Specific RT-PCR Research Articles

Confirmation of highly pathogenic avian influenza H5N1 in skuas, Antarctica 2024

From December 2023 to March 2024, a surveillance program aiming to detect Highly Pathogenic Avian Influenza (HPAI) H5N1 was conducted on Antarctica territories, specifically at Fildes Peninsula (King George Island, Maritime Antarctic), and James Ross Island. At Fildes Peninsula, samples from marine birds and mammals were collected from four accessible sampling locations with significant animal colonies: Ardley Island, hosting a large concentration of Gentoo penguins (Pygoscelis papua); Ardley Cove, where small groups of likely non-breeding Chinstrap penguins (Pygoscelis antarcticus) were present; seal haul-out sites of Southern elephant (Mirounga leonina) and Weddell (Leptonycotes wedellii); and, a nesting site of Southern giant petrels (Macronectes giganteus). Additionally, six samples were collected from five dead skuas near the Lachman lakes on James Ross Island (63.7989S, 57.8105W) on March 3, 2024. Despite collecting a total of 943 samples from Fildes Peninsula, all results tested negative for HPAI, and no animals displayed clinical signs or behaviors consistent with HPAI infection. However, all skua samples from James Ross Island tested positive for HPAI H5N1 clade 2.3.4.4 by specific real-time RT-PCR reactions, confirming the first recorded HPAI-related mortality event in Antarctica (south of 60°S), specifically in skuas. Further research is necessary to genetically characterize the virus and better understand the role of skuas in viral dissemination in Antarctica.

First report of iris yellow spot virus infecting shallot (Allium cepa var. aggregatum) in Australia

AbstractIn 2019, symptoms typical of infection by iris yellow spot virus (IYSV; family Tospoviridae, genus Orthotospovirus) were observed on shallot (A. cepa var. aggregatum) grown at the Gatton Research Facility, Department of Agriculture and Fisheries, Queensland. Initial testing by DAS-ELISA was positive for IYSV, and subsequent specific RT-PCR amplifying part of the RdRp (L-segment) and sequencing of amplicons confirmed the IYSV infection. This is the first record of IYSV infection of shallot in Australia.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of American Plum Line Pattern Virus.

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3-5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries.

Abstract P138: Diet Influences the Expression of Renal Sodium-Glucose Cotransporters in Juvenile Male Rats

Poor nutrition and metabolic dysfunction are becoming more prevalent among children, as evident by increasing childhood obesity in recent years. The kidney sodium-glucose cotransporter 2 (SGLT2) is a contributor to overall metabolic homeostasis, with inhibition of SGLT2 significantly reducing circulating glucose and lipids. The study objective was to determine if diet influences the expression of renal glucose transporters in young rats. Weanling male Wistar Kyoto rats received 1) standard laboratory chow, 2) AIN-93G diet, or 3) a high-fat diet (60% kcal, HFD). Each dietary group was then further subdivided to receive either filtered water or 5% (w/v) sucrose solution ad libitum . Four weeks later, animals were euthanized and kidneys were collected and processed. mRNA expression of Sglt1 , Sglt2 , Sqstm1 , and Nlrp3 was determined using gene specific primers and RT-PCR. Renal SGLT2 protein expression was visualized using immunohistochemistry and quantified using Imagej software. Diet significantly influenced the renal expression of both Sglt1 ( P =0.005) and Sglt2 (P=0.046), while beverage did not have a significant effect on either ( P =0.7 and P =0.4, respectively). Rats fed the HFD had more than 30% reduction in Sglt1 mRNA expression as compared to rats fed chow, and ~20% reduction in Sglt2 as compared to rats fed AIN. Preliminary data suggest that diet and beverage significantly altered renal protein expression of SGLT2, with rats fed chow having the lowest renal expression that was slightly increased with sugar consumption (1.5% versus 2.7%, respectively). Rats fed AIN had the greatest expression, but was drastically reduced in rats consuming sugar (4% versus 1.2%, respectively). To determine if autophagy and inflammasome activation is involved, the expression of Sqstm1 and Nlrp3 was measured by RT-PCR. Similarly, rats fed HFD had the lowest expression of both Sqstm1 ( P<0.001) and Nlrp3 ( P=0.03). Sqstm1 and Nlrp3 mRNA expression was decreased with sugar consumption in rats fed chow and AIN, whereas Sqstm1 ( P =0.01) and Nlrp3 ( P <0.001) mRNA expression was increased in rats fed HFD with sugar beverage. Our data suggest that diet significantly influences renal glucose transporter expression in juvenile rats in a way that may be mediated by autophagic impairment. The modulatory effect of sugar consumption on SGLT2 protein, Sqstm1, and Nlrp3 was diet-dependent, highlighting the importance of nutrition to renal physiology within weeks after weaning.

An all-out assault on a dominant resistance gene: Local emergence, establishment, and spread of strains of tomato spotted wilt orthotospovirus (TSWV) that overcome Sw-5b-mediated resistance in fresh market and processing tomatoes in California.

Tomato spotted wilt orthotospovirus (TSWV) causes substantial economic loss to tomato production, and the Sw-5b resistance gene is widely deployed for management. Here, we show (i) the emergence of resistance-breaking (RB) TSWV strains in processing and fresh market tomato production in California over the past ten years, and (ii) evolutionary relationships with RB strains from other areas. A specific RT-PCR test was used to show the C118Y RB strain that emerged in Fresno County in 2016 quickly became predominant in the central production area and remained so through this study. In 2021, the C118Y strain was detected in the Northern production area, and was predominant in 2022. However, in 2023, the C118Y strain was unexpectedly detected in fewer spotted wilt samples from resistant varieties. This was due to emergence of the T120N RB strain, previously known to occur in Spain. A specific RT-PCR test was developed and used to show that the T120N RB strain was predominant in Colusa and Sutter counties (detected in 75-80% of samples), and detected in ~50% of samples from Yolo County. Pathogenicity tests confirmed California isolates of the T120N strain infected Sw-5b tomato varieties and induced severe symptoms. Another RB strain, C118F, was associated with spotted wilt samples of Sw-5 varieties from fresh market tomato production in southern California. Phylogenetic analyses with complete NSm sequences revealed that the C118Y and T120N RB strains infecting resistant processing tomato in California emerged locally, whereas those from fresh market production were more closely related to isolates from Mexico. Thus, widespread deployment of this single dominant resistance gene in California has driven the local emergence of multiple RB strains in different tomato production areas and types. These results further emphasize the need for ongoing monitoring for RB strains, and identification of sources of resistance to these strains.

Prevalence of coronaviruses in European bison (Bison bonasus) in Poland

Coronaviruses have been confirmed to infect a variety of species, but only one case of associated winter dysentery of European bison has been described. The study aimed to analyze the prevalence, and define the impact on the species conservation, the source of coronavirus infection, and the role of the European bison in the transmission of the pathogen in Poland. Molecular and serological screening was performed on 409 European bison from 6 free-ranging and 14 captive herds over the period of 6 years (2017–2023). Presence of coronavirus was confirmed in one nasal swab by pancoronavirus RT-PCR and in 3 nasal swab samples by bovine coronavirus (BCoV) specific real time RT-PCR. The detected virus showed high (> 98%) homology in both RdRp and Spike genes to BCoV strains characterised recently in Polish cattle and strains isolated from wild cervids in Italy. Antibodies specific to BCoV were found in 6.4% of tested samples, all originating from free-ranging animals. Seroprevalence was higher in adult animals over 5 years of age (p = 0.0015) and in females (p = 0.09). Our results suggest that European bison play only a limited role as reservoirs of bovine-like coronaviruses. Although the most probable source of infections in the European bison population in Poland is cattle, other wild ruminants could also be involved. In addition, the zoonotic potential of bovine coronaviruses is quite low.

Identification and molecular characterization of two novel viruses from Allamanda cathartica in China.

Allamanda cathartica is an ornamental medicinal plant that grows widely in the tropics. In the present study, two novel viruses, Allamanda chlorotic virus A (AlCVA) and Allamanda chlorotic virus B (AlCVB), were identified in an A. cathartica plant with interveinal chlorosis by ribosomal RNA-depleted total-RNA sequencing. Phylogenetic analysis and sequence comparisons confirmed that AlCVA and AlCVB belong to the families Closteroviridae and Betaflexiviridae, respectively. Long, flexuous, filamentous virus particles approximately 12 nm in diameter and 784-2291 nm in length were observed using transmission electron microscopy. A specific RT-PCR assay was used to demonstrate a consistent association of viral infection with symptoms.

Evaluation of Mastadenovirus and Rotavirus Presence in Phyllostomid, Vespertilionid, and Molossid Bats Captured in Rio Grande do Sul, Southern Brazil.

Bat-borne viruses may affect public health and the global economy. These mammals have a wide geographical distribution and unique biological, physiological, and immunogenic characteristics, allowing the dissemination of many known and unknown viruses. Enteric viruses, such as adeno (AdV) and rotaviruses, are recognized as the main causative agents of disease and outbreaks. In the present study, the presence of viruses from Adenoviridae and Reoviridae families was evaluated in molossid, phyllostomid, and vespertilionid bats captured in Rio Grande do Sul, Southern Brazil, between September 2021 and July 2022. Sixty bat rectal swabs were analyzed by PCR. Eight (13.3%) samples were positive for adenovirus and classified as human mastadenovirus C (HAdV-C) (three samples) and HAdV-E (five samples) by sequencing followed by phylogenetic analysis. All samples were negative in rotavirus specific RT-PCR. This is the first study to describe the presence of HAdV in samples of Glossophaga soricina, Eptesicus brasiliensis, and Histiotus velatus. Furthermore, the presence of HAdV-E in bats was reported, which is unusual and may suggest that other HAdV genotypes, in addition to HAdV-C, may also be harbored by wild animals. The data generated in the present study reinforces the importance of eco-surveillance of viral agents related to diseases in humans and wild animals. In addition, it is essential to identify possible new hosts or reservoirs that increase the risk of spillover and dissemination of infectious pathogens, helping to prevent and control zoonotic diseases.

Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance.

The major arboviruses mainly belong to the Bunyaviridae, Togaviridae, and Flaviviridae families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R2 = 0.99, for DENV-2 and DENV-3; R2 = 0.98, for DENV-4; and CHIKV; R2 = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10-1 copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 101 for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.

Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extract.

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.

Emergence of enterovirus D68 in a Norwegian paediatric population 2012-2022

BackgroundEnterovirus D68 (EV-D68) primarily causes respiratory infection, occasionally manifesting with neurological symptoms. Outbreak reports have been published from various countries including Norway, but a longitudinal study on EV-D68 prevalence in Northern Europe is lacking.MethodsRespiratory samples from children ≤14 years received at Oslo University Hospital in the years 2012-2022 were examined for EV-D68. Samples from 2012-2015 were retrospectively screened using a semi-specific RT-PCR, with positive samples confirmed by an EV-D68 specific RT-PCR. Samples from 2016-2022 underwent routine diagnostics with the EV-D68 specific RT-PCR.ResultsAmong the 22,911 samples tested, EV-D68 was detected in 338 samples (324 patients). Most EV-D68 cases occurred in August to December. The highest detection rate was recorded in 2014, 2016 and 2022 (6.0%, 7.8% and 6.6% of samples from August-December). Lower frequencies were observed in 2018 and 2019 (1.0% and 2.4%), and in the years before the 2014 outbreak (2012: 1.3%, 2013: 0.8%). Few cases were identified in 2020-2021. Children aged 0-1 years accounted for 40%, and 0-4 years for 78%, of the EV-D68 positive patients. Most of the patients with EV-D68 (83%) were hospitalised.DiscussionAlso in Norway, EV-D68 has caused outbreaks with significant disease burden, especially among the youngest children. The detection rate varies, with a trend towards biennial outbreaks, except for low numbers in 2018 and during the COVID-19 restrictions (2020-2021). Due to its potential for severe respiratory illness and significant neurological complications, conducting EV-D68 testing is essential both for diagnosing clinically suspected cases, and for monitoring the disease burden.

Enhancing CAR-T cells: unleashing lasting impact potential with phytohemagglutinin activation in in vivo leukemia model.

Chimeric antigen receptor T (CAR-T) cell therapy holds great promise as an innovative immunotherapeutic approach for cancer treatment. To optimize the production and application of CAR-T cells, we evaluated the in vivo stability and efficacy capacities of CAR-T cells developed under different conditions. In this study, CAR-T cells were activated using Phytohemagglutinin (PHA) or anti-CD3&anti-CD28 and were compared in an in vivo CD19+B-cell cancer model in mouse groups. Our results demonstrated that CAR-T cells activated with PHA exhibited higher stability and anti-cancer efficacy compared to those activated with anti-CD3&anti-CD28. Specifically, CAR19BB-T cells activated with PHA exhibited continuous proliferation and long-term persistence without compromising their anti-cancer efficacy. Kaplan-Meier survival analysis revealed prolonged overall survival in the CAR-T cell-treated groups compared to the only tumor group. Furthermore, specific LTR-targeted RT-PCR analysis confirmed the presence of CAR-T cells in the treated groups, with significantly higher levels observed in the CAR19BB-T (PHA) group compared to other groups. Histopathological analysis of spleen, kidney, and liver tissue sections indicated reduced inflammation and improved tissue integrity in the CAR-T cell-treated groups. Our findings highlight the potential benefits of using PHA as a co-stimulatory method for CAR-T cell production, offering a promising strategy to enhance their stability and persistence. These results provide valuable insights for the development of more effective and enduring immunotherapeutic approaches for cancer treatment. CAR-T cells activated with PHA may offer a compelling therapeutic option for advancing cancer immunotherapy in clinical applications.

Characterization of a New Toti-like Virus in Sea Bass, Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is the main species reared in Mediterranean aquaculture. Its larval stage, which is very sensitive and highly affected by sanitary and environmental conditions, is particularly scrutinized in hatcheries. Recently, a Mediterranean sea bass farm had to deal with an abnormal increase in mortality, especially between 20 and 35 days post-hatching (dph). Biological investigations led to the observation of cytopathic effects on three different fish cell lines after almost 3 weeks of culture at 14 °C in contact with homogenized affected larvae, suggesting the presence of a viral agent. High-throughput sequencing revealed a 6818-nucleotide-long RNA genome with six putative ORFs, corresponding to the organization of viruses belonging to the Totiviridae family. This genome clustered with the newly described and suggested Pistolvirus genus, sharing 45.5% to 37.2% nucleotide identity with other piscine toti-like viruses such as Cyclopterus lumpus toti-like virus (CLuTLV) or piscine myocarditis virus (PMCV), respectively. Therefore, we propose to name this new viral agent sea bass toti-like virus (SBTLV). Specific real-time RT-PCR confirmed the presence of the viral genome in the affected larval homogenate from different production batches and the corresponding cell culture supernatant. Experimental infections performed on sea bass fingerlings did not induce mortality, although the virus could be detected in various organs and a specific immune response was developed. Additional studies are needed to understand the exact involvement of this virus in the mortality observed in hatcheries and the potential associated cofactors.

Rapid and Accurate Diagnosis of Dermatophyte Infections Using the DendrisCHIP® Technology.

Dermatophytosis is a superficial fungal infection with an ever-increasing number of patients. Culture-based mycology remains the most commonly used diagnosis, but it takes around four weeks to identify the causative agent. Therefore, routine clinical laboratories need rapid, high throughput, and accurate species-specific analytical methods for diagnosis and therapeutic management. Based on these requirements, we investigated the feasibility of DendrisCHIP® technology as an innovative molecular diagnostic method for the identification of a subset of 13 pathogens potentially responsible for dermatophytosis infections in clinical samples. This technology is based on DNA microarray, which potentially enables the detection and discrimination of several germs in a single sample. A major originality of DendrisCHIP® technology is the use of a decision algorithm for probability presence or absence of pathogens based on machine learning methods. In this study, the diagnosis of dermatophyte infection was carried out on more than 284 isolates by conventional microbial culture and DendrisCHIP®DP, which correspond to the DendrisCHIP® carrying oligoprobes of the targeted pathogens implicated in dermatophytosis. While convergence ranging from 75 to 86% depending on the sampling procedure was obtained with both methods, the DendrisCHIP®DP proved to identify more isolates with pathogens that escaped the culture method. These results were confirmed at 86% by a third method, which was either a specific RT-PCR or genome sequencing. In addition, diagnostic results with DendrisCHIP®DP can be obtained within a day. This faster and more accurate identification of fungal pathogens with DendrisCHIP®DP enables the clinician to quickly and successfully implement appropriate antifungal treatment to prevent the spread and elimination of dermatophyte infection. Taken together, these results demonstrate that this technology is a very promising method for routine diagnosis of dermatophytosis.

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/μL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Surveillance, epidemiology, and impact of the coronavirus disease 2019 interventions on the incidence of enterovirus infections in Nanchang, China, 2010-2022.

Pathogen spectrum of Hand, foot and mouth disease (HFMD) has substantially changed in the past decade in China. Growing evidence has indicated that anti-COVID-19 nonpharmaceutical interventions (NPIs) can support control of various infectious diseases, including intestinal diseases. In this study, HFMD cases were enrolled from sentinel hospitals of Nanchang, Jiangxi province, and enteroviruses were genotyped using specific real time RT-PCR. We systematically characterized the epidemiology of HFMD based on the continuous molecular surveillance and estimated the impact of COVID-19 intervention on HFMD incidence using seasonal autoregressive integrated moving average (ARIMA) models. A total of 10247 HFMD cases were included during 2010-2022, of which 6121 enterovirus (EV)-positive cases (59.7%) were identified by real-time RT-PCR. Over 80% cases were associated with EV-A71 and coxsackievirus A16 (CVA16) during 2010-2012, while the type distribution significantly changed as CVA6 emerged to be dominant, accounting for 22.6%-59.6% during 2013-2022. It was observed that the prevalence patterns of EV-A71 and CVA16 were similar and both of them peaked in the second quarter and then leveled off. However, CVA6 was generally prevalent around the fourth quarter, demonstrating a staggered prevalence during 2010-2019. During the COVID-19 epidemic, the seasonal HFMD epidemic peak was restrained, and the ARIMA analysis indicated that the COVID-19 intervention had mitigated EV transmission during the first COVID-19 outbreak in early 2020. In addition, bivariate Spearman's cross-correlation coefficients were estimated for the major types CVA6, CVA16 and EV-A71. Our analyses indicated the possible existence of correlations among CVA6, CVA16 and EV-A71 prevalence in the epidemiological level. Taken together, the type distribution of HFMD has substantially changed over the last decade and CVA6 and CVA16 are currently the most predominant types co-circulating in Nanchang. The anti-COVID-19 NPIs significantly reduced the incidence of EV infections.

Cross-sectional assessment of a history of SARS-CoV-2 infections using IgG

IntroductionSevere acute respiratory syndrome, coronavirus type 2 (SARS-CoV-2) has become a global threat for every healthcare system, and the coronavirus disease 2019 (COVID-19) pandemic has resulted in over 3 million deaths worldwide. SARS-CoV-2 brings symptoms such as fever, cough, shortness of breath, headache, sore throat and loss of taste or smell. Diagnostic of COVID-19 may include specific RT-PCR for viral ribonucleic acid detection, and ELISA testing for virus-derived spike protein or nucleocapsid.AimThe aim of this study was to measure the antinucleocapsid level of SARS-CoV-2 IgG to identify the number of asymptomatic cases of COVID-19 after infection in a population of workers from a production company.Material and methodsHuman anti-SARS-CoV-2(N) IgG ELISA kit was used to determine serum IgG level. Study includes 107 individuals (48% female, 52% male) in different ages (18–60 years).Results and discussionOf 107 tested individuals in 80 (74.7%) cases SARS-CoV-2(N)-specific IgG antibodies were detected, with an average antibody concentration of the whole study group 4.08 µg/mL (<i>n</i> = 107 with the range 0.59–7.91 µg/mL; <i>n</i> = 80 were included in the study with the sensitivity of the method above 2.344 µg/mL). In only 9 cases, SARS-CoV-2 infection was confirmed before using the PCR test. Our data underscore the need for a population study in Poland to test the proportion of asymptomatic IgG positive for SARS-CoV-2 individuals.ConclusionsThis study indicates that within studied sample large proportion of asymptomatic people have undergone SARS-CoV-2 infection and suggests that isolation of only symptomatic patients would not stop the transmission of the virus.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

Severe Neurologic Disease in a Horse Caused by Tick-Borne Encephalitis Virus, Austria, 2021.

As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encephalitis viruses with RT-qPCR, RT-PCR, and qPCR, respectively. TBEV-specific amplification products were subjected to Sanger sequencing. In addition, a direct fluorescent antibody test for rabies was performed. Clinical and patho-histological findings are reported. Using specific RT-qPCR and RT-PCR assays, TBEV nucleic acids were demonstrated in brain tissue samples. Sequencing revealed the Western (formerly Central) European subtype of TBEV as the etiological agent. A high titer of TBEV-specific neutralizing antibodies was found in the serum. RNAscope in situ hybridization revealed TBEV RNA confined to neuronal cell bodies and processes. No other pathogens or nucleic acids thereof could be detected. Diagnostic procedures need to be carried out early after the onset of neurological signs to allow for a final etiological diagnosis of acute TBEV infections in horses.

Analysis of interleukin 7 and platelet-derived growth factor-BB mRNA expression as potential markers in erythema nodosum leprosum.

Erythema nodosum leprosum (ENL) is an immunological complication of leprosy characterized by acute inflammation of the skin, nerves, and other organs. Identifying laboratory parameters is important for early diagnosis of leprosy reactions. Various cytokine biomarkers have been examined and only a few studies have reported on angiogenesis in leprosy. This study aims to understand the pathomechanism of ENL by examining IL-7 and platelet-derived growth factor (PDGF)-BB mRNA expression that can be the development and consideration of new effective therapies to prevent reactions, recurrences, and defects in leprosy. The study used a cross-sectional analytic design. Sampling was done by peripheral blood from the patient and measuring mRNA expression with specific primers RT-PCR. The expression of mRNA IL-7 and PDGF-BB was significantly different between multibasilar patients without reaction and with ENL reaction, where there was an increased expression in ENL patients. This could be used as the development of potential biomarkers in ENL and development of new therapeutic intervention pathways in ENL.