Isolation of PCV3 from Perinatal and Reproductive Cases of PCV3-Associated Disease and In Vivo Characterization of PCV3 Replication in CD/CD Growing Pigs

Viruses

2020 Feb 16

Mora-D�az J, Pi�eyro P, Shen H, Schwartz K, Vannucci F2, Li G, Arruda B, Gim�nez-Lirola L
PMID: 32079070 | DOI: 10.3390/v12020219

Porcine circovirus 3 (PCV3) has been identified as a putative swine pathogen with a subset of infections resulting in stillborn and mummified fetuses, encephalitis and myocarditis in perinatal, and periarteritis in growing pigs. Three PCV3 isolates were isolated from weak-born piglets or elevated stillborn and mummified fetuses. Full-length genome sequences from different passages and isolates (PCV3a1 ISU27734, PCV3a2 ISU58312, PCV3c ISU44806) were determined using metagenomics sequencing. Virus production in cell culture was confirmed by qPCR, IFA, and in situ hybridization. In vivo replication of PCV3 was also demonstrated in CD/CD pigs (n = 8) under experimental conditions. Viremia, first detected at 7 dpi, was detected in all pigs by 28 dpi. IgM antibody response was detected between 7-14 dpi in 5/8 PCV3-inoculated pigs but no IgG seroconversion was detected throughout the study. Pigs presented histological lesion consistent with multi systemic inflammation characterized by myocarditis and systemic perivasculitis. Viral replication was confirmed in all tissues by in situ hybridization. Clinically, all animals were unremarkable throughout the study. Although the clinical relevance of PCV3 remains under debate, this is the first isolation of PCV3 from perinatal and reproductive cases of PCV3-associated disease and in vivo characterization of PCV3 infection in a CD/CD pig model

Activation of STAT3 signaling pathway in the kidney of COVID-19 patients

Journal of nephrology

2021 Oct 09

Salem, F;Li, XZ;Hindi, J;Casablanca, NM;Zhong, F;El Jamal, SM;Haroon Al Rasheed, MR;Li, L;Lee, K;Chan, L;He, JC;
PMID: 34626364 | DOI: 10.1007/s40620-021-01173-0

Acute kidney injury is common in patients with COVID-19, however mechanisms of kidney injury remain unclear. Since cytokine storm is likely a cause of AKI and glomerular disease, we investigated the two major transcription factors, STAT3 and NF-kB, which are known to be activated by cytokines.This is an observational study of the postmortem kidneys of 50 patients who died with COVID-19 in the Mount Sinai Hospital during the first pandemic surge. All samples were reviewed under light microscopy, electron microscopy, and immunofluorescence by trained renal pathologists. In situ hybridization evaluation for SARS-CoV-2 and immunostaining of transcription factors STAT3 and NF-kB were performed.Consistent with previous findings, acute tubular injury was the major pathological finding, together with global or focal glomerulosclerosis. We were not able to detect SARS-CoV-2 in kidney cells. ACE2 expression was reduced in the tubular cells of patients who died with COVID-19 and did not co-localize with TMPRSS2. SARS-CoV-2 was identified occasionally in the mononuclear cells in the peritubular capillary and interstitium. STAT3 phosphorylation at Tyr705 was increased in 2 cases in the glomeruli and in 3 cases in the tubulointerstitial compartments. Interestingly, STAT3 phosphorylation at Ser727 increased in 9 cases but only in the tubulointerstitial compartment. A significant increase in NF-kB phosphorylation at Ser276 was also found in the tubulointerstitium of the two patients with increased p-STAT3 (Tyr705).Our findings suggest that, instead of tyrosine phosphorylation, serine phosphorylation of STAT3 is commonly activated in the kidney of patients with COVID-19.

Field infection of a gilt and its litter demonstrates vertical transmission and effect on reproductive failure caused by porcine circovirus type 3 (PCV3)

BMC veterinary research

2021 Apr 08

Vargas-Bermúdez, DS;Vargas-Pinto, MA;Mogollón, JD;Jaime, J;
PMID: 33832500 | DOI: 10.1186/s12917-021-02862-5

PCV3 is a member of the Circovirus family, associated with disease and mortality in pigs. It is not clear whether PCV3 putatively causes clinical symptoms and disease. In the present case, we reported a gilt infected with PCV3 associated with reproductive failures, vertical transmission, tissue lesions, viral replication by in situ hybridization, and the hypothesis that some strains of PCV3 clade one are associated with reproductive failures at the field level. In May 2019, a pig farm in Colombia reported increased reproductive failures, and the presence of PCV3 in gilts and sows was established in a single form or coinfections, mainly with PCV2 and PPV7. Ten sows with a single infection with PCV3 were found, and one gilt with a pre-farrowing serum viral load above 103 was studied. This gilt was followed up during the pre-farrowing, farrowing period and on her litter for 6 weeks. During dystocic farrowing, a mummy and ten piglets were released, including two weak-born piglets. The highest viral loads for PCV3 were found in the mummy and the placenta. In the weak-born piglets, there were viral loads both in serum and in tissues, mainly in the mesenteric ganglia and lung. Replication of PCV3 in these tissues was demonstrated by in situ hybridizations. PCV3 was also found in the precolostrum sera of piglets and colostrum, showing vertical transmission. The viral load in piglets decreased gradually until week six of life. The viral genome's complete sequencing was made from the mummy, and its analysis classified it as PCV3 clade one. This report confirms that PCV3 can cause disease at the field level, and putatively, in this case, we find the generation of reproductive failures. The ability of PCV3 to cause disease as a putative pathogen may be associated with the viral load present in the pig and the strain that is affecting the farm. For this case, we found that viral loads above 103 (4.93 log genomic copies / mL) in the gilt were associated with clinical manifestation and that some PCV3 strains belonging to clade one are more associated with the reproductive presentation.

Evidence of vertical transmission of SARS-CoV-2 and interstitial pneumonia in second trimester twin stillbirth in asymptomatic woman. Case report and review of the literature

American journal of obstetrics & gynecology MFM

2022 Feb 04

Patanè, L;Cadamuro, M;Massazza, G;Pirola, S;Stagnati, V;Comerio, C;Carnelli, M;Arosio, M;Callegaro, AP;Tebaldi, P;Rigoli, E;Gianatti, A;Morotti, D;
PMID: 35131495 | DOI: 10.1016/j.ajogmf.2022.100589

Data on the vertical transmission rate of COVID-19 in pregnancy are limited, while data reporting mother-fetal transmission in the second trimester of pregnancy are controversial. We described a case of second trimester twin stillbirth in a woman positive for SARS-CoV-2 in which, despite the absence of respiratory syndrome, placental and fetal markers of infection were detected. The patient developed a clinical chorioamnionitis and spontaneously delivered two stillborn infants. Placental histology and immunohistochemistry demonstrated SARS-CoV-2 infection mostly within the syncytiotrophoblast and the fetal autopsy showed development of interstitial pneumonia. Our findings demonstrate that, in utero vertical transmission is possible, also in asymptomatic SARS-CoV-2 pregnant women and that infection can lead to severe morbidity in the second trimester of pregnancy.

Multisystem screening reveals SARS-CoV-2 in neurons of the myenteric plexus and in megakaryocytes

The Journal of pathology

2022 Feb 02

Gray-Rodriguez, S;Jensen, MP;Otero-Jimenez, M;Hanley, B;Swann, OC;Ward, PA;Salguero, FJ;Querido, N;Farkas, I;Velentza-Almpani, E;Weir, J;Barclay, WS;Carroll, MW;Jaunmuktane, Z;Brandner, S;Pohl, U;Allinson, K;Thom, M;Troakes, C;Al-Sarraj, S;Sastre, M;Gveric, D;Gentleman, S;Roufosse, C;Osborn, M;Alegre-Abarrategui, J;
PMID: 35107828 | DOI: 10.1002/path.5878

SARS-CoV-2, the causative agent of COVID-19, typically manifests as a respiratory illness although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, as well as frequent thrombotic events, are increasingly recognised. How this maps onto SARS-CoV-2 organ tropism at the histological level, however, remains unclear. Here, we perform a comprehensive validation of a monoclonal antibody against the SARS-CoV-2 nucleocapsid protein (NP) followed by systematic multisystem organ immunohistochemistry analysis of the viral cellular tropism in tissue from 36 patients, 16 post-mortem cases and 16 biopsies with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 status from the peaks of the pandemic in 2020 and four pre-COVID post-mortem controls. SARS-CoV-2 anti-NP staining in the post-mortem cases revealed broad multiorgan involvement of the respiratory, digestive, haematopoietic, genitourinary and nervous systems, with a typical pattern of staining characterised by punctate paranuclear and apical cytoplasmic labelling. The average time from symptom onset to time of death was shorter in positively versus negatively stained post-mortem cases (mean = 10.3 days versus mean = 20.3 days, p = 0.0416, with no cases showing definitive staining if the interval exceeded 15 days). One striking finding was the widespread presence of SARS-CoV-2 NP in neurons of the myenteric plexus, a site of high ACE-2 expression, the entry receptor for SARS-CoV-2, and one of the earliest affected cells in Parkinson's disease. In the bone marrow, we observed viral SARS-CoV-2 NP within megakaryocytes, key cells in platelet production and thrombus formation. In 15 tracheal biopsies performed in patients requiring ventilation, there was a near complete concordance between immunohistochemistry and PCR swab results. Going forward, our findings have relevance to correlating clinical symptoms to the organ tropism of SARS-CoV-2 in contemporary cases as well as providing insights into potential long-term complications of COVID-19. This article is protected by

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates

Signal transduction and targeted therapy

2021 Dec 24

Zhao, H;Wang, TC;Li, XF;Zhang, NN;Li, L;Zhou, C;Deng, YQ;Cao, TS;Yang, G;Li, RT;Huang, YJ;Li, YG;Zhang, YM;Li, FX;Zhou, YR;Jiang, YH;Lu, XS;Sun, SH;Cheng, ML;Gu, KP;Zhang, M;Ma, QQ;Yang, X;Ying, B;Gao, YW;Qin, CF;
PMID: 34952914 | DOI: 10.1038/s41392-021-00861-4

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.

Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs

Animals : an open access journal from MDPI

2021 May 24

De Conti, ER;Resende, TP;Marshall-Lund, L;Rovira, A;Vannucci, FA;
PMID: 34073660 | DOI: 10.3390/ani11061520

Porcine circovirus type 3 (PCV3) has been recently described as a potential cause of abortions and systemic vasculitis in pigs. Although the virus has been detected by real-time PCR in several porcine tissues from countries worldwide, PCV3-associated diseases have not been satisfactorily clarified. The objective of this study was to investigate the association between the presence of PCV3 mRNA detected by in situ hybridization (ISH) within histological lesions and PCV3 DNA detected by real-time PCR in naturally infected pigs. A total of 25 PCV3 PCR-positive cases were analyzed. Formalin-fixed tissues from these cases were evaluated for histologic lesions and for ISH-RNA positive signals for PCV3. The most frequent tissue type with histopathologic lesions was heart, 76.2%, with lymphoplasmacytic myocarditis and epicarditis as the most frequent lesions observed. Lymphoplasmacytic interstitial pneumonia was also a frequent finding, 47.6%. There were also lesions in kidney, liver, spleen and lymph nodes. PCV3-ISH-RNA positive signals were mostly observed in association with lymphoplasmacytic inflammatory infiltrate in various tissues, including arteries. Based on our results, the minimum set of specimens to be submitted for histopathology and mRNA in situ hybridization to confirm or exclude a diagnosis of PCV3 are heart, lung and lymphoid tissues (i.e., spleen and lymph nodes), especially for differential diagnosis related with PCV2-associated diseases.

Tissue-Based SARS-Cov-2 Detection in Fatal COVID-19 Infections: Sustained Direct Viral-Induced Damage is Not Necessary to Drive Disease Progression

Human pathology

2021 May 04

El Jamal, SM;Pujadas, E;Ramos, I;Bryce, C;Grimes, ZM;Amanat, F;Tsankova, NM;Mussa, Z;Olson, S;Salem, F;Miorin, L;Aydillo, T;Schotsaert, M;Albrecht, RA;Liu, WC;Marjanovic, N;Francoeur, N;Sebra, R;Sealfon, SC;García-Sastre, A;Fowkes, M;Cordon-Cardo, C;Westra, WH;
PMID: 33961839 | DOI: 10.1016/j.humpath.2021.04.012

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in-situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative PCR (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple organ pathogenic pro-inflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.

Adventitial Microcirculation Is a Major Target of SARS-CoV-2-Mediated Vascular Inflammation

Biomolecules

2021 Jul 20

Vasuri, F;Ciavarella, C;Collura, S;Mascoli, C;Valente, S;Degiovanni, A;Gargiulo, M;Capri, M;Pasquinelli, G;
| DOI: 10.3390/biom11071063

We report the case of a 77-year-old woman affected by coronavirus disease-19 (COVID-19) who developed an occlusive arterial disease of the lower limb requiring a left leg amputation. We studied the mechanisms of vascular damage by SARS-CoV-2 by means of a comprehensive multi-technique in situ analysis on the diseased popliteal arterial district, including immunohistochemistry (IHC), transmission electron microscopy (TEM) and miRNA analysis. At histological analyses, we observed a lymphocytic inflammatory infiltrate, oedema and endothelialitis of adventitial vasa vasorum while the media was normal and the intima had only minor changes. The vasa vasorum expressed the ACE2 receptor and factor VIII; compared with the controls, VEGFR2 staining was reduced. TEM analyses showed endothelial injury and numerous Weibel-Palade bodies in the cytoplasm. No coronavirus particle was seen. IL-6 protein and mRNA, together with miR-155-5p and miRs-27a-5p, which can target IL-6, were significantly increased compared with that in the controls. Our case report suggests an involvement of adventitial artery microcirculation by inflammation in the course of COVID-19. Without evident signs of current infection by SARS-CoV-2, endothelial cells show a spectrum of structural and functional alterations that can fuel the cardiovascular complications observed in people infected with SARS-CoV-2.

Novel Morbillivirus as Putative Cause of Fetal Death and Encephalitis among Swine

Emerging infectious diseases

2021 Jul 01

Arruda, B;Shen, H;Zheng, Y;Li, G;
PMID: 34152961 | DOI: 10.3201/eid2707.203971

Morbilliviruses are highly contagious pathogens. The Morbillivirus genus includes measles virus, canine distemper virus (CDV), phocine distemper virus (PDV), peste des petits ruminants virus, rinderpest virus, and feline morbillivirus. We detected a novel porcine morbillivirus (PoMV) as a putative cause of fetal death, encephalitis, and placentitis among swine by using histopathology, metagenomic sequencing, and in situ hybridization. Phylogenetic analyses showed PoMV is most closely related to CDV (62.9% nt identities) and PDV (62.8% nt identities). We observed intranuclear inclusions in neurons and glial cells of swine fetuses with encephalitis. Cellular tropism is similar to other morbilliviruses, and PoMV viral RNA was detected in neurons, respiratory epithelium, and lymphocytes. This study provides fundamental knowledge concerning the pathology, genome composition, transmission, and cellular tropism of a novel pathogen within the genus Morbillivirus and opens the door to a new, applicable disease model to drive research forward.

Pathogenic Characterization of a Porcine Circovirus Type 3 Isolate from Heilongjiang, China

Disease Markers

2021 Jun 24

Wang, M;Yu, Y;Wu, J;Meng, F;Tang, Y;Wang, S;Wang, Y;Cui, H;He, X;Tu, Y;Wang, G;Cai, X;
| DOI: 10.1155/2021/9434944

The clinical outcome of porcine circovirus 3 (PCV3) infection is still controversial. Herein, a novel PCV3 isolate (PCV3-China/DB-1/2017) with the molecular characterization of 24A and 27K in the Cap protein was used to inoculate three-week-old cesarean-derived, colostrum-deprived piglets. The nine PCV3 DB-1 inoculated piglets exhibited no obvious clinical symptoms or macroscopic lesions. PCV3 displayed a broad histotropism, including the heart, liver, spleen, lung, kidney, brain, lymph nodes, and tonsil, and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs. From 7 days after PCV3 DB-1 inoculation, the piglets showed obvious IgG antibody responses against PCV3 rCap-VLPs. The cumulative results demonstrated that PCV3 trend to low pathogenicity.

	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
EnEm	Probe targets exons n and m
En-Em	Probe 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

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