scholarly journals A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis

2013 ◽  
Vol 103 (5) ◽  
pp. 488-500 ◽  
Author(s):  
Avijit Roy ◽  
Nandlal Choudhary ◽  
Leon M. Guillermo ◽  
Jonathan Shao ◽  
Ananthakrishnan Govindarajulu ◽  
...  
Keyword(s):  
Rna Virus ◽  
Phylogenetic Analyses ◽  
Pcr Primers ◽  
Diagnostic Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Causal Organism ◽  
Putative Coat Protein ◽  
Citrus Leprosis ◽  
Novel Virus

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3′ untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

scholarly journals Characterization of a Novel Emaravirus Affecting Ash Species (Fraxinus spp.) in Europe

Forests ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1574
Author(s):  
Thomas R. Gaskin ◽  
Max Tischendorf ◽  
Ines Günther ◽  
Marius Rehanek ◽  
Carmen Büttner ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Phylogenetic Analyses ◽  
Leaf Tissue ◽  
Spherical Particles ◽  
Infected Leaf ◽  
Rt Pcr ◽  
Glycoprotein Precursor ◽  
A Genome ◽  
Leaf Deformation ◽  
Novel Virus

We identified a novel virus in diseased European ash (Fraxinus excelsior) and manna ash (F. ornus) trees exhibiting chlorotic ringspots, mottle and leaf deformation such as curling and shoestring symptoms. High-throughput sequencing (HTS, Illumina RNASeq) of total RNA isolated from diseased leaf material in combination with RT-PCR-based amplification techniques and Sanger sequencing determined five complete genome segments, each encoding a single open reading frame. Sequence analyses of RNA1–RNA5 revealed a genome organization typical for emaraviruses, i.e., (i) conserved and complementary terminal 5′and 3′termini of each genome segment (ii) proteins showing significant homologies to the RNA-dependent RNA polymerase (RdRP) encoded by RNA1, the glycoprotein precursor (GPP) encoded by RNA2, the viral nucleocapsid protein (N, RNA3), the movement protein (MP, RNA4), and a protein of 26 kDA (P26, RNA5) highly similar to proteins of unknown function encoded by other emaraviruses. Furthermore, we identified spherical particles (double-membrane bodies, DMB) of different sizes (70–80 nm in diameter) which are typical for emaraviruses exclusively in virus-infected leaf tissue exhibiting mottle and leaf deformation. Sequence comparison and phylogenetic analyses confirmed the identified novel virus as a new member of the genus Emaravirus. We established a species-specific RT-PCR detection protocol and could associate the observed disease symptoms with the infection of the novel emaravirus in F. excelsior and F. ornus. Therefore, we propose the name ash shoestring-associated emaravirus (ASaV). Investigation of ASaV-infected sample trees originating from different locations in Switzerland, Germany, Italy and Sweden provided a wide geographical distribution of the virus in affected ash species. To our knowledge, this is the first confirmation of an emaravirus affecting ash tree species with shoestring symptoms of leaves in Europe.

scholarly journals Circulation of bluetongue virus in goats in the Karamoja region of Uganda

2013 ◽  
Vol 84 (1) ◽  
Author(s):  
Elijah N. Mulabbi ◽  
Chrisostom Ayebazibwe ◽  
Samuel Majalija ◽  
Carrie A. Batten ◽  
Christopher A.L. Oura
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
Bluetongue Virus ◽  
Rna Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
High Seroprevalence ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Karamoja Region

The presence of bluetongue virus (BTV) in indigenous goats from the Karamoja region of northern Uganda was investigated. A total of 300 goats were sampled (serum and whole blood) from five districts within the Karamoja region. The samples were analysed for the presence of bluetongue (BT) antibodies using a commercial Enzyme-linked immunosorbent assay (ELISA) and for the presence of BTV viral RNA by real-time Reverse transcription polymerase chain reaction (RT-PCR), because BTV is an RNA virus. Of the 300 goats tested, 269 (90%) were positive for BTV antibodies, indicating high levels of BTV circulation within the region. Out of the 150 whole blood samples tested for the presence of the virus by real-time RT-PCR, 84 (56%) were positive for BTV RNA. This study, which is the first of its kind in Uganda, showed a high seroprevalence of BT antibodies and active circulation of BTV in a high proportion of goats in the Karamoja region.

scholarly journals Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains

2007 ◽  
Vol 88 (10) ◽  
pp. 2811-2823 ◽  
Author(s):  
P. P. C. Mertens ◽  
N. S. Maan ◽  
G. Prasad ◽  
A. R. Samuel ◽  
A. E. Shaw ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Rna Virus ◽  
Pcr Primers ◽  
Virus Genome ◽  
Rapid Identification ◽  
Rt Pcr ◽  
Perfect Agreement ◽  
Virus Serotype ◽  
Primary Determinant ◽  
Protective Antibodies

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 ° S and 5 ° N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

scholarly journals TNF-α levels in respiratory samples are associated with SARS-CoV-2 infection.

2021 ◽  
Author(s):  
Matias Javier Pereson ◽  
Maria Noel Badano ◽  
Natalia Aloisi ◽  
Roberto Chuit ◽  
Maria Marta Braco ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serum Levels ◽  
Pcr Primers ◽  
Tnf Alpha ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Entry Site ◽  
Disease Outcomes ◽  
Tnf Α ◽  
And Gender

Purpose: Increased serum levels of IL-6 and TNF-alpha have been proposed as biomarkers for COVID-19 progression. However, the role and the implication of these cytokines in SARS-CoV-2 infection remain controversial. The aim of this study was to measure levels of IL-6 and TNF-alpha in swab samples from individuals with symptoms compatible with COVID-19 and analyze their association with SARS-CoV-2 presence. Methods: SARS-CoV-2 detection was performed using the CDC (USA) real-time RT-PCR primers, probes and protocols. Cytokine concentrations were measured using commercial reagents based on enzyme linked immunosorbent assay (ELISA). Results: TNF-alpha median levels were greater in COVID19 (+) symptomatic group (5.88 (1.36 - 172.1) pg/ml) compared to COVID19 (-) symptomatic individuals (2.87 (1.45 - 69.9) pg/ml) (p=0.0003). No significant differences were shown in IL-6 median values between COVID-19 (+) and (-) symptomatic patients (5.40 (1.7 - 467) pg/ml and 6.07 (1.57 - 466.6) pg/ml respectively). In addition, increased TNF-alpha; levels (greater than 10 pg/ml), but not IL-6, were associated with SARS-CoV-2 presence (OR= 5.7; p=0.006; 95% CI= 1,551 to 19,11). Conclusions: IL-6 concentration showed high levels in swabs from some symptomatic patients, suggesting the presence of immune response at viral entry site. However, IL-6 levels were independent from SARS-CoV-2 presence and viral load, individual's age and gender. On the contrary, TNF-alpha evaluation confirmed the presence of inflammatory response but mostly related to COVID-19. More studies are required in order to characterize the cytokine profile expressed at the site of infection of SARS-CoV-2 and its implications in disease outcomes.

scholarly journals Genetic Recombination during Coinfection of Two Mutants of Human Respiratory Syncytial Virus

2003 ◽  
Vol 77 (20) ◽  
pp. 11201-11211 ◽  
Author(s):  
Kirsten M. Spann ◽  
Peter L. Collins ◽  
Michael N. Teng
Keyword(s):  
Respiratory Syncytial Virus ◽  
Genetic Recombination ◽  
Rna Virus ◽  
Pcr Primers ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Syncytial Virus ◽  
And Control ◽  
Optimized Conditions

ABSTRACT Recombination between coinfecting viruses had not been documented previously for a nonsegmented negative-strand RNA virus (mononegavirus). We investigated the potential of intermolecular recombination by respiratory syncytial virus (RSV) by coinfecting HEp-2 cells with two recombinant RSV (rRSV) mutants lacking either the G gene (ΔG/HEK) or the NS1 and NS2 genes (ΔNS1/2). These viruses replicate inefficiently and form pinpoint plaques in HEp-2 cells. Therefore, potential recombined viruses with a growth and/or plaque formation advantage should easily be identified and differentiated from the two parental viruses. Further identification of potential recombinants was aided by the inclusion of point mutation markers in the F and L genes of ΔG/HEK and the design of reverse transcription-PCR (RT-PCR) primers capable of detecting these markers. Independent coinfections and control single infections by these two rRSV mutants were performed. In one of six coinfections, an RSV variant was identified that produced plaques slightly larger than those of wild-type RSV in HEp-2 cells. RT-PCR and sequencing provided evidence that this variant was a recombined RSV (rec-RSV). The rec-RSV appeared to have been generated by a polymerase jump from the ΔG/HEK genome to that of ΔNS1/2 and back again in the vicinity of the SH-G-F genes. This apparently involved nonhomologous and homologous recombination events, respectively. The recombined genome was identical to that of the ΔG/HEK mutant except that all but the first 12 nucleotides of the SH gene were deleted and replaced by an insert consisting of the last 91 nucleotides of the G gene and its downstream intergenic region. This insert could have come only from the coinfecting ΔNS1/2 virus. This resulted in the formation of a short chimeric SH:G gene. Northern and Western blot analysis confirmed that the rec-RSV did not express the normal SH and G mRNAs and proteins but did express the aberrant SH:G mRNA. This provides an experimental demonstration of intermolecular recombination yielding a viable, helper-independent mononegavirus. However, the isolation of only a single rec-RSV under these optimized conditions supports the idea that RSV recombination is rare indeed.

scholarly journals Comparison and analysis of various complementary diagnosis methods for the current situation and problems of COVID-19 diagnosis

2021 ◽  
Author(s):  
Hee Jin Huh ◽  
Seok Lae Chae ◽  
Dong-Min Kim
Keyword(s):  
Diagnostic Tests ◽  
Diagnostic Methods ◽  
Current Status ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fluorescence Immunoassay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Patient Symptom ◽  
Study Population ◽  
Polymerase Chain

AbstractWe evaluated and compared the diagnostic performance of fluorescence immunoassay (FIA) and two types of serological diagnostic tests: enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for detection of SARS-CoV-2 antigen and antibody to diagnose COVID-19 infections. This study is aimed to analyze and compare the current status and problems of COVID-19 diagnosis and various alternative diagnostic methods that are viable. The enrolled subjects in our study population were tested with real-time polymerase chain reaction (RT-PCR). ELISA and immunochromatographic diagnostic kit were used to diagnose 362 positive and 3010 negative SARS-CoV-2 specimens, and antigen fluorescence immunoassay kit was used on 62 positive and 70 negative SARS-CoV-2 RT-PCR confirmed samples for diagnosis. As a result, categorizing by the patient symptom onset days, PCL COVID19 Total Ab EIA (ELISA) showed the sensitivity of 93.4% from 15 to 21 days, 94.2% from over 22 days, and the specificity of 99.97%. PCL COVID19 IgG/IgM Rapid Gold (ICA) had a sensitivity of 86.9%, 97.4%, and the specificity of 98.14% respectively. PCL COVID19 Ag Rapid FIA sensitivity was 93.8% from 0 to 7 days, 71.4% from 8 to 12 days and specificity was 98.57%. In conclusion, COVID-19 Ab ELISA and ICA, and COVID-19 Ag FIA are all complementary and applicable diagnostic methods to resolve the current problems of COVID-19 diagnosis.

African Origin of Human T-Lymphotropic Virus Type 2 (HTLV-2) Supported by a Potential New HTLV-2d Subtype in Congolese Bambuti Efe Pygmies

1998 ◽  
Vol 72 (5) ◽  
pp. 4327-4340 ◽  
Author(s):  
Anne-Mieke Vandamme ◽  
Marco Salemi ◽  
Marianne Van Brussel ◽  
Hsin-Fu Liu ◽  
Kristel Van Laethem ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Phylogenetic Analyses ◽  
Enzyme Linked Immunosorbent Assay ◽  
African Origin ◽  
Entire Genome ◽  
T Lymphotropic Virus ◽  
Tax Protein ◽  
Human T Cell

ABSTRACT We identified a potential new subtype within human T-cell lymphotropic virus type 2 (HTLV-2), HTLV-2d, present in members of an isolated Efe Bambuti Pygmy tribe. Two of 23 Efe Pygmies were HTLV-2 seropositive, with HTLV-2 Western blot and enzyme-linked immunosorbent assay reactivities. From one of them the entire genome of the HTLV-2 strain Efe2 could be amplified and sequenced. In all gene regions analyzed, this strain was the most divergent HTLV-2 strain, differing by 2.4% (tax/rex) to 10.7% (long terminal repeat) from both subtypes HTLV-2a and HTLV-2b, yet major functional elements are conserved. The similarity between the HTLV-2 Efe2 Gag and Env proteins and the corresponding HTLV-2a and -2b proteins is consistent with the observed serological reactivity. In the proximal pX region, one of the two alternative splice acceptor sites is abolished in HTLV-2 Efe2. Another interesting feature of this potential new subtype is that it has a Tax protein of 344 amino acids (aa), which is intermediate in length between the HTLV-2a Tax protein (331 aa) and the HTLV-2b and -2c Tax proteins (356 aa) and similar to the simian T-cell lymphotropic virus type 2 (STLV-2) PP1664 Tax protein. Together these two findings suggest a different phenotype for the HTLV-2 Efe2 strain. Phylogenetic analyses confirmed that the Pygmy Efe2 strain potentially belonged to a new and quite divergent subtype, HTLV-2d. When the STLV-2 bonobo viruses PP1664 and PanP were used as an outgroup, it was clear that the Pygmy HTLV-2 Efe2 strain had the longest independent evolution and that HTLV-2 evolution is consistent with an African origin.

scholarly journals Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 66
Author(s):  
Zoltán László ◽  
Péter Pankovics ◽  
Gábor Reuter ◽  
Attila Cságola ◽  
Ádám Bálint ◽  
...  
Keyword(s):  
Novel Species ◽  
Phylogenetic Analyses ◽  
Interspecies Transmission ◽  
Background Information ◽  
Type Ii ◽  
Rt Pcr ◽  
Faecal Samples ◽  
The Family ◽  
Domestic Livestock

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain “neglected” genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species “Bopivirus B” in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine–caprine interspecies transmission of certain bopiviruses.

scholarly journals SARS-CoV-2 IgG Antibodies Seroprevalence and Sera Neutralizing Activity in MEXICO: A National Cross-Sectional Study during 2020

2021 ◽  
Vol 9 (4) ◽  
pp. 850
Author(s):  
José Esteban Muñoz-Medina ◽  
Concepción Grajales-Muñiz ◽  
Angel Gustavo Salas-Lais ◽  
Larissa Fernandes-Matano ◽  
Constantino López-Macías ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Neutralizing Antibodies ◽  
Herd Immunity ◽  
Cross Sectional Study ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Sectional ◽  
Neutralizing Activity

Until recently, the incidence of COVID-19 was primarily estimated using molecular diagnostic methods. However, the number of cases is vastly underreported using these methods. Seroprevalence studies estimate cumulative infection incidences and allow monitoring of transmission dynamics, and the presence of neutralizing antibodies in the population. In February 2020, the Mexican Social Security Institute began conducting anonymous unrelated sampling of residual sera from specimens across the country, excluding patients with fever within the previous two weeks and/or patients with an acute respiratory infection. Sampling was carried out weekly and began 17 days before Mexico’s first officially confirmed case. The 24,273 sera obtained were analyzed by chemiluminescent-linked immunosorbent assay (CLIA) IgG S1/S2 and, later, positive cases using this technique were also analyzed to determine the rate of neutralization using the enzyme-linked immunosorbent assay (ELISA). We identified 40 CLIA IgG positive cases before the first official report of SARS-CoV-2 infection in Mexico. The national seroprevalence was 3.5% in February and 33.5% in December. Neutralizing activity among IgG positives patients during overall study period was 86.1%. The extent of the SARS-CoV-2 infection in Mexico is 21 times higher than that reported by molecular techniques. Although the general population is still far from achieving herd immunity, epidemiological indicators should be re-estimated based on serological studies of this type.

scholarly journals Hepatitis E Virus Occurrence in Pigs Slaughtered in Italy

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 277
Author(s):  
Eleonora Chelli ◽  
Elisabetta Suffredini ◽  
Paola De Santis ◽  
Dario De Medici ◽  
Santina Di Bella ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Phylogenetic Analyses ◽  
Hepatitis E ◽  
Rt Pcr ◽  
High Seroprevalence ◽  
Genotype 3 ◽  
Sporadic Cases ◽  
Wild Boar Meat ◽  
Small Clusters ◽  
Frequent Exposure

In Europe, foodborne transmission has been clearly associated to sporadic cases and small clusters of hepatitis E in humans linked to the consumption of contaminated pig liver sausages, raw venison, or undercooked wild boar meat. In Europe, zoonotic HEV-genotype 3 strains are widespread in pig farms but little information is available on the prevalence of HEV positive pigs at slaughterhouse. In the present study, the prevalence of HEV-RNA positive pigs was assessed on 585 animals from 4 abattoirs located across Italy. Twenty-one pigs (3.6%) tested positive for HEV in either feces or liver by real-time RT-PCR. In these 21 pigs, eight diaphragm muscles resulted positive for HEV-RNA. Among animals collected in one abattoir, 4 out of 91 plasma tested positive for HEV-RNA. ELISA tests for the detection of total antibodies against HEV showed a high seroprevalence (76.8%), confirming the frequent exposure of pigs to the virus. The phylogenetic analyses conducted on sequences of both ORF1 and ORF2 fragments, shows the circulation of HEV-3c and of a novel unclassified subtype. This study provides information on HEV occurrence in pigs at the slaughterhouse, confirming that muscles are rarely contaminated by HEV-RNA compared to liver, which is the most frequently positive for HEV.

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