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Probes for INS

ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

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The same strain of Piscine orthoreovirus (PRV-1) is involved with the development of different, but related, diseases in Atlantic and Pacific Salmon in British Columbia

arXiv preprint

2018 Apr 23

Di Cicco E, Ferguson HW, Kaukinen KH, Schulze AD, Li S, Tabata A, Gunther OP, Mordecai G, Suttle CA, Miller KM.
PMID: - | DOI: 10.1139/facets-2018-0008

Piscine orthoreovirus Strain PRV-1 is the causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar). Given its high prevalence in net pen salmon, debate has arisen on whether PRV poses a risk to migratory salmon, especially in British Columbia (BC) where commercially important wild Pacific salmon are in decline. Various strains of PRV have been associated with diseases in Pacific salmon, including erythrocytic inclusion body syndrome (EIBS), HSMI-like disease, and jaundice/anemia in Japan, Norway, Chile and Canada. We examine the developmental pathway of HSMI and jaundice/anemia associated with PRV-1 in farmed Atlantic and Chinook (Oncorhynchus tshawytscha) salmon in BC, respectively. In situ hybridization localized PRV-1 within developing lesions in both diseases. The two diseases showed dissimilar pathological pathways, with inflammatory lesions in heart and skeletal muscle in Atlantic salmon, and degenerative-necrotic lesions in kidney and liver in Chinook salmon, plausibly explained by differences in PRV load tolerance in red blood cells. Viral genome sequencing revealed no consistent differences in PRV-1 variants intimately involved in the development of both diseases, suggesting that migratory Chinook salmon may be at more than a minimal risk of disease from exposure to the high levels of PRV occurring on salmon farms.

Isolation of infectious Lloviu virus from Schreiber's bats in Hungary

Nature communications

2022 Mar 31

Kemenesi, G;Tóth, GE;Mayora-Neto, M;Scott, S;Temperton, N;Wright, E;Mühlberger, E;Hume, AJ;Suder, EL;Zana, B;Boldogh, SA;Görföl, T;Estók, P;Lanszki, Z;Somogyi, BA;Nagy, Á;Pereszlényi, CI;Dudás, G;Földes, F;Kurucz, K;Madai, M;Zeghbib, S;Maes, P;Vanmechelen, B;Jakab, F;
PMID: 35361761 | DOI: 10.1038/s41467-022-29298-1

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.
γδ T cells and the immune response to respiratory syncytial virus infection.

Vet Immunol Immunopathol.

2016 Feb 21

McGill JL, Sacco RE.
PMID: 26923879 | DOI: 10.1016/j.vetimm.2016.02.012

γδ T cells are a subset of nonconventional T cells that play a critical role in bridging the innate and adaptive arms of the immune system. γδ T cells are particularly abundant in ruminant species and may constitute up to 60% of the circulating lymphocyte pool in young cattle. The frequency of circulating γδ T cells is highest in neonatal calves and declines as the animal ages, suggesting these cells may be particularly important in the immune system of the very young. Bovine respiratory syncytial virus (BRSV) is a significant cause of respiratory infection in calves, and is most severe in animals under one year of age. BRSV is also a significant factor in the development of bovine respiratory disease complex (BRDC), the leading cause of morbidity and mortality in feedlot cattle. Human respiratory syncytial virus (RSV) is closely related to BRSV and a leading cause of lower respiratory tract infection in infants and children worldwide. BRSV infection in calves shares striking similarities with RSV infection in human infants. To date, there have been few studies defining the role of γδ T cells in the immune response to BRSV or RSV infection in animals or humans, respectively. However, emerging evidence suggests that γδ T cells may play a critical role in the early recognition of infection and in shaping the development of the adaptive immune response through inflammatory chemokine and cytokine production. Further, while it is clear that γδ T cells accumulate in the lungs during BRSV and RSV infection, their role in protection vs. immunopathology remains unclear. This review will summarize what is currently known about the role of γδ T cells in the immune response to BRSV and BRDC in cattle, and where appropriate, draw parallels to the role of γδ T cells in the human response to RSV infection.

The Raccoon Polyomavirus Genome and Tumor Antigen Transcription are Stable and Abundant in Neuroglial Tumors.

J Virol. 2014 Aug 27. pii: JVI.01912-14.

Brostoff T, Dela Cruz FN Jr, Church ME, Woolard KD, Pesavento PA.
PMID: 25165109 | DOI: JVI.01912-14.

Abstract Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including SV40, murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (non-integrated) in neuroglial tumors. Here we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further co-localized TAg DNA and mRNA by in situ hybridization, and found that the majority of tumor cells showed positive staining. Lastly, we examined stability of the viral genome and TAg transcription by quantitative reverse-transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly two log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplant of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors, and a novel mechanism of stability for a polyomavirus in cancer. IMPORTANCE: The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors, but was low or undetectable in non-tumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen proteins, we cultured primary raccoon tumor cells and passaged them in mice, confirming the non-integrated state of the virus and the maintenance of viral protein transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.
Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys

Nat Microbiol.

2017 Jul 17

Zeng X, Blancett CD, Koistinen KA, Schellhase CW, Bearss JJ, Radoshitzky SR, Honnold SP, Chance TB, Warren TK, Froude JW, Cashman KA, Dye JM, Bavari S, Palacios G, Kuhn JH, Sun MG.
PMID: 28715405 | DOI: 10.1038/nmicrobiol.2017.113

Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013-2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68+ cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence.

Disruption of latent HIV in vivo during the clearance of actinic keratosis by ingenol mebutate.

JCI Insight.

2019 Apr 04

Jiang G, Maverakis E, Cheng MY, Elsheikh MM, Deleage C, Méndez-Lagares G, Shimoda M, Yukl SA, Hartigan-O'Connor DJ, Thompson GR 3rd, Estes JD, Wong JK, Dandekar S.
PMID: 30944245 | DOI: 10.1172/jci.insight.126027

Actinic keratosis (AK) is a precancerous skin lesion that is common in HIV-positive patients. Without effective treatment, AKs can progress to squamous cell carcinoma. Ingenol mebutate, a PKC agonist, is a US Food and Drug Administration-approved (FDA-approved) topical treatment for AKs. It can induce reactivation of latent HIV transcription in CD4+ T cells both in vitro and ex vivo. Although PKC agonists are known to be potent inducers of HIV expression from latency, their effects in vivo are not known because of the concerns of toxicity. Therefore, we sought to determine the effects of topical ingenol mebutate gel on the HIV transcription profile in HIV-infected individuals with AKs, specifically in the setting of suppressive antiretroviral therapy (ART). We found that AKs cleared following topical application of ingenol mebutate and detected marginal changes in immune activation in the peripheral blood and in skin biopsies. An overall increase in the level of HIV transcription initiation, elongation, and complete transcription was detected only in skin biopsies after the treatment. Our data demonstrate that application of ingenol mebutate to AKs in ART-suppressed HIV-positive patients can effectively cure AKs as well as disrupt HIV latency in the skin tissue microenvironment in vivo without causing massive immune activation.

Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells

Hepatology (Baltimore, Md.)

2022 Jun 19

Zhao, K;Guo, F;Wang, J;Zhong, Y;Yi, J;Teng, Y;Xu, Z;Zhao, L;Li, A;Wang, Z;Chen, X;Cheng, X;Xia, Y;
PMID: 35718932 | DOI: 10.1002/hep.32622

Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells.Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pre-genomic RNA (pgRNA), covalently closed circular DNA (cccDNA) and different relaxed circular DNA (rcDNA) intermediates were produced in vitro. The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine hepatic cells. HBV replication-competent plasmid, cccDNA and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo. In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine but not in human hepatic cells.Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.This article is protected by
Zika virus infects renal proximal tubular epithelial cells with prolonged persistency and cytopathic effects

Emerg Microbes Infect.

2017 Aug 23

Chen J, Yang YF, Chen J, Zhou X, Dong Z, Chen T, Yang Y, Zou P, Jiang B, Hu Y, Lu L, Zhang X, Liu J, Xu J, Zhu T.
PMID: 28831192 | DOI: 10.1038/emi.2017.67

Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain-Barré syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients' urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients' urine.

Pathogenesis of varicelloviruses in primates

J Pathol. 2015 Jan;235(2):298-311.

Ouwendijk WJ, Verjans GM.
PMID: 25255989 | DOI: 10.1002/path.4451.

Varicelloviruses in primates comprise the prototypic human varicella-zoster virus (VZV) and its non-human primate homologue, simian varicella virus (SVV). Both viruses cause varicella as a primary infection, establish latency in ganglionic neurons and reactivate later in life to cause herpes zoster in their respective hosts. VZV is endemic worldwide and, although varicella is usually a benign disease in childhood, VZV reactivation is a significant cause of neurological disease in the elderly and in immunocompromised individuals. The pathogenesis of VZV infection remains ill-defined, mostly due to the species restriction of VZV that impedes studies in experimental animal models. SVV infection of non-human primates parallels virological, clinical, pathological and immunological features of human VZV infection, thereby providing an excellent model to study the pathogenesis of varicella and herpes zoster in its natural host. In this review, we discuss recent studies that provided novel insight in both the virus and host factors involved in the three elementary stages of Varicellovirus infection in primates: primary infection, latency and reactivation.
SIV persistence in cellular and anatomic reservoirs in ART-suppressed infant rhesus macaques

J Virol.

2018 Jul 11

Mavigner M, Habib J, Deleage C, Rosen E, Mattingly C, Bricker K, Kashuba A, Amblard F, Schinazi RF, Jean S, Cohen J, McGary C, Paiardini M, Wood MP, Sodora DL, Silvestri G, Estes J, Chahroudi A.
PMID: 29997216 | DOI: 10.1128/JVI.00562-18

Worldwide, nearly two million children are infected with HIV, with breastfeeding accounting for the majority of contemporary HIV transmissions. Antiretroviral therapy (ART) has reduced HIV-related morbidity and mortality but is not curative. The main barrier to a cure is persistence of latent HIV in long-lived reservoirs. However, our understanding of the cellular and anatomic sources of the HIV reservoir during infancy and childhood is limited. Here, we developed a pediatric model of ART suppression in orally SIV-infected rhesus macaque (RM) infants, with measurement of virus persistence in blood and tissues after 6-9 months of ART. Cross-sectional analyses were conducted to compare SIV RNA and DNA levels in adult and infant RMs naïve to treatment and on ART. We demonstrate efficient viral suppression following ART initiation in SIV-infected RM infants with sustained undetectable plasma viral loads in the setting of heterogeneous penetration of ART into lymphoid and gastrointestinal tissues and low drug levels in the brain. We further show reduction in SIV RNA and DNA on ART in lymphoid tissues of both infant and adult RMs, but stable (albeit low) levels of SIV RNA and DNA in the brains of viremic and ART-suppressed infants. Finally, we report a large contribution of naïve CD4+ T-cells to the total CD4 reservoir of SIV in blood and lymph nodes of ART-suppressed RM infants, that differs from what we show in adults. These results reveal important aspects of HIV/SIV persistence in infants and provide insight into strategic targets for cure interventions in a pediatric population.IMPORTANCE While antiretroviral therapy (ART) can reduce HIV replication, the virus cannot be eradicated from an infected individual and our incomplete understanding of HIV persistence in reservoirs greatly complicates the generation of a cure for HIV. Given the immaturity of the infant immune system, it is of critical importance to study HIV reservoirs specifically in this population. Here, we established a pediatric animal model to simulate breastfeeding transmission and study SIV reservoirs in rhesus macaques (RM) infants. Our study demonstrates that ART can be safely administered to infant RM for prolonged periods of time and efficiently controls viral replication in this model. SIV persistence was shown in blood and tissues with a similar anatomic distribution of SIV reservoirs in infant and adult RMs. However, in the peripheral blood and lymph nodes, a higher contribution of the naïve CD4+ T-cells to the SIV reservoir was observed in infants compared to adults.

Prevalence of HPV infection in head and neck carcinomas shows geographical variability: a comparative study from Brazil and Germany

Virchows Archiv (2015): 1-9.

Hauck F, Oliveira-Silva M, Dreyer JH, Ferreira Perrusi VJ, Arcuri RA, Hassan R, Bonvicino CR, Barros MHM, Niedobitek G.
PMID: 25820374 | DOI: 10.1007/s00428-015-1761-4

Rising prevalence rates of high-risk human papillomaviruses (hrHPV) infection in oropharyngeal carcinoma (up to 80 %) have been reported in North America and Scandinavia. We have analysed 424 German and 163 Brazilian head and neck squamous cell carcinomas (HNSCC) from the oral cavity (OSCC), oropharynx (OPSCC) and hypopharynx (HPSCC) using p16 immunohistochemistry, HPV DNA PCR and sequencing, hrHPV DNA in situ hybridisation (ISH) and hrHPV E6/E7 RNA ISH. In the German series, 52/424 cases (12.3 %) were p16-positive/hrHPV-positive (OSCC 3.8 % [10/265], OPSCC 34.4 % [42/122], HPSCC 0 % [0/37]). In addition, there were 9 cases that were p16-positive/hrHPV-negative (5 OPSCC and 4 OSCC). In the Brazilian series, the overall hrHPV DNA prevalence by PCR was 11.0 % ([18/163]; OSCC 6 % [5/83], OPSCC 15.5 % [11/71], HPSCC 22.2 % [2/9]). Ten of these cases were hrHPV-positive/p16-positive. The remaining 8 hrHPV-positive/p16-negative cases were also negative in both ISH assays. Furthermore, 5 p16-positive/hrHPV-negative cases (2 OPSCC and 3 OSCC) were identified. In both series, HPV16 was by far the most common HPV type detected. We confirm that regardless of geographical origin, the highest hrHPV prevalence in HNSCC is observed in oropharyngeal carcinomas. The proportion of HPV-associated OPSCC was substantially higher in the German cohort than in the Brazilian series (34.4 vs. 15.5 %), and in both groups, the prevalence of hrHPV in OPSCC was much lower than in recent reports from North America and Scandinavia. We suggest, therefore, that it may be possible to define areas with high (e.g. USA, Canada, Scandinavia), intermediate (e.g. Germany) and low (e.g. Brazil) prevalences of HPV infection in OPSCC.
Expression of host defense peptides in the intestine of Eimeria-challenged chickens.

Poult Sci.

2017 May 17

Su S, Dwyer DM, Miska KB, Fetterer RH, Jenkins MC, Wong EA.
PMID: 28521031 | DOI: 10.3382/ps/pew468

Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.

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Description
sense
Example: Hs-LAG3-sense
Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron#
Example: Mm-Htt-intron2
Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan
Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)
A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp
Example: Hs-PDGFB-No-XMm
Does not cross detect with the species (Sp)
XSp
Example: Rn-Pde9a-XMm
designed to cross detect with the species (Sp)
O#
Example: Mm-Islr-O1
Alternative design targeting different regions of the same transcript or isoforms
CDS
Example: Hs-SLC31A-CDS
Probe targets the protein-coding sequence only
EnEmProbe targets exons n and m
En-EmProbe targets region from exon n to exon m
Retired Nomenclature
tvn
Example: Hs-LEPR-tv1
Designed to target transcript variant n
ORF
Example: Hs-ACVRL1-ORF
Probe targets open reading frame
UTR
Example: Hs-HTT-UTR-C3
Probe targets the untranslated region (non-protein-coding region) only
5UTR
Example: Hs-GNRHR-5UTR
Probe targets the 5' untranslated region only
3UTR
Example: Rn-Npy1r-3UTR
Probe targets the 3' untranslated region only
Pan
Example: Pool
A mixture of multiple probe sets targeting multiple genes or transcripts

Enabling research, drug development (CDx) and diagnostics

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