An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues

The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species' genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine

A single-amino-acid substitution in the HA protein changes the replication and pathogenicity of the 2009 pandemic A (H1N1) influenza viruses in vitro and in vivo

BACKGROUND: The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and since then it spread world wide over a short period of time. Although the virus infection is generally associated with mild disease and a relatively low mortality, it is projected that mutations in specific regions of the viral genome, especially within the receptor binding domain of the hemagglutinin (HA) protein could result in more virulent virus stains, leading to a more severe pandemic. RESULTS: Here, we found that a single amino acid substitution of Asp-to-Gly at position 222 in the HA protein of the A (H1N1) virus occurred after two passage propagation in the allantoic cavities of chicken embryonated eggs, and this single residue variation dramatically increased the viral replication ability in MDCK cells and pathogenicity in BALB/c mice. CONCLUSIONS: A substitution of Asp-to-Gly at position 222 in the HA protein was prone to occur under positive selection pressures, and this single amino acid mutation could dramatically increase the virus replication ability in vitro and pathogenicity in vivo. Our finding offers a better understanding of the transmission and evolution of the 2009 pandemic A (H1N1) virus and brings attention to further potentially severe influenza pandemic that may result from cross-host evolution of the influenza viruses

Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>Pathogenic yersiniae, including <it>Y. pestis</it>, share a type III secretion system (T3SS) that is composed of a secretion machinery, a set of translocation proteins, a control system, and six Yop effector proteins including YpkA and YopJ. The cyclic AMP receptor protein (CRP), a global regulator, was recently found to regulate the laterally acquired genes (<it>pla </it>and <it>pst</it>) in <it>Y. pestis</it>. The regulation of T3SS components by CRP is unknown.</p> <p>Results</p> <p>The <it>sycO</it>, <it>ypkA </it>and <it>yopJ </it>genes constitute a single operon in <it>Y. pestis</it>. CRP specifically binds to the promoter-proximate region of <it>sycO</it>, and represses the expression of the <it>sycO-ypkA-yopJ </it>operon. A single CRP-dependent promoter is employed for the <it>sycO-ypkA-yopJ </it>operon, but two CRP binding sites (site 1 and site 2) are detected within the promoter region. A CRP box homologue is found in site 1 other than site 2. The determination of CRP-binding sites, transcription start site and core promoter element (-10 and -35 regions) promotes us to depict the structural organization of CRP-dependent promoter, giving a map of CRP-promoter DNA interaction for <it>sycO-ypkA-yopJ</it>.</p> <p>Conclusion</p> <p>The <it>sycO-ypkA-yopJ </it>operon is under the direct and negative regulation of CRP in <it>Y. pestis</it>. The <it>sycO-ypkA-yopJ </it>promoter-proximate regions are extremely conserved in <it>Y. pestis</it>, <it>Y. pseudotuberculosis </it>and <it>Y. enterocolitica</it>. Therefore, data presented here can be generally applied to the above three pathogenic yersiniae.</p

The Effects of ATIR Blocker on the Severity of COVID-19 in Hypertensive Inpatients and Virulence of SARS-CoV-2 in Hypertensive hACE2 Transgenic Mice.

Angiotensin-converting enzyme 2 (ACE2) is required for the cellular entry of the severe acute respiratory syndrome coronavirus 2. ACE2, via the Ang-(1-7)-Mas-R axis, is part of the antihypertensive and cardioprotective effects of the renin-angiotensin system. We studied hospitalized COVID-19 patients with hypertension and hypertensive human(h) ACE2 transgenic mice to determine the outcome of COVID-19 with or without AT1 receptor (AT1R) blocker treatment. The severity of the illness and the levels of serum cardiac biomarkers (CK, CK-BM, cTnI), as well as the inflammation markers (IL-1, IL-6, CRP), were lesser in hypertensive COVID-19 patients treated with AT1R blockers than those treated with other antihypertensive drugs. Hypertensive hACE2 transgenic mice, pretreated with AT1R blocker, had increased ACE2 expression and SARS-CoV-2 in the kidney and heart, 1 day post-infection. We conclude that those hypertensive patients treated with AT1R blocker may be at higher risk for SARS-CoV-2 infection. However, AT1R blockers had no effect on the severity of the illness but instead may have protected COVID-19 patients from heart injury, via the ACE2-angiotensin1-7-Mas receptor axis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12265-021-10147-3

Adaption of Seasonal H1N1 Influenza Virus in Mice

The experimental infection of a mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. The mouse adaption of human influenza A virus can result in mutations in the HA and other proteins, which is associated with increased virulence in mouse lungs. In this study, a mouse-adapted seasonal H1N1 virus was obtained through serial lung-to-lung passages and had significantly increased virulence and pathogenicity in mice. Genetic analysis indicated that the increased virulence of the mouse-adapted virus was attributed to incremental acquisition of three mutations in the HA protein (T89I, N125T, and D221G). However, the mouse adaption of influenza A virus did not change the specificity and affinity of receptor binding and the pH-dependent membrane fusion of HA, as well as the in vitro replication in MDCK cells. Notably, infection with the mouse adapted virus induced severe lymphopenia and modulated cytokine and chemokine responses in mice. Apparently, mouse adaption of human influenza A virus may change the ability to replicate in mouse lungs, which induces strong immune responses and inflammation in mice. Therefore, our findings may provide new insights into understanding the mechanisms underlying the mouse adaption and pathogenicity of highly virulent influenza viruses

Genomic Polymorphism of the Pandemic A (H1N1) Influenza Viruses Correlates with Viral Replication, Virulence, and Pathogenicity In Vitro and In Vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus

Animal Models for Tuberculosis in Translational and Precision Medicine

Tuberculosis (TB) is a health threat to the global population. Anti-TB drugs and vaccines are key approaches for TB prevention and control. TB animal models are basic tools for developing biomarkers of diagnosis, drugs for therapy, vaccines for prevention and researching pathogenic mechanisms for identification of targets; thus, they serve as the cornerstone of comparative medicine, translational medicine, and precision medicine. In this review, we discuss the current use of TB animal models and their problems, as well as offering perspectives on the future of these models

Evaluation and Analysis of Soil Temperature Data over Poyang Lake Basin, China

Soil temperature reflects the impact of local factors, such as the vegetation, soil, and atmosphere of a region. Therefore, it is important to understand the regional variation of soil temperature. However, given the lack of observations with adequate spatial and/or temporal coverage, it is often difficult to use observational data to study the regional variation. Based on the observational data from Nanchang and Ganzhou stations and ERA-Interim/Land reanalysis data, this study analyzed the spatiotemporal distribution characteristics of soil temperature over Poyang Lake Basin. Four soil depths were examined, 0–7, 7–28, 28–100, and 100–289 cm, recorded as ST1, ST2, ST3, and ST4, respectively. The results showed close correlations between observation data and reanalysis data at different depths. Reanalysis data could reproduce the main spatiotemporal distributions of soil temperature over the Poyang Lake Basin but generally underestimated their magnitudes. Temporally, there was a clear warming trend in the basin. Seasonally, the temperature increase was the most rapid in spring and the slowest in summer, except for ST4, which increased the fastest in spring and the slowest in winter. The temperature increase was faster for ST1 than the other depths. The warming trend was almost the same for ST2, ST3, and ST4. An abrupt change of annual soil temperature at all depths occurred in 1997, and annual soil temperatures at all depths were abnormally low in 1984. Spatially, annual soil temperature decreased with latitude, except for the summer ST1. Because of the high temperature and precipitation in summer, the ST1 values were higher around the lake and the river. The climatic trend of soil temperature generally increased from south to north, which was opposite to the distribution of soil temperature. These findings provide a basis for understanding and assessing the variation of soil temperature in the Poyang Lake Basin

The double-sided effects of Mycobacterium Bovis bacillus Calmette–Guérin vaccine

Bacillus Calmette–Guérin (BCG), the only vaccine proven to be effective against tuberculosis (TB), is the most commonly used vaccine globally. In addition to its effects on mycobacterial diseases, an increasing amount of epidemiological and experimental evidence accumulated since its introduction in 1921 has shown that BCG also exerts non-specific effects against a number of diseases, such as non-mycobacterial infections, allergies and certain malignancies. Recent Corona Virus Disease 2019 (COVID-19) outbreak has put BCG, a classic vaccine with significant non-specific protection, into the spotlight again. This literature review briefly covers the diverse facets of BCG vaccine, providing new perspectives in terms of specific and non-specific protection mechanisms of this old, multifaceted, and controversial vaccine