A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Development of a Potential Penside Colorimetric LAMP Assay Using Neutral Red for Detection of African Swine Fever Virus.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on the global pig industry. The penside tests for detecting PCV3 are critical for assessing the epidemiological status and working out disease prevention and control programs due to the unavailability of a commercial vaccine. A one-step molecular assay based on visual loop-mediated isothermal amplification (vLAMP) was developed for simple and rapid detection of PCV3. We compared its sensitivity and specificity with TaqMan quantitative real-time polymerase chain reaction (qPCR) and applied the developed assay in the epidemiological study of (n = 407) pooled swine sera collected from almost the entire mainland China during the years 2017-2018. We also explored the feasibility of the vLAMP assay for detecting raw samples without a prior DNA isolation step to expand its application capability. Results showed that the vLAMP assay could reliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equal to that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positive detection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35% (152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detection method without genome extraction, the results kept satisfactory specificity (100%) but displayed lower sensitivity (100% for CT < 32), indicating the direct detection is not sensitive enough to discriminate the samples with low viral loads. The one-step vLAMP is a convenient, rapid, and cost-effective diagnostic for penside detection and will enable the epidemiological surveillance of PCV3, which has widely spread in mainland China.

Full-genome analysis and pathogenicity of a genetically distinct Russian PRRSV-1 Tyu16 strain.

Porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) strains from Eastern Europe have a high diversity. All three known subtypes (1, 2, 3) of PRRSV-1 have been detected in Russia. There are two different groups of viruses belonging to the subtype 1: pan-European subtype 1 strains, and insufficiently studied Russian strains. The main objective of this study was to characterize the full genomic structure of the atypical Tyu16 strain of the Russian group subtype 1 PRRSV-1 and to assess its pathogenicity. Complete sequencing of the Tyu16 strain revealed that it did not belong to any existing subtype. Comparison of the whole genome sequence of the Tyu16 strain with that of PRRSV-1 prototype strains revealed 78.1 % (subtype 1 Lelystad), 78.1 % (subtype 2 WestSib13) and 77.7 % (subtype 3 Lena) nucleotide identity level, respectively. The coding sequence of different parts of the Tyu16 strain genome demonstrated a varying percentage identity to the different reference PRRSV-1 strains, which may indicate recombination events in its evolutionary history. We assume that among PRRSV-1 isolates, the Tyu16 is the closest relative to the common ancestor of PRRSV-1 and PRRSV-2. Low pathogenicity of the Tyu16 was demonstrated by experimental infection of 70-day-old piglets. Infected animals showed fever not exceeding 7 days, dyspnea in two out of five pigs and reduced weight gain. The virus shedding was undetectable and viremia was at low level.

First detection and full genome sequence of porcine circovirus type 3 in Russia.

Porcine circovirus type 3 (PCV3) was firstly detected in 2016 in USA. Later PCV3 was discovered in Asia, Europe, and South America. The present investigation demonstrates for the first time the circulation of PCV3 among pigs in Russia. The viruses were detected at two geographically distant unrelated commercial farms with records of reproductive failure (abortions, stillbirth), porcine dermatitis, and nephropathy syndrome (PDNS). The two farms were located in the region of Smolensk (western part of Russia) and the region of Tyumen (West Siberia, Russia). We investigated samples collected from pigs of different ages. We performed PCR for the PCV3 DNA detection. The DNA of PCV3 was detected in serum, kidney, heart, spleen, pleural effusion, and peritoneal cavity fluid samples. Two viral genomes were sequenced and the corresponding strains were named PCV3-RU/SM17 (the strain from Smolensk region) and PCV3-RU/TY17 (the strain from Tyumen region). The full genome sequences of both strains were 2000 nucleotides in length and contained at least two ORFs, encoding the Cap and Rep proteins. Full sequence alignment revealed a 99.3% identity between the PCV3-RU/SM17 and PCV3-RU/TY17 strains. Molecular analysis showed that the two strains from Russia are highly homologous to viruses identified in other countries, with a 98.5-99.6% homology for PCV3-RU/TY17 and 97.9-99.0 for PCV3-RU/SM17. The PCV3-RU/SM17 and PCV3-RU/TY17 strains were found to form a monophyletic group in a phylogenetic tree based on PCV3 complete genome sequences.

Genetic and pathogenic characterization of a Russian subtype 2 PRRSV-1 isolate.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory problems. Data about the virulence and pathogenicity of subtype 2 PRRSV-1 strains are limited. The main purposes of this investigation were to characterize the full genome sequence of the subtype 2 PRRSV-1 WestSib13 strain and to compare the pathogenicity with that of the subtype 1 PRRSV-1 Lelystad strain. Comparison of the whole genome sequence of the WestSib13 strain with that of PRRSV-1 prototype strains revealed a 76.2% (subtype 1 Lelystad virus) and 79.0% (subtype 3 Lena virus) identity, respectively The virulence and pathogenicity of the European subtype 2 PRRSV strain WestSib13 and the European subtype 1 PRRSV strain Lelystad were compared in 3-week-old piglets upon inoculation of 105.4 TCID50 of virus. Non-infected animals (control group) as well as animals infected with the Lelystad strain were clinically healthy until 14days post challenge. In contrast, animals infected with the WestSib13 strain demonstrated dyspnea starting at 3days post-inoculation (dpi). All piglets in this group died between 5 and 8 dpi. During that period, fever was not observed in WestSib13-infected animals. Viremia was detected in animals from both infected groups starting from 2 dpi. Viral loads in serum and lungs upon euthanasia were significantly higher (3 log10) in the WestSib13-infected than in the LV-infected animals. Taken together, this study provides the full genome sequence and the unusual virological and clinical outcome (high level viremia without fever) of the novel WestSib13 strain.

The successful immune response against hepatitis C nonstructural protein 5A (NS5A) requires heterologous DNA/protein immunization.

The aim of this study was to evaluate the immunogenicity of NS5A protein of human hepatitis C virus (HCV) when delivered as naked DNA (NS5A DNA), or recombinant protein (rNS5A). DBA/2J mice received NS5A DNA, rNS5A, or NS5A DNA/rNS5A in different prime-boost combinations with a peptidoglycan Immunomax((R)). The weakest response was induced after rNS5A prime and NS5A DNA boost; rNS5A alone induced an immune response with a strong Th2-component; and NS5A DNA alone, a relatively weak secretion of IL-2 and IFN-gamma. The most efficient was co-injection of NS5A DNA and rNS5A, which induced a significant increase in CD4(+) and CD8(+) T-cell counts, anti-NS5A antibodies, specific T-cell proliferation, and proinflammatory cytokine production in vitro against a broad spectrum of NS5A epitopes. Administration of the mixture of adjuvanted DNA and protein immunogens can be selected as the best regimen for further preclinical HCV-vaccine trials.

Hepatitis C virus core protein transforms murine fibroblasts by promoting genomic instability.

The oncogenic potential of hepatitis C virus (HCV) core protein has been demonstrated, but the precise mechanism of cell transformation triggered by HCV core is still unclear. This study shows that constitutive expression of HCV core protein (core) in NIH 3T3 murine fibroblasts triggers malignant transformation. At the preneoplastic stage, clones that expressed HCV core constitutively demonstrated genomic instability seen as disruption of the mitotic spindle cell checkpoint leading to increased ploidy. Transformation was completed by the loss of DNA and resistance to apoptosis induced by serum starvation. Simultaneously, cells acquired a capacity for anchorage independent growth and absence of contact inhibition. Inoculation of these transformed cells into severe combined immune deficiency (SCID) mice led to formation of solid core-expressing tumors. Transformation and tumorigenicity of core-expressing cell lines coincided with a 5- to 10-fold repression of endogenous p53 transactivation. Thus, long-term HCV core expression alone is sufficient for complete transformation of immortal fibroblasts that can then induce tumors in a susceptible host. This data suggests that malignant transformation by HCV core may occur through primary stress, induction of genomic instability, and further HCV core-induced rescue of surviving mutated cells.

DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus.

Nonstructural protein 3 (NS3) of human hepatitis C virus (HCV) is a conserved multi-functional protein essential for replication and translation of viral RNA and polyprotein processing. Early T-cell response against NS3 is capable of restricting viremia. We aimed at characterizing the immunogenicity in gene immunization of the conserved regions of NS3 critical for protein folding and activity. C57BL/6 mice were injected with NS3 gene of Russian HCV 1b isolate 274933RU. Immunization did not exert any overt histological changes and had no long-term effects on the immune status of NS3 gene-recipients. The immune response in NS3 gene-recipients was screened by antibody ELISA, T-cell proliferation test and immune assays for specific cytokine production. T-lymphocytes of NS3 gene-recipients proliferated in response to peptides representing conserved regions of protease and ATPase/helicase. Stimulated T-lymphocytes produced IL-2, and in response to protease-derived peptides, also IFN-gamma. Potent and long-lasting antibody response was raised against conserved NS3 regions including "Greek-key" motif of protease, motifs II, V and polynucleotide-binding domains of ATPase/helicase. Thus, gene immunization effectively targeted conserved regions critical for NS3 protease and helicase function. In type and specificity, immune response of NS3 gene-immunized mice mimicked immunity achieved in the acute self-limiting HCV infection of human and primates and in virus-exposed healthy individuals, indicating promiscuity of NS3 as immunogen.