Detection Of Viral Genome Research Articles

Reduction of foot-and-mouth disease virus transmission in cattle vaccinated one or two weeks before challenge using a commercial polyvalent vaccine

Immediate vaccination of the most susceptible and epidemiological relevant animals is a crucial part of control measures that facilitate virus elimination in case of entry of foot-and-mouth disease (FMD). The objective of this study was to evaluate the effect of cattle vaccination 7 and 14 days prior challenge using a vaccine commonly applied in systematic vaccination campaigns against transmission of FMD virus (FMDV). Transmission of FMDV was investigated in three groups of ten cattle each: one non-vaccinated group and two groups that were either vaccinated 7 days (−7/vaccinated group) or 14 days (−14/vaccinated group) before intranasal (IN) inoculation. Five cattle heads from each group were inoculated using the IN-route with the A/Argentina/2001 FMDV strain, while the remaining five cattle heads of each group were contact-exposed to inoculated cattle. Clinical signs were recorded; virus isolation and genome detection by RT-PCR were carried out on oesophageal–pharyngeal fluid (OPF) and blood. Neutralizing antibody titers and antibodies against non-structural proteins (NSP) of FMDV were also determined. Results suggest that the experimental design, virus challenge dose, and virus infectivity were appropriate and that the virus had been transmitted to naïve calves. Under the outlined experimental conditions, vaccination 7 and 14 days prior to challenge induced full clinical protection against virus inoculation. Moreover, −7/ or −14/vaccinated calves that had been contact-exposed to −7/ or −14/vaccinated IN-challenged calves, did not become infected. Consequently, no virus transmission occurred from vaccinated and subsequently infected calves to cohabitating vaccinated calves (R = 0). According to our results, early vaccination during an outbreak is effective as virus transmission can be significantly reduced using a vaccine commercially available, routinely applied in systematic vaccination campaigns.

Cytomegalovirus, Epstein-Barr virus, and human herpesvirus-6 infections in patients with myalgic еncephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling multisystem chronic disease. The etiology and pathogenesis of ME/CFS are unknown. Infections of cytomegalovirus (CMV), Epstein‐Barr virus (EBV), and human herpesvirus‐6 (HHV‐6) are suspected as etiological agents for ME/CFS. This study aims to estimate prevalence and type (active/latent) of EBV, CMV, and HHV‐6 infections in Bulgarian ME/CFS patients. In the study were included 58 patients with ME/CFS and 50 healthy controls. Virus‐specific antibodies were detected by enzyme‐linked immunosorbent assay and viral genomic sequences in peripheral blood mononuclear cell (PBMCs) and plasma samples by nested polymerase chain reaction (PCR). We did not observe any significant differences in virus‐specific immunoglobulin G and immunoglobulin M positivity rates between patients with ME/CFS and control group. In ME/CFS plasma samples, EBV DNA was found in 24.1%, CMV DNA in 3.4%, and HHV‐6 DNA in 1.7% of samples. EBV DNA was detected in 4%, and CMV and HHV‐6 DNA were not found in plasma samples of controls. The frequency of viral genome detection in PBMCs of patients and controls was 74% vs 78% for CMV, 81% vs 84% for EBV, and 82.8% vs 82% for HHV‐6. The difference in frequency of EBV active infection in ME/CFS and control group was statistically significant (P = .0027). No ME/CFS and control individuals with active CMV and HHV‐6 infection were observed. In conclusion, this study using both serological and PCR‐based techniques for distinguishing between active and latent infection showed high rate of active EBV infection among patients with ME/CFS indicating that at least in a subset of cases, EBV is important factor for the development of disease.

Myocardial strain on admission predicts disease severity in infants hospitalized for bronchiolitis.

To assess cardiac function in infants with bronchiolitis and the association with disease severity and outcomes. Cardiac function may be impaired in bronchiolitis and represent an early predictor of disease severity. A prospective cohort study. Infants with suspected bronchiolitis were included. All cases received antigen detection and viral genome detection from nasal lavage or swabs and echocardiography within 24 hours from admission. Systolic and diastolic function in left ventricle (LV) and right ventricle (RV) were assessed using longitudinal strain (LS), a measure of myocardial deformation. Pulmonary artery pressures were estimated using tricuspid regurgitation jet (TR), when present, and end-systolic eccentricity index (EI ES). Main outcomes (duration of respiratory support, length of stay [LOS], and type of respiratory support) were collected. Data were compared to normative existing data, and a group of healthy infants, matched in age. Twenty-eight infants with bronchiolitis and 10 healthy comparators were included. Cases with bronchiolitis showed significantly lower values of RV LS and LV LS compared to healthy comparators (LV: p0.04 and RV: P < .001). Ten infants (36%) had a normal biventricular function, nine (32%) had LV impairment, and nine (32%) had a biventricular impairment. No significant differences were found in TR and EI ES. Infants with biventricular impairment demonstrated a significant increase in LOS (p0.04) and higher levels of respiratory support compared to the healthy comparators (P = .03). Bronchiolitis is associated with myocardial impairment. Cardiac function is related to disease severity and outcome.

Nationwide prevalence of Torque teno sus virus 1 and k2a in pig populations in Japan.

Because broad genetic diversity has recently been detected in Torque teno sus viruses (TTSuV1 and TTSuVk2), the viral genome detection method needs to be improved to understand the prevalence of these viruses. Here, we established single PCR-based detection methods for the TTSuV1 and TTSuVk2a genomes with newly designed primer pairs and applied them to investigate the prevalence of TTSuV1 and TTSuVk2a in Japanese pig populations. The results revealed that 98.2% and 81.7% of the pig farms tested positive for the TTSuV1 and TTSuVk2a genomes, respectively, indicating that both TTSuV1 and TTSuVk2a are widespread in Japan.

Detection and characterization of Ilheus and Iguape virus genomes in historical mosquito samples from Southern Brazil

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.

Mosquito-Independent Transmission of West Nile virus in Farmed Saltwater Crocodiles (Crocodylus porosus).

West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2–17 dpi and in-contact animals 11–21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.

Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Viral serology in “infarct-like” acute myocarditis: Useful or useless?

The most common cause of myocarditis is viral infection, especially from cardiotropic viruses (Parvovirus B19, Human Herpesvirus 6 and Coxsackie) resulting in a destruction of the myocardium. Moreover, a serological analysis is not systematic and their value remains unclear. The objectives of this study were to assess the viral serology of the patients with acute myocarditis and the relation with clinical and CMR parameters at the acute phase and at distance. All cases of infarct-like acute myocarditis confirmed by CMR from April 2012 to January 2018 were included in a prospective study at Dijon University Hospital. A blood sampling including viral serology is obtained at the moment of the diagnostic. One-year follow-up included a complete clinical and rhythmic evaluation, and a CMR control. Among the patients with “infarct like” acute myocarditis, serological analyses were performed in 62 patients and cardiotropic viruses are found in 29 patients. No statistical difference was observed regarding demographic and clinical parameters. However, concerning CMR parameters, the extent of myocarditis is more important in the “cardiotropic viruses” group at the acute phase (10.4 vs. 7.3 – P = 0.05) and at three months (6.3 vs. 2.7 – P = 0.007) compared to the “no cardiotropic viruses” group. At one year, there was no significant difference between the two groups regarding CMR parameters. Cardiotropic viruses (HHV6, Parvovirus B19 and Coxsackie virus) were found in almost 50% of cases in “infarct like” acute myocarditis. According the presence or absence of cardiotropic viruses, CMR parameters were different. Furthermore, use of quantitative PCR (detection of viral genomes) or detection of viral DNA and RNA via in situ hybridization would be a challenge for the future identification of viruses in myocarditis.

Persistence of Viral RNA, Pneumocyte Syncytia and Thrombosis Are Hallmarks of Advanced COVID-19 Pathology

Background: COVID-19 is a deadly pulmonary disease with peculiar characteristics, which include variable clinical course and thrombophilia. A thorough understanding of the pathological correlates of the disease is still missing.Methods: Here we report the systematic analysis of 41 consecutive post-mortem samples from individuals who died of COVID-19. Histological analysis is complemented by immunohistochemistry for cellular and viral antigens and the detection of viral genomes by in situ RNA hybridization.Findings: COVID-19 is characterized by extensive alveolar damage (41/41 of patients) and thrombosis of the lung micro- and macro-vasculature (29/41, 71%). Thrombi were in different stages of organization, consistent with their local origin. Pneumocytes and endothelial cells contained viral RNA even at the later stages of the disease. An additional feature was the common presence of a large number of dysmorphic pneumocytes, often forming syncytial elements (36/41, 87%). Despite occasional detection of virus-positive cells, no overt signs of viral infection were detected in other organs, which showed non-specific alterations.Interpretation: COVID-19 is a unique interstitial pneumonia characterized by extensive lung thrombosis, long-term persistence of viral RNA in pneumocytes and endothelial cells, along with the presence of infected cell syncytia. Several of COVID-19 features might be consequent to the persistence of virus-infected cells for the duration of the disease.Funding: This work was supported by a King’s Together Rapid COVID-19 Call grant from King’s College London. MG is supported by the European Research Council (ERC) Advanced Grant 787971 "CuRE" and by Programme Grant RG/19/11/34633 from the British Heart Foundation.Declaration of Interests: The authors declare no conflict of interests.Ethics Approval Statement: This study was approved by the competent Joint Ethical Committee of the Regione Friuli Venezia Giulia, Italy.

Comparison of colorimetric loop-mediated isothermal amplification kit and reverse transcription-polymerase chain reaction in the diagnosis of peste des petits ruminants in sheep and goats in Southeast Nigeria

Background and Aim:Peste des petits ruminants (PPR) is an acute, extremely contagious transboundary viral disease of small ruminants with severe economic consequences, caused by PPR virus. Cost-effective and rapid diagnosis of the disease is essential for prompt management and control. This study aimed to compare the application of a commercial colorimetric loop-mediated isothermal amplification (cLAMP) kit and reverse transcriptase-polymerase chain reaction (RT-PCR) in the diagnosis of PPR in sheep and goats in Southeast Nigeria.Materials and Methods:Nasal swab samples were collected from West African Dwarf sheep and goats showing clinical signs suggestive of PPR (n=80) and those without any clinical signs (n=140) of the disease. The diagnosis was achieved through detection of PPR viral genome in the samples using a cLAMP kit and RT-PCR. cLAMP assay was done directly on nasal swab samples without ribosomal nucleic acid extraction. A set of six primers targeting the matrix gene protein was used for the cLAMP assay.Results:PPR viral genome was detected by both cLAMP and RT-PCR in 51 (63.8%) of the 80 samples from sheep and goats with signs suggestive of PPR while 14 (10%) of those without signs tested positive for PPR by both assay methods. There was a 100% agreement in the cLAMP and RT-PCR results. However, cLAMP was a faster, easier, and less expensive method compared to RT-PCR.Conclusion:The cLAMP assay demonstrates the potential for a point of care diagnosis in the field and a valuable diagnostic tool in areas with poor electricity supply as well as in a less equipped diagnostic laboratory. Since the reagents are affordable, cLAMP can be a diagnostic tool of choice in the detection and surveillance of PPR virus in countries with limited resources.

Covid-19 pandemic: History, aetiology, epidemiology, vaccinology and societal impact

Covid-19 pandemic: History, aetiology, epidemiology, vaccinology and societal impact

A novel avian isolate of hepatitis E virus from Pakistan

BackgroundAvian hepatitis E virus (aHEV) has been associated with hepatitis-splenomegaly syndrome (HSS) in chickens along with asymptomatic subclinical infection in many cases. So far, four genotypes have been described, which cause infection in chickens, specifically in broiler breeders and layer chickens. In the present study, we isolated and identified two novel aHEV strains from the bile of layer chickens in Pakistan evincing clinical symptoms related to HSS.MethodologyHistology of liver and spleen tissues was carried out to observe histopathological changes in these tissues. Bile fluid and fecal suspensions were used for viral RNA isolation through MegNA pure and Trizol method which was further used for viral genome detection and characterization by cDNA synthesis and amplification of partial open reading frame (ORF) 1, ORF2 and complete ORF3. The bioinformatics tools; Molecular Evolutionary Genetics Analysis version 6.0 (MEGA 6), Mfold and ProtScale were used for phylogenic analysis, RNA secondary structure prediction and protein hydropathy analysis, respectively.ResultsSequencing and phylogenetic analysis on the basis of partial methyltranferase (MeT), helicase (Hel) domain, ORF2 and complete ORF3 sequence suggests these Pakistani aHEV (Pak aHEV) isolates may belong to a Pakistani specific clade. The overall sequence similarity between the Pak aHEV sequences was 98–100%. The ORF1/ORF3 intergenic region contains a conserved cis-reactive element (CRE) and stem-loop structure (SLS). Analysis of the amino acid sequence of ORF3 indicated two hydrophobic domains (HD) and single conserved proline-rich domain (PRD) PREPSAPP (PXXPXXPP) with a single PSAP motif found in C-terminal. Amino acid changes S15 T, A31T, Q35H and G46D unique to the Pak aHEV sequences were found in the N-terminal region of ORF3.ConclusionsOur data suggests that Pak aHEV isolates may represent a novel Pakistani clade and high sequence homology to each other support the supposition they may belong to a monophyletic clade circulating in the region around Pakistan. The data presented in this study provide further information for aHEV genetic diversity, genotype mapping, global distribution and epidemiology.

P5556Impact of viral genome detection in endo-myocardial biopsy of arrhythmogenic cardiomyopathy substrate

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is a genetically inherited cardiomyopathy characterized by myocardial fibro-fatty replacement. A pathogenetic role of viral myocardial infections in ACM natural history has been proposed over the years, although no definitive conclusion has been reached yet. Purpose To describe viral genome presence into a cohort of ACM biopsy proven patients (pts) and its impact on clinical features and outcome. Methods A cohort of all ACM pts undergoing an invasive third level evaluation at our institution was enrolled. All pts underwent a cardiac magnetic resonance (MR), an invasive electrophysiological study (EPS) with endo-cavitary electro-anatomical mapping (EAM), and a EAM guided endo-myocardial biopsy (EMB). Viral genome research through molecular biology techniques was performed on all biopsied samples. According to arrhythmic risk evaluation, a trans-catheter ablation (TCA) and/or an internal cardioverter device (ICD) implant was performed. Clinical arrhythmic presentation, MR data, arrhythmia inducibility at EPS, EAM and EMB characteristic, and arrhythmic events at a 12-month follow up visit were retrieved in all pts and compared between the viral genome positive (v+ACM) and negative group (v-ACM). Results Forty-five pts were enrolled in our study (48±13 years; 66% male); the EMB samples of 7 (15%) pts presented a lymphocytic infiltrate and tested positive for viral genome (n=3 B19 Parvovirus; n=2 for Citomegalovirus; n=2 for Ebstein-Bar Virus) [Figure1]. At arrhythmic presentation, complex ventricular arrhythmias (NSVT, SVT and FV) were more frequent in the v+ACM group (86% vs 50%; p=0.039). Both left and right ventricular ejection fraction at MR resulted more depressed in the v+ACM group (44±7 vs 52±2 and 47±2 vs 52±2; p=0.047 and p=0.041). Complex ventricular arrhythmia inducibility at EPS was more frequent in v+ACM (72% vs 34%; p=0.032), while no differences in pathological potentials rate and extension at unipolar and bipolar EAM were found. TCA was performed in 55% and 57% and an ICD was implanted in 29% and 42% in the v+ACM and v-ACM group respectively. No differences in 12-months arrhythmic event rates (39% vs 42%) between the two groups were described. EBM at different magnifications/stains Conclusion In our cohort a viral infection super-imposed to the fibrofatty infiltration was found in 15% of the patients. ACM pts testing positive for viral genome at the EMB had a more severe arrhythmic disease presentation, a more impaired heart function, and a higher rate of complex ventricular arrhythmias at disease presentation, but seemed to respond as well as viral genome negative ACM to ablative and pharmacological treatment

Impact of genetic diversity on biological characteristics of Usutu virus strains in Africa

Usutu virus (USUV) previously restricted to Africa where it caused mild infections, emerged in 2001 in Europe and caused more severe infections among birds and humans with neurological forms, suggesting an adaptation and increasing virulence. This evolution suggests the need to better understand USUV transmission patterns for assessing risks and to develop control strategies. Phylogenetic analysis conducted in Africa showed low genetic diversity of African USUV strains except for one human and the USUV subtype (USUVsub) strains, which exhibited a deletion in the 3′UTR and nucleotide substitutions throughout the genome. Here we analyzed their viral replication in vitro in mosquito and mammalian cells, and vector competence of Culex quinquefasciatus, compared to a reference strain. Growth kinetics of the different strains showed comparable replication rates however variations in replication and translation efficiency were observed. Vector competence analysis showed that all strains were able to infect Culex quinquefasciatus the main peridomestic Culex species in Africa, with detection of USUV viral genomes and infectious particles. Dissemination and transmission were observed only for USUVsub, but infectious particles were not detected in Culex quinquefasciatus saliva. Our findings suggest that genetic variability can affect USUV in vitro replication in a cell type-dependent manner and in vivo in mosquitoes. In addition, the results show that Culex quinquefasciatus is not competent for the USUV strains analyzed here and also suggest an aborted transmission process for the USUVsub, which requires further investigations.

Investigation about the Occurrence of Transmission Cycles of Arbovirus in the Tropical Forest, Amazon Region.

Because of its ecological characteristics, the Caxiuanã National Forest (FLONA) is a potential area of arbovirus circulation. The present study aimed to investigate the occurrence of arbovirus transmission cycles at FLONA de Caxiuanã. Five field trips were performed to capture mosquitoes and sylvatic vertebrates. For these vertebrates, we attempted viral isolation by cell monolayer inoculation from blood, and hemagglutination inhibition and further seroneutralization assays from sera. For mosquitoes, we performed tests of viral genome detection. A total of 338 vertebrates were captured, and the greatest representative was birds (251/74.26%). A total of 16,725 mosquitoes were captured, distributed among 56 species. There were no viruses isolated by newborn mouse inoculation. Among birds, antibodies against Ilheus virus were the most prevalent. Catu virus, Caraparu virus, and Mucambo virus were the most prevalent among mammals and reptiles. Fragments of Mucambo virus, Ilheus virus, Bussuquara virus, and Rocio virus genome were detected in a pool of mosquito samples. These results of the study suggest the occurrence of arbovirus transmission cycles in the FLONA of Caxiuanã. The proximity of human populations with elements, involved in transmission cycles, makes surveillance necessary in this population to avoid dispersion of arboviruses to naïve locations.

Impact of nucleic acid extraction platforms on hepatitis virus genome detection

Detection and quantification of viral nucleic acids are important for diagnosing current viral infections and monitoring response to antiviral therapy. Automated nucleic acid extraction and purification platforms are routinely used during the first step in these processes in clinical and research laboratories. Here, we compare the extraction efficiencies of four MagNA Pure magnetic bead-based nucleic acid extraction platforms and associated kits using samples positive for nucleic acids from HAV, HBV, HCV, HDV, and HEV. These five hepatitis viruses are diverse in their virion structures and type of nucleic acid that compose their genomes. We found that the most efficient nucleic acid extraction platform and corresponding kit, when averaged across all tested viruses, was the MagNA Pure 96, which yielded twice as much detectable nucleic acid as the other platforms. However, the relative efficiencies of the different platforms varied by virus type, suggesting that an extraction platform that is more efficient for one virus type will not necessarily function better with a different virus type. Our results show that the choice of a nucleic acid extraction platform influences the sensitivity of the methodology and has the potential to generate false-negative results especially in samples with low levels of viral nucleic acids.

Тест-система для выявления генома вируса заразного узелкового дерматита крупного рогатого скота методом ПЦР в реальном времени

Тест-система для выявления генома вируса заразного узелкового дерматита крупного рогатого скота методом ПЦР в реальном времени

High-throughput isolation of giant viruses using high-content screening

The race to discover and isolate giant viruses began 15 years ago. Metagenomics is counterbalancing coculture, with the detection of giant virus genomes becoming faster as sequencing technologies develop. Since the discovery of giant viruses, many efforts have been made to improve methods for coculturing amebas and giant viruses, which remains the key engine of isolation of these microorganisms. However, these techniques still lack the proper tools for high-speed detection. In this paper, we present advances in the isolation of giant viruses. A new strategy was developed using a high-throughput microscope for real-time monitoring of cocultures using optimized algorithms targeting infected amebas. After validating the strategy, we adapted a new tabletop scanning electron microscope for high-speed identification of giant viruses directly from culture. The speed and isolation rate of this strategy has raised the coculture to almost the same level as sequencing techniques in terms of detection speed and sensitivity.

Three asymptomatic animal infection models of hemorrhagic fever with renal syndrome caused by hantaviruses.

Hantaan virus (HTNV) and Puumala virus (PUUV) are rodent-borne hantaviruses that are the primary causes of hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. The development of well characterized animal models of HTNV and PUUV infection is critical for the evaluation and the potential licensure of HFRS vaccines and therapeutics. In this study we present three animal models of HTNV infection (hamster, ferret and marmoset), and two animal models of PUUV infection (hamster, ferret). Infection of hamsters with a ~3 times the infectious dose 99% (ID99) of HTNV by the intramuscular and ~1 ID99 of HTNV by the intranasal route leads to a persistent asymptomatic infection, characterized by sporadic viremia and high levels of viral genome in the lung, brain and kidney. In contrast, infection of hamsters with ~2 ID99 of PUUV by the intramuscular or ~1 ID99 of PUUV by the intranasal route leads to seroconversion with no detectable viremia, and a transient detection of viral genome. Infection of ferrets with a high dose of either HTNV or PUUV by the intramuscular route leads to seroconversion and gradual weight loss, though kidney function remained unimpaired and serum viremia and viral dissemination to organs was not detected. In marmosets a 1,000 PFU HTNV intramuscular challenge led to robust seroconversion and neutralizing antibody production. Similarly to the ferret model of HTNV infection, no renal impairment, serum viremia or viral dissemination to organs was detected in marmosets. This is the first report of hantavirus infection in ferrets and marmosets.

Development and Validation of Heminested RT-PCR and qRT-PCR Assays for Comprehensive Detection of Rabies Virus in the Suspected Rabid Brain and Saliva Samples

Background: Rabies virus (RV) is one of the most dangerous zoonotic disease and major public health problems in most of the world, especially underdeveloped countries. Rabies is preventable by proper vaccination, even shortly after exposure. Today, it seems a fast, sensitive, and reliable rabies diagnostic method is required, which might reduce the financial burden of inappropriate diagnosis. Objectives: The aim of this study was to develop and validate two molecular techniques, including heminested RT-PCR and qRT-PCR assays, for comprehensive detection of rabies virus in the suspected rabid brain and saliva samples. Methods: In this study, we developed qRT-PCR as a fast, sensitive, and specific method for rapid detection of rabies virus in brain and saliva samples. Also, the sensitivity and specificity of the method were compared with heminested RT-PCR test and direct fluorescent antibody (dFA) as a serologic gold standard method of World Health Organization (WHO) and MIT (mouse inoculation test) as a confirmatory test. Results: A combination of compatible primers based on RNA-dependent RNA polymerase (L) gene of the Pasteur virus fixed strain (PV) (accession number. M13215) was used for developing the qRT-PCR assay. Primer and probes were designed according to other Iran circulating viruses genomes that were available in public databases (GenBank). The clinical sensitivities of qRT-PCR and heminested RT-PCR methods were determined 97.14% and 94.3%, respectively. In addition, the clinical specificities of qRT-PCR and heminested RT-PCR methods were determined 93.75% and 88.24%, respectively. Also, the analytical sensitivities of qRT-PCR and heminested RT-PCR methods were about 5 × 102 and 5 × 103 FFU/mL, respectively. Conclusions: In this study, qRT-PCR assay as a diagnostic molecular method with high sensitivity and specificity was developed for the detection of the rabies virus genome in both brain and saliva samples. Therefore, this rapid, accurate, and cost-effective detection and quantification method may be used as an investigative tool, which can be valid for detection of target viral genome in the research and diagnosis field.