Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains. MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs). RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals. CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

Identifying Inconclusive Data in the SARS-CoV-2 Molecular Diagnostic Using Nucleocapsid Phosphoprotein Gene as Target

ABSTRACT Context: The gold standard test to identify the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in coronavirus disease 2019 (COVID-19) patients is the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), but the inconclusive data and presence of false positive diagnosis remain the major problem of this approach. Objective: To compare the fitness of two primers sets to the SARS-CoV-2 nucleocapsid phosphoprotein (NP) gene in the molecular diagnosis of COVID-19, we verify the inconclusive data and confidence of high cycle threshold (Ct) values in the SARS-CoV-2 detection. Design: The 970 patient samples were tested using United States Centers for Disease Control and Prevention protocol. We compared the fitness of two primers sets to two different regions of NP gene. In addition, we check the consistency of positive samples with high Ct values by retesting extracted SARS-CoV-2 RNA or by second testing of patients. Results: The N1 and N2 displayed similar fitness during testing with no differences between Ct values. Then, we verified security range Cts related to positive diagnostic with Ct above 34 failing in 21/32 (65.6%) after retesting of samples. The samples patients with Ct above 34.89 that were doubly positive revealed a low sensitivity (52.4%) and specificity (63.6%) of the test in samples with Ct above 34. Conclusions: It is secure to use one primer set to the NP gene to identify SARS-CoV-2 in samples. However, samples with high Ct values may be considered inconclusive and retested to avoid false positive diagnosis.

Genetic and antigenic characterization of an expanding H3 influenza A virus clade in US swine visualized by Nextstrain

Defining factors that influence spatial and temporal patterns of influenza A virus (IAV) is essential to inform vaccine strain selection and strategies to reduce the spread of potentially zoonotic swine-origin IAV. The relative frequency of detection of the H3 phylogenetic clade 1990.4.a (colloquially known as C-IVA) in US swine declined to 7% in 2017, but increased to 32% in 2019. We conducted phylogenetic and phenotypic analyses to determine putative mechanisms associated with increased detection. We created an implementation of Nextstrain to visualize the emergence, spatial spread, and genetic evolution of H3 IAV-S, identifying two C-IVA clades that emerged in 2017 and cocirculated in multiple US states. Phylodynamic analysis of the HA gene documented low relative genetic diversity from 2017 to 2019, suggesting clonal expansion. The major H3 C-IVA clade contained an N156H amino acid substitution, but HI assays demonstrated no significant antigenic drift. The minor HA clade was paired with the NA clade N2-2002B prior to 2016, but acquired and maintained N2-2002A in 2016, resulting in a loss in antigenic cross-reactivity between N2-2002B and -2002A containing H3N2 strains. The major C-IVA clade viruses acquired a nucleoprotein (NP) of the H1N1pdm09 lineage through reassortment in replacement of the North American swine lineage NP. Instead of genetic or antigenic diversity within the C-IVA HA, our data suggest that population immunity to H3 2010.1, along with antigenic diversity of the NA and acquisition of the H1N1pdm09 NP gene likely explain the re-emergence and transmission of C-IVA H3N2 in swine.

The Investigation of Canine Distemper Virus in Different Diagnosis Materials of Dogs using Molecular and Pathological Methods, Northeastern Turkey

Background: Canine distemper virus (CDV) is highly contagious disease that affects dogs despite several control measures. This study was aimed at investigating the presence of CDV nucleic acid in different clinical and tissue materials, from naturally infected dogs, by reverse transcriptase-polymerase chain reaction (RT-PCR) and to molecularly characterize distemper strains according to the partial Nucleoprotein (NP) gene sequence. Furthermore, tissue samples under went histopathological examination for distemper infection. Methods: A total of 202 different diagnosis materials were collected from dogs (n=60) in the Kars region in northeastern Turkey. The samples were tested for CDV using RT-PCR with primers designed for the CDV NP gene. Samples determined as positive for CDV (n=7) were sequenced. Tissue samples underwent histopathological examination. Result: Most of the cases were in animals aged 0-6 months. The most common clinical finding was severe respiratory system infection. This finding was accompanied by gastrointestinal and nervous system infections. CDV nucleic acid was detected in 112 of 202 materials by RT-PCR. According to RT-PCR results, positivity rates of 88.2% (30/34), 72.2% (13/18), 60% (3/5), 55.5% (10/18), 55.5% (10/18), 51.6% (16/31), 45.5% (5/11), 37.8% (14/37) and 36.7% (11/30) were detected in nasal swab, lung, footpad, kidney, spleen, rectal swab, cerebrospinal fluids (CSF), leuokocyte and cerebellum samples, respectively. Viral nucleic acids were detected at higher rates in nasal swabs. The phylogenetic assessment of the amplicon sequences revealed a 97.7%-100% similarity among the Turkish CDV strains, which are independent from vaccine strains, were found to be more closely related to the European lineage. Intranuclear and intracytoplasmic inclusion bodies were detected by histopathology. This is the first study to investigate CDV in naturally infected dogs from northeastern Turkey and to provide novel and updated epidemiological information.

Comparison and Evaluation of Real-Time Taqman PCR for Detection and Quantification of Ebolavirus

Given that ebolavirus causes severe and frequently lethal disease, its rapid and accurate detection using available and validated methods is essential for controlling infection. Real-time reverse-transcription PCR (RT-PCR) has proven to be an invaluable tool for ebolaviruses diagnostics. Many assays with different targets have been developed, but they have not been externally compared or validated, and limits of detection are not uniformly reported. Here we compared and evaluated the sensitivity, reproducibility and specificity of 23 in-house assays under the same conditions. Our results showed that these assays were highly gene- and species- specific when evaluated using in vitro RNA transcripts and viral RNA, and the potential limits of detection were uniformly reported ranging from 102 to 106 in vitro synthesized RNA transcripts copies perμL and 1–100 TCID50/mL. The comparison of these assays indicated that those targeting the more conservative NP gene could be the better option for EVD case definition and quantitative measurement because of its higher sensitivity for the same species. Our analysis could contribute to the standardization of ebolavirus detection and quantification assays, which can offer a better understanding of the meaning of results across laboratories and time points, as well as make them easy to implement, especially under outbreak conditions.

Pleiotropic Regulatory Genes as a Tool for Streptomyces Strains Bioprospecting and Improvement

: Many taxa within class Actinobacteria, most notably genus Streptomyces, are known for the abundant presence of specialized biosynthetic pathways that convert essential cellular metabolites (amino acids, acyl moieties such as acetyl-CoA, nucleotides etc) into diverse natural products (NPs). NPs remain one of the pillars of modern pharmaceutical industry, and use of NPs as antibiotics is perhaps the most notable example of the commercial success of NPs. Nowadays, as humankind faces a formidable challenge to counter the rise of antimicrobial resistance and viral infections, there is renewed interest in streptomycetes as a source of novel NPs. This prompted the investigation of a variety of approaches to discover novel NPs and to improve the production of known ones. The focus of this review is on the use of regulatory genes to discover novel NPs. The two-layered scheme of regulation of NP biosynthesis is described and terms referring to cryptic NP gene cluster are detailed. Major players in global regulatory network are described as well as how their manipulation may be used to access the secondary metabolomes of Streptomyces and Actinobacteria in general. The value of studying the NP regulation in the era of synthetic biology is summarized in the last section.

Molecular detection and whole genome characterization of Canine Parainfluenza type 5 in Thailand

Parainfluenza virus type 5 (PIV-5) causes respiratory infection in several animal species and humans. Canine parainfluenza virus type 5 (CPIV-5) causes respiratory disease in domestic dogs worldwide. In this study, we conducted a cross-sectional survey of CPIV-5 in dogs with respiratory symptoms from small animal hospitals in Thailand from November 2015 to December 2018. Our results showed that 32 out of 571 nasal swab samples (5.6%) were positive for CPIV-5 by RT-PCR specific to the NP gene. To characterize the viruses, three representative CPIV-5 were subjected to whole genome sequencing, and an additional ten CPIV-5 were subjected to HN, F, SH and V/P gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai CPIV-5 was closely related to the CPIV-5 isolated from China and Korea. In conclusion, this study constitutes a whole genome characterization of CPIV-5 from dogs in Thailand. The surveillance of CPIV-5 should be further investigated at a larger scale to determine the dynamics, distribution and potential zoonotic transmission of CPIV-5.

Abstract 17325: Obesity, Blood Pressure, and Diurnal Variation in Natriuretic Peptides: A Clinical Trial

Background: Obese individuals have disturbed blood pressure (BP) rhythmicity and relative natriuretic peptide (NP) deficiency. The relationship of diurnal variation in NP levels and 24h BP rhythm is not known. Furthermore, mechanisms behind difference in circulating NPs in healthy obese and lean individuals has not been explored. We conducted a prospective clinical trial to evaluate 1) the diurnal rhythmicity of NPs and its relationship with 24-hour BP rhythm between healthy lean and obese individuals, and 2) elucidate mechanism behind NP deficiency in obese. Methods: Healthy, normotensive, lean (BMI:18.5-25 kg/m 2 ) and obese (BMI:30-45 kg/m 2 ) individuals aged 18-40 years, underwent 24-hour standardized inpatient protocol involving ambulatory BP monitoring (ABPM), controlled light intensity, and 10 blood draws, following 5-days of standardized diet. NP gene expression was evaluated in a cohort of 37 healthy donor heart tissues obtained from the MAGNet repository. Results: Among 52 participants screened, a total of 40 participants (18 lean; 22 obese) were enrolled. Diurnal variation in MRproANP levels was seen in both lean and obese individuals (p for rhythmicity=0.001). The mesor of the NP rhythm was 15% (8.5-21.6%) lower in obese. The diurnal variation in MRproANP was in antiphase with diurnal variation of systolic BP (p<0.001) ( Figure ). Obese participants had lower 24h renin levels (p=0.06) and higher nocturnal sodium excretion (p=0.08). Among obese, there was lower expression of NP production genes ( NPPA, NPPB) (p<0.05) , and higher expression of clearance gene ( NPR3 ) (p<0.001) in heart tissue. Conclusions: In a mechanistic human trial, we elucidate key neurohormonal differences in rhythm and evidence of poor salt handling in obese. Decreased production and increased clearance may contribute to the NP deficiency in obese. Targeting the diurnal NP-BP rhythm axis may reduce the cardiovascular risk burden, specifically in obese individuals.

A HIGHLY SENSITIVE REAL-TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION FOR DETECTING NUCLEOCAPSID PROTEIN GENE OF BOTH CLASSES I AND II OF NEWCASTLE DISEASE VIRUS

Newcastle disease virus (NDV) is a worldwide viral agent that infects over 200 species of birds and is responsible for outbreaks of ND. Although a series of real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been developed for detecting different genes of NDV, diagnostic sensitivity and efficiency still can be improved. This study describes a nucleocapsid protein gene rRT-PCR screening assay based on TaqMan technology for the detection of divergent NDV strains. All 23 representative NDV strains of classes I and II in the tested panel were detected using the NP-gene rRT-PCR assay, whereas eight class I and two class II NDV isolates cannot be detected by the USDA-validated matrix-gene assay. The detection limit of the NP-gene assay was approximately 10[Formula: see text] EID[Formula: see text]/mL. The new assay also demonstrated a high degree of specificity with no false-positive results of 35 non-NDV viruses. A total of 146 clinical specimens were also tested and the NP-gene assay gave high relative sensitivity (100%) and specificity (96.61%) when compared with virus isolation. This NP-gene rRT-PCR assay offers a sensitive, specific and rapid assay for detecting both class I and II NDV and can be used alongside with the existing diagnostic assays for this notifiable disease agent.

Single-cell transcriptomic evidence for dense intracortical neuropeptide networks

Seeking new insights into the homeostasis, modulation and plasticity of cortical synaptic networks, we have analyzed results from a single-cell RNA-seq study of 22,439 mouse neocortical neurons. Our analysis exposes transcriptomic evidence for dozens of molecularly distinct neuropeptidergic modulatory networks that directly interconnect all cortical neurons. This evidence begins with a discovery that transcripts of one or more neuropeptide precursor (NPP) and one or more neuropeptide-selective G-protein-coupled receptor (NP-GPCR) genes are highly abundant in all, or very nearly all, cortical neurons. Individual neurons express diverse subsets of NP signaling genes from palettes encoding 18 NPPs and 29 NP-GPCRs. These 47 genes comprise 37 cognate NPP/NP-GPCR pairs, implying the likelihood of local neuropeptide signaling. Here, we use neuron-type-specific patterns of NP gene expression to offer specific, testable predictions regarding 37 peptidergic neuromodulatory networks that may play prominent roles in cortical homeostasis and plasticity.