directions for vaccine development.

Identification of DAGLA as an autoantibody target in cerebellar ataxia

BackgroundWe aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis.MethodsSerum and cerebrospinal fliud (CSF) samples from four index patients were subjected...

Association between serum antinuclear antibody and rheumatoid arthritis.

The relationship between serum antinuclear antibody (ANA) and rheumatoid arthritis (RA) remains unknown. Therefore, we aimed to evaluate whether serum ANA was associated with an increased risk of RA in a case-control study. Patients with rheumatoid arthritis hospitalized at Shandong Provincial Hospital from January 2018 to December 2022 were recruited as the case group, and patients with other types of arthritis and healthy people at the same time were taken as the control group. Antinuclear antibody (ANA) was detected by indirect immunofluorescence assays. Propensity score matching was employed to construct a cohort of patients exhibiting comparable baseline characteristics. The relationship between serum ANA and the risk of rheumatoid arthritis was analyzed by logistic regression analysis. A total of 1,175 patients with RA and 1,662 control subjects were included in this study. After adjusting for potential confounding factors in the propensity-score matched cohort, the risk of RA gradually increased with rising of ANA titers. When ANA titers were divided into three groups (1:100, 1:320, and 1:1,000), the OR (95% CI) for ANA titers from low to high was 3.95 (3.01, 5.18), 16.63 (9.44, 29.30), and 17.34 (9.53, 31.54), respectively, compared to those when ANA was negative. The ANA patterns closely related to the occurrence of RA include nuclear homogeneous, nuclear speckled, and cytoplasmic speckled. Among them, the positive rate of nuclear homogeneous was the highest, which accounted for 42.64%. The OR (95% CI) of ANA patterns including nuclear homogeneous, nuclear speckled, and cytoplasmic speckled was 16.81 (11.46, 24.65), 3.40 (2.49, 4.63), and 3.09 (1.77, 5.40), respectively. There was a curve relation between ANA titer and RA, and the higher the ANA titer, the higher the probability of RA. However, there was no statistical difference in probability of RA for 1:320 versus 1:1,000 ANA titers. The most important kind of ANA pattern in the blood of RA patients was nuclear homogeneous. These findings suggest that ANA may be a novel risk marker for RA.

Medium-chain chlorinated paraffins (MCCPs) induce renal cell aging and ferroptosis.

Medium-chained chlorinated paraffins (MCCPs) are a class of chlorinated derivatives of straight-chain n-alkanes with complex compositions, which are widely used in industry. The chlorinated paraffins (CPs) are divided into short chain chlorinated paraffins (SCCPs), medium chain chlorinated paraffins (MCCPs) and long chain chlorinated paraffins (LCCPs). SCCPs have been banned due to their severe bioaccumulation and biotoxicity. Therefore, MCCPs are used as a substitute for SCCPs. However, the toxicological data of MCCPs are still very limited. For this, we systematically investigated the toxicological impact of MCCPs on a renal cell model in the current study. Our work provides basic research data for analyzing the toxicological effects of MCCPs, suggesting that MCCPs should be restricted in their usage. A series of biochemical experiments was performed, including Western blot, indirect immunofluorescence assay, and ELISA was performed to analyze the toxicological effects of MCCPs. Two renal cell lines were used as a model for assessing the toxicological effects of MCCPs. Cell proliferation assays showed that MCCPs could inhibit the proliferation of kidney cells in a dose-dependent manner. Further studies showed that MCCPs induced ferroptosis in kidney cells by evaluating a series of ferroptosis marker molecules. Additionally, MCCPs induced inflammatory response and premature senescence in HEK293 and NRK-52E cells. Molecular mechanism experiments showed that ferroptosis induced by MCCPs emerged as a significant contributor to premature aging of kidney cells. The current study provides basic research data to analyze the toxicological effects of MCCPs and their toxicity mechanisms. It also provides a theoretical basis for the assessment of the potential ecological risk of MCCPs, as well as basic experimental data for the rational and standardized use of MCCPs.

Paraneoplastic Calmodulin Kinase-Like Vesicle-Associated Protein (CAMKV) Autoimmune Encephalitis.

To report an autoimmune paraneoplastic encephalitis characterized by immunoglobulin G (IgG) antibody targeting synaptic protein calmodulin kinase-like vesicle-associated (CAMKV). Serum and cerebrospinal fluid (CSF) samples harboring unclassified antibodies on murine brain-based indirect immunofluorescence assay (IFA) were screened by human protein microarray. In 5 patients with identical cerebral IFA staining, CAMKV was identified as top-ranking candidate antigen. Western blots, confocal microscopy, immune-absorption, and mass spectrometry were performed to substantiate CAMKV specificity. Recombinant CAMKV-specific assays (cell-based [fixed and live] and Western blot) provided additional confirmation. Of 5 CAMKV-IgG positive patients, 3 were women (median symptom-onset age was 59 years; range, 53-74). Encephalitis-onset was subacute (4) or acute (1) and manifested with: altered mental status (all), seizures (4), hyperkinetic movements (4), psychiatric features (3), memory loss (2), and insomnia (2). Paraclinical testing revealed CSF lymphocytic pleocytosis (all 4 tested), electrographic seizures (3 of 4 tested), and striking MRI abnormalities in all (mesial temporal lobe T2 hyperintensities [all patients], caudate head T2 hyperintensities [3], and cortical diffusion weighted hyperintensities [2]). None had post-gadolinium enhancement. Cancers were uterine adenocarcinoma (3 patients: poorly differentiated or neuroendocrine-differentiated in 2, both demonstrated CAMKV immunoreactivity), bladder urothelial carcinoma (1), and non-Hodgkin lymphoma (1). Two patients developed encephalitis following immune checkpoint inhibitor cancer therapy (atezolizumab [1], pembrolizumab [1]). All treated patients (4) demonstrated an initial response to immunotherapy (corticosteroids [4], IVIG [2]), though 3 died from cancer. CAMKV-IgG is a biomarker of immunotherapy-responsive paraneoplastic encephalitis with temporal and extratemporal features and uterine cancer as a prominent oncologic association. ANN NEUROL 2024.

Development of monoclonal antibodies against oropouche virus and its applicability to immunohistochemical diagnosis

Orthobunyavirus oropouche ense virus (OROV), the causative agent of Oropouche fever, is widely dispersed in Brazil and South America, causing sporadic outbreaks. Due to the similarity of initial clinical symptoms caused by OROV with other arboviruses found in overlapping geographical areas, differential diagnosis is challenging. As for most neglected tropical diseases, there is a shortage of reagents for diagnosing and studying OROV pathogenesis. We therefore developed and characterized mouse monoclonal antibodies and, one of them recognizes the OROV nucleocapsid in indirect immunofluorescent (IFA) and immunohistochemistry (IHC) assays. Considering that it is the first monoclonal antibody produced for detecting OROV infections, we believe that it will be useful not only for diagnostic purposes but also for performing serological surveys and epidemiological surveillance on the dispersion and prevalence of OROV in Brazil and South America.

Serosurvey of Coxiella burnetii in Police Officers and Working Dogs in Brazil: Case Report and One Health Implications.

Although the Coxiella burnetii infection has been investigated in dogs, its role in human transmission remains to be fully established, particularly in close and daily human-dog contact settings, such as in Police K-9 Units. Accordingly, this study aimed to assess anti-C. burnetii antibodies in clinically healthy police officers by an in-house indirect immunofluorescence assay (IFA), and working dogs by a commercial IFA Kit, from the State Special Operations Battalion, Paraná, Southern Brazil. Overall, 1/18 (5.5%) police officers and 9/30 (30.0%; CI 95% 16.66-47.88) dogs tested seropositive to anti-C. burnetii IgG antibodies. To date, this is the highest prevalence of Q fever seropositivity among military dogs worldwide. Despite the low sampling rate, a statistically significant association was found between seropositivity and female dogs (p = 0.0492). Further studies with larger sample sizes should be conducted to establish the prevalence of Q Fever in other Brazilian K-9 Units. In summary, this study is the first to conduct a concomitant serosurvey of police officers and working dogs, and its findings should be considered a warning for cross-exposure and transmission of Coxiella burnetii among Police K-9 Units in Brazil and worldwide.

Isolation and Identification of Severe Fever with Thrombocytopenia Syndrome Virus from Farmed Mink in Shandong, China

Severe fever with thrombocytopenia syndrome (SFTS) virus, recently named Bandavirus dabieense, belongs to the genus Bandavirus of family Phenuiviridae, and it causes SFTS in humans with clinical symptoms including fever, thrombocytopenia, gastrointestinal symptoms, and leukocytopenia. However, there are few reports on the pathogenesis of SFTSV in animals. This study first isolated the SFTSV strain SD22-2 from sick-farmed mink. Viral metagenomics was used to detect SFTSV nucleotide in the clinical specimens obtained from symptomatic minks. Then, we isolated the virus using Vero and DH82 cells, and Real-Time Quantitative PCR (RT-qPCR), indirect immunofluorescence assay, transmission electron microscopy, and Western blotting identified it. Meanwhile, phylogenetic analysis based on partial L, M, and S segment sequences indicated that the mink-origin SFTSV strain SD22-2 belonged to genotype D and was genetically close to the HB2016-003 strain isolated from humans. Taken together, we isolated and identified an SFTSV from farmed mink that may be the reservoir hosts of SFTSV. We should pay more attention to farmed minks and biosecurity practices, and active surveillance at fur farms must be reviewed and enhanced.

Exposure of small ruminants and humans to Coxiella burnetii in the semi-arid region of Northeastern Brazil.

Q fever is a zoonotic disease caused by the obligate intracellular pathogen Coxiella burnetii, for which domestic ruminants are the primary source of infection in humans. Herein, we investigated the presence of C. burnetii in humans, sheep, and goats in the semi-arid region of northeastern Brazil. The presence of anti-C. burnetii antibodies was surveyed using indirect immunofluorescence assay, and detection of C. burnetii DNA was performed by polymerase chain reaction (PCR). Anti-C. burnetii antibodies were detected in 60% of farms, 4.8% of goats, 1.5% of sheep, and 4.5% of human samples. PCR was positive in 18.9% of blood samples, 7.7% of milk samples, and 7.7% of vaginal mucus samples. A DNA sequence of a C. burnetii DNA sample extracted from the goat vaginal mucus showed 99.2-99.4% nucleotide identity with other strains previously reported in Brazil. These results indicate that C. burnetii is present in the surveyed area, where it poses a risk to both public and animal health. These findings indicate an urgent need for educative actions to protect population, as well as better training of veterinarians to detect and report Q fever.

A Zoonotic Strain of Rocahepevirus ratti Hepatitis E Virus Does Not Replicate Efficiently within Human Placental JEG-3 Cells

Paslahepevirus balayani and Rocahepevirus ratti are genetically diverse species of hepatitis E virus [HEV]. Previously, only members of the Paslahepevirus genus were known to infect humans but recently some Rocahepevirus members have been found to be infectious to both immunocompromised and immunocompetent humans. Paslahepevirus balayani genotypes (gt) 1, 2, and 4 are known for their detrimental effects during pregnancy, causing pregnancy-related disorders. Recent findings have demonstrated the ability of Paslahepevirus balayani gt3 to replicate within placental cell lines, suggesting a direct effect on the placenta and fetus. To study whether zoonotic rat HEV strains possess a similar human-host placental tropism, we utilized JEG-3 cells to understand the replicative ability of an infectious clone of a recently reported strain of Rocahepevirus ratti, the LCK-3110 strain. Infectious cDNA clones of Pasla-, Avi-, and Rocahepevirus were transcribed and then, transduced into JEG-3 cells. Cells were harvested, and cell lysates were used for testing infectivity. Five days post-transfection or after inoculation onto naive HepG2/C3A cells, the cells were analyzed for infection. Replication in transduced JEG-3 cells and the infection potential in HepG2/C3A cells were assessed via an indirect immunofluorescence assay and a flow-cytometry assay. We found that the Rocahepevirus ratti LCK-3110 strain did not have efficient replication in JEG-3 cell cultures.

Epidemiology of Murine Typhus in Taiwan from 2013 to 2020.

Murine typhus is a flea-borne disease caused by Rickettsia typhi infection. The disease is a notifiable infectious disease in Taiwan. Specimens from suspected cases are required to be sent to the Taiwan Centers for Disease Control and Prevention for laboratory diagnosis. In this study, 204 cases of murine typhus were identified by bacterial isolation, real-time polymerase chain reaction, or indirect immunofluorescence assay between 2013 and 2020. The average incidence rate was 0.11/100,000 person-years (95% CI: 0.08-0.13). Murine typhus occurred throughout the year, but it was most prevalent in summer (May to August). The majority of patients were males (75%), residents of Kaohsiung city (31%), and worked in agriculture, forestry, fishing, and animal husbandry (27%). Fever was the most common symptom, present in 95.6% of patients, followed by headache (41%), myalgia (33%), and liver dysfunction (33%). Only 13% of patients had a rash. Up to 80% of cases were among hospitalized patients, and 43% of patients developed severe manifestations. Serological assays also indicated coinfection events. Seven patients showed a 4-fold increase in antibody titers against Orientia tsutsugamushi (N = 2), Coxiella burnetii (n = 2), and Leptospira (N = 3). In conclusion, murine typhus is an endemic and important zoonotic rickettsial disease in Taiwan that cannot be ignored. Further epidemiological surveillance and clinical characteristics should be continuously investigated to prevent and control murine typhus.

First report of Ralstonia pseudosolanacearum causing bacterial wilt on Rosa sp. in Greece.

In March 2021, a sample of nine-month-old, non-grafted, diseased rose (Rosa sp.) plants was sent by a grower to the Benaki Phytopathological Institute for examination. The plants exhibited symptoms of dieback with black necrosis of pruned shoots, brown discoloration of shoot and root vascular tissues, and whitish slime exudation on cutting wounds of the shoots. The symptoms resembled those caused by Ralstonia pseudosolanacearum (Tjou-Tam-Sin et al. 2016). According to the sample's information sheet, the sample had been collected in a commercial greenhouse rose crop for cut flowers with a 10% disease incidence in the area of Troizinia-Methana (Regional Unit of Islands, Greece). Microscopic examination of symptomatic shoot and root vascular tissues revealed masses of bacterial cells streaming out of them. Sections of symptomatic tissues were suspended in water and in the resulting suspension, bacteria of the R. solanacearum species complex (RSSC) were detected by an indirect immunofluorescence (IF) assay using polyclonal antibodies (Plant Research International, the Netherlands) and a qPCR assay (RS-I-F/RS-II-R primers, RSP-55T probe) (Vreeburg et al. 2016). Furthermore, colonies with typical characteristics of RSSC were isolated from vascular tissues of shoots and roots on non-selective (NA) and semi-selective (mSMSA) media (EPPO 2022), and their identification as RSSC was confirmed by the above-mentioned IF and qPCR assays. Also, the isolates were assigned to: i) biovar 3, based on their ability to metabolize three disaccharides (maltose, lactose, D(+) cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) producing acid (EU 2006) and ii) phylotype I, by multiplex conventional PCR (Opina et al. 1997; Fegan and Prior 2005). A representative isolate was selected for sequencing part of the genes: 16S rDNA (1464bp), mutS (729bp) and egl (795bp) with GenBank Accession Nos. OR102443, OR683617 and OR702781, respectively. Blast analysis of these sequences showed 100% identity with those of various RSSC strains (e.g. GenBank Ac. Nos. CP025741.1, CP021762.1, MF141029.1, respectively). The obtained egl sequence conforms with the characteristics of phylotype I based on the DNA barcoding tool (EPPO 2021) and is 100% identical to that of the Dutch strain PD7216 (MF141029.1) reported to be sequevar I-33 (Bergsma-Vlami et al. 2018). The pathogenicity of two isolates was tested by inoculating: i) tomato seedlings (cv. 'Belladona') at their stem between the cotyledons and the first true leaf (EU 2006) and b) rose plants (cv. 'Aqua' and 'Papa Meilland') at their shoot base (Tjou-Tam-Sin et al. 2016), with bacterial suspensions in water (108 cfu/ml). The inoculated plants were maintained at a day/night temperature about 28/20°C with tomato plants exhibiting leaf wilting (7-17 dpi) and rose plants exhibiting chlorosis and necrosis of leaves (17 dpi). The pathogen was re-isolated on mSMSA from both artificially infected plant species and identified by the IF assay described above, thus fulfilling Koch's postulates. This is the first diagnosis in Greece of: i) rose plants infected by a Ralstonia species and ii) a crop infected by R. solanacearum phylotype I that corresponds to the R. pseudosolanacearum species (EPPO 2022). Official phytosanitary measures imposed in the affected area include an annual survey of rose crops for the presence of this pathogen, aiming at an early detection and prevention of its spread in such a highly valued ornamental crop.

Titanium Dioxide Nanoparticles: A Novel Approach for Inhibiting Human Papillomavirus

Nanotechnology products such as titanium dioxide nanoparticles (TiO2-NPs) can be used for viral infections because of their unique characteristics. The current study aimed to determine the impact of TiO2-NPs on HPV type 1 and 2 infections. The characterization of these NPs was performed using dynamic light scattering (DLS), field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), and X-ray diffraction (XRD). The MTT assay was used to determine the toxic impacts of TiO2-NPs on BHK-21 cells. The efficiency of TiO2-NPs was performed using several parameters, including TCID50 and RT-PCR assays. An indirect immunofluorescence assay (IFA) was performed to estimate the inhibitory impact of TiO2-NPs on viral antigen expression, and Acyclovir was used as a reference medicine. When the human papilloma type 1 and 2 viruses exposed to TiO2-NPs at high doses (100 μg/mL) produced 0.3, 1.1, 2.3, and 3.3 log10 TCID50 decreases in infective virus load when compared with control viruses (P<0.0001), these TiO2-NPs doses were related to 24.9%, 35.1%, 47.2%, 59.5%, and 66.6% inhibition percentages that were determined depending on the viral titer as compared to virus control. It is concluded that TiO2-NPs have strong potential for the treatment of face and labial lesions caused by papillomaviruses 1 and 2 and could be used in topical formulations.

Nano-sized microplastics exposure induces skin cell senescence via triggering the mitochondrial localization of GSDMD

Nano-sized microplastic pollution is distributed worldwide. Nano-sized microplastics can enter the blood through the digestive tract, and then transported to various tissues and organs of the body, resulting in a series of toxicological effects. In addition, nano-sized microplastics can penetrate the skin barrier. However, the toxicological effects of nano-sized microplastics on the skin are still not completely understood. Two skin cell lines were used as in vitro models to investigate the toxicological effects of nano-sized microplastics on skin cells and their potential molecular mechanisms. First, cellular behavioral research results showed that nano-sized microplastics can be internalized into skin cells in a time- and dose-dependent manner. Further experiments using western blotting, indirect immunofluorescence, and ELISA assays demonstrated that nano-sized microplastics cause an increase in skin cell inflammation levels. Additionally, our research showed that nano-sized microplastics caused skin cell senescence damage by evaluating aging-marker molecules such as p16 and p21. Subsequently, we studied the potential molecular mechanism by which nano-sized microplastics cause pathological skin injury and found that they induce mitochondrial oxidative stress, depolarize the mitochondrial membrane potential, and recruit GSDMD to the mitochondria. Subsequently, mtDNA enters the cytoplasm via GSDMD pores, which then activates the AIM2 Inflammasome. Ultimately, it causes a series of biochemical reactions such as inflammation and aging in cells. In an in vivo model, we tested the effect of nano-sized microplastics on skin regeneration and found that they acted as an inhibitor to skin regeneration and aggravated the inflammatory reaction of the skin. Overall, our results provide new evidence of the skin toxicity of nano-sized microplastics. This study provides a theoretical foundation for further research on the potential toxicological effects of nano-sized microplastics on the skin.

Structure elucidation and antiviral activity of a cold water-extracted mannogalactofucan Ts1-1A from Trametes sanguinea against human cytomegalovirus in vitro

In this study, we purified a partially acetylated heteropolysaccharide (Ts1-1A) from the fruit bodies of Trametes sanguinea Lloyd through cold water extraction and serial chromatographic separation. The purified polysaccharide Ts1-1A (12.8 kDa) was characterized as a branched mannogalactofucan with a backbone of alternately connected 1,3-linked α-Fucp and 1,6-linked α-Galp, which was partially substituted by non-reducing end units of β-Manp at O-2 and O-3 positions of 1,6-linked α-Galp. Ts1-1A showed pronounced anti-human cytomegalovirus activity at the concentration of 200 and 500 μg/mL in systematical assessments including morphological changes, western blotting, qPCR, indirect immunofluorescence and tissue culture infective dose assays. Moreover, Ts1-1A exerted its antiviral activity at two distinct stages of viral proliferation manifesting as significantly inhibiting viral protein (IE1/2 and p52) expression and reducing viral gene (UL123, UL44 and UL32) replication in the HCMV-infected WI-38 cells. At viral attachment stage, Ts1-1A interacted with HCMV and prevented HCMV from attaching to its host cells. While at early phase of viral replication stage, Ts1-1A suppressed HCMV replication by downregulating NQO1 and HO-1 proteins related to oxidative stress as an antioxidant. To sum up, Ts1-1A is a promising anti-HCMV agent which could be developed for HCMV infection prevention and therapy.

Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells.

The Mammalian Target of Rapamycin (mTOR) signaling pathway regulates protein phosphorylation and exerts control over major cellular processes. mTOR is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb), which is encoded by the Rheb1 and Rheb-like-1 (RhebL1) genes. There is currently a paucity of information on the role of RhebL1, and specifically its involvement in viral infection. In the present study we investigated the role of RhebL1 during human influenza A/NWS/33 (NWS/33) (H1N1) virus infection of rhesus monkey-kidney (LLC-MK2) cells and human type II alveolar epithelial (A549) cells. To assess the efficiency of NWS/33 virus replication, the expression of viral nucleoprotein was examined by indirect immunofluorescence (IIF) and the viral yield by fifty percent tissue culture infectious dose assay. An RNA-mediated RNA interference approach was used to investigate the role of RhebL1 during NWS/33 infection. RhebL1 expression was evaluated by IIF, Western blotting, and enzyme-linked immunosorbent assays. A two-tailed Student's t-test was applied to evaluate differences between groups. RhebL1 was differentially expressed in the cell models used in this study. Silencing of the RhebL1 gene led to increased NWS/33 virus infection in A549 cells, but not in LLC-MK2 cells. Moreover, the expression of hyperphosphorylated cytokeratin 8, a marker of NWS/33 virus infection efficiency, increased in A549 cells depleted of RhebL1 but remained almost unchanged in LLC-MK2 cells. These are the first results showing involvement of the endogenous RhebL1 protein during viral infection. Our data suggests that RhebL1 exerts a host cell-dependent modulatory role during influenza virus infection. RhebL1 appears to be a restrictive factor against NWS/33 virus replication in A549 cells, but not in LLC-MK2.

Approaching the complexity of Crimean-Congo hemorrhagic fever virus serology: A study in swine

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results.

SARS-CoV and SARS-CoV -2 cross-reactive antibodies in domestic animals and wildlife in Nigeria suggest circulation of sarbecoviruses

Anthropogenic exposure of domestic animals, as well as wildlife, can result in zoonotic transmission events with known and unknown pathogens including sarbecoviruses. During the COVID-19 pandemic, SARS-CoV-2 infections in animals, most likely resulting from spill-over from humans, have been documented worldwide. However, only limited information is available for Africa. The anthropozoonotic transmission from humans to animals, followed by further inter- and intraspecies propagation may contribute to viral evolution, and thereby subsequently alter the epidemiological patterns of transmission. To shed light on the possible role of domestic animals and wildlife in the ecology and epidemiology of sarbecoviruses in Nigeria, and to analyze the possible circulation of other, undiscovered, but potentially zoonotic sarbecoviruses in animals, we tested 504 serum samples from dogs, rabbits, bats, and pangolins collected between December 2020 and April 2022. The samples were analyzed using an indirect multi-species enzyme-linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of SARS-CoV and SARS-CoV -2, respectively. ELISA reactive sera were further analyzed by highly specific virus neutralization test and indirect immunofluorescence assay for confirmation of the presence of antibodies. In this study, we found SARS-CoV reactive antibodies in 16 (11.5%) dogs, 7 (2.97%) rabbits, 2 (7.7%) pangolins and SARS-CoV-2 reactive antibodies in 20 (13.4%) dogs, 6 (2.5%) rabbits and 2 (7.7%) pangolins, respectively. Interestingly, 2 (2.3%) bat samples were positive only for SARS-CoV RBD reactive antibodies. These serological findings of SARS-CoV and/or SARS-CoV-2 infections in both domestic animals and wildlife indicates exposure to sarbecoviruses and requires further One Health-oriented research on the potential reservoir role that different species might play in the ecology and epidemiology of coronaviruses at the human-animal interface.