Comparative Evaluation of Standard RT-PCR Assays and Commercial Real-Time RT-PCR Kits for Detection of Lassa Virus.

Lassa virus (LASV) is a causative agent of hemorrhagic fever epidemic in West Africa. In recent years, it has been transmitted several times to North America, Europe, and Asia. Standard reverse transcription (RT)-PCR and real-time RT-PCR are extensively used for early detection of LASV. However, the high nucleotide diversity of LASV strains complicates the development of appropriate diagnostic assays. Here, we analyzed LASV diversity clustered with geographic location and evaluated the specificity and sensitivity of two standard RT-PCR methods (GPC RT-PCR/1994 and 2007) and four commercial real-time RT-PCR kits (namely, Da an, Mabsky, Bioperfectus, and ZJ) to detect six representative LASV lineages using in vitro synthesized RNA templates. The results showed that the GPC RT-PCR/2007 assay had better sensitivity compared to the GPC RT-PCR/1994 assay. The Mabsky and ZJ kits were able to detect all RNA templates of six LASV lineages. Contrastingly, the Bioperfectus and Da an kits failed to detect lineages IV and V/VI. The limit of detection for lineage I with the Da an, Bioperfectus, and ZJ kits were significantly higher than that of the Mabsky kit at an RNA concentration of 1 × 1010 to 1 × 1011 copies/mL. The Bioperfectus and Da an kits detected lineages II and III at an RNA concentration of 1 × 109 copies/mL, higher than that of the other kits. In conclusion, the GPC RT-PCR/2007 assay and the Mabsky kit were suitable assays for the detection of LASV strains based on good analytical sensitivity and specificity. IMPORTANCE Lassa virus (LASV) is a significant human pathogen causing hemorrhagic fever in West Africa. Increased traveling around the world raises the risk of imported cases to other countries. The high nucleotide diversity of LASV strains clustered with geographic location complicates the development of appropriate diagnostic assays. In this study, we showed that the GPC reverse transcription (RT)-PCR/2007 assay and the Mabsky kit are suitable for detecting most LASV strains. Future assays for molecular detection of LASV should be based on specific countries/regions along with new variants.

Emergence of an ancient and pathogenic mammarenavirus.

Emerging zoonoses of wildlife origin caused by previously unknown agents are one of the most important challenges for human health. The Qinghai-Tibet Plateau represents a unique ecological niche with diverse wildlife that harbours several human pathogens and numerous previously uncharacterized pathogens. In this study, we identified and characterized a novel arenavirus (namely, plateau pika virus, PPV) from plateau pikas (Ochotona curzoniae) on the Qinghai-Tibet Plateau by virome analysis. Isolated PPV strains could replicate in several mammalian cells. We further investigated PPV pathogenesis using animal models. PPV administered via an intraventricular route caused trembling and sudden death in IFNαßR-/- mice, and pathological inflammatory lesions in brain tissue were observed. According to a retrospective serological survey in the geographical region where PPV was isolated, PPV-specific IgG antibodies were detected in 8 (2.4%) of 335 outpatients with available sera. Phylogenetic analyses revealed that this virus was clearly separated from previously reported New and Old World mammarenaviruses. Under the co-speciation framework, the estimated divergence time of PPV was 77-88 million years ago (MYA), earlier than that of OW and NW mammarenaviruses (26-34 MYA).

RNA Virus Diversity in Birds and Small Mammals From Qinghai-Tibet Plateau of China.

Most emerging and re-emerging viruses causing infectious diseases in humans and domestic animals have originated from wildlife. However, current knowledge of the spectrum of RNA viruses in the Qinghai-Tibet Plateau in China is still limited. Here, we performed metatranscriptomic sequencing on fecal samples from 56 birds and 91 small mammals in Tibet and Qinghai Provinces, China, to delineate their viromes and focused on vertebrate RNA viruses. A total of 184 nearly complete genome RNA viruses belonging to 28 families were identified. Among these, 173 new viruses shared <90% amino acid identity with previously known viral sequences. Several of these viruses, such as those belonging to genera Orthonairovirus and Hepatovirus, could be zoonotic viruses. In addition, host taxonomy and geographical location of these viruses showed new hosts and distribution of several previously discovered viruses. Moreover, 12 invertebrate RNA viruses were identified with <40% amino acid identity to known viruses, indicating that they belong to potentially new taxa. The detection and characterization of RNA viruses from wildlife will broaden our knowledge of virus biodiversity and possible viral diseases in the Qinghai-Tibet Plateau.

Discovery and Evolution of a Divergent Coronavirus in the Plateau Pika From China That Extends the Host Range of Alphacoronaviruses.

Although plateau pikas are the keystone species in the plateau ecosystem of the Qinghai Province of China, little is known about their role in the evolution and transmission of viral pathogens, especially coronaviruses. Here, we describe the characterization and evolution of a novel alphacoronavirus, termed plateau pika coronavirus (PPCoV) P83, which has a prevalence of 4.5% in plateau pika fecal samples. In addition to classical gene order, the complete viral genome contains a unique nonstructural protein (NS2), several variable transcription regulatory sequences and a highly divergent spike protein. Phylogenetic analysis indicates that the newly discovered PPCoV falls into the genus Alphacoronavirus and is most closely related to rodent alphacoronaviruses. The co-speciation analysis shows that the phylogenetic trees of the alphacoronaviruses and their hosts are not always matched, suggesting inter-species transmission is common in alphacoronaviruses. And, PPCoV origin was estimated by molecular clock based on membrane and RNA-dependent RNA polymerase encoding genes, respectively, which revealed an apparent discrepancy with that of co-speciation analysis. PPCoV was detected mainly in intestinal samples, indicating a potential enteric tropism for the virus. Overall, this study extends the host range of alphacoronaviruses to a new order (Lagomorpha), indicating that plateau pikas may be the natural reservoir of PPCoV and play an important and long-term role in alphacoronavirus evolution.

Beta- and Novel Delta-Coronaviruses Are Identified from Wild Animals in the Qinghai-Tibetan Plateau, China.

Outbreaks of severe virus infections with the potential to cause global pandemics are increasingly concerning. One type of those commonly emerging and re-emerging pathogens are coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2). Wild animals are hosts of different coronaviruses with the potential risk of cross-species transmission. However, little is known about the reservoir and host of coronaviruses in wild animals in Qinghai Province, where has the greatest biodiversity among the world's high-altitude regions. Here, from the next-generation sequencing data, we obtained a known beta-coronavirus (beta-CoV) genome and a novel delta-coronavirus (delta-CoV) genome from faecal samples of 29 marmots, 50 rats and 25 birds in Yushu Tibetan Autonomous Prefecture, Qinghai Province, China in July 2019. According to the phylogenetic analysis, the beta-CoV shared high nucleotide identity with Coronavirus HKU24. Although the novel delta-CoV (MtCoV) was closely related to Sparrow deltacoronavirus ISU42824, the protein spike of the novel delta-CoV showed highest amino acid identity to Sparrow coronavirus HKU17 (73.1%). Interestingly, our results identified a novel host (Montifringilla taczanowskii) for the novel delta-CoV and the potential cross-species transmission. The most recent common ancestor (tMRCA) of MtCoVs along with other closest members of the species of Coronavirus HKU15 was estimated to be 289 years ago. Thus, this study increases our understanding of the genetic diversity of beta-CoVs and delta-CoVs, and also provides a new perspective of the coronavirus hosts.

Anti-inflammatory Mechanism Involved in 4-Ethylguaiacol-Mediated Inhibition of LPS-Induced Inflammation in THP-1 Cells.

4-Ethylguaiacol, a common aroma compound of baijiu (a traditional Chinese alcoholic beverage), was assessed for its potential anti-inflammatory effects in an LPS-induced THP-1 cell model. To characterize the effect of 4-ethylguaiacol on the LPS-induced inflammatory response, the mRNA and protein expression of the TLR4-MAPKs-NF-κB-IκBα-AP-1, Nrf2-HO-1, and AMPK-SIRT1 pathways were monitored by ELISA, real-time PCR, and Western blotting. On the basis of the result, 4-ethylguaiacol exerted anti-inflammatory effects at doses of 10, 100, and 500 µM (the concentration of 4-ethylguaiacol in gujinggong baijiu is in the range of 1044 ± 44 to 1661 ± 63 µg/L) and significantly mitigated LPS-induced inflammation via activation of the Nrf2-HO-1 and AMPK-SIRT1 pathways and inhibition of NF-κB and AP-1 activation, thereby markedly inhibiting the activation of inflammasomes and down-regulating the production of inflammatory cytokines. These results indicated that 4-ethylguaiacol could reverse LPS-induced inflammatory responses and is a natural, potent anti-inflammatory component in baijiu.

Marmota himalayana in the Qinghai-Tibetan plateau as a special host for bi-segmented and unsegmented picobirnaviruses.

Wildlife has been considered the main source of novel viruses causing emerging infectious diseases. Marmota himalayana is endemic to the Qinghai-Tibetan Plateau, China. Here, based on a high-throughput method using Illumina RNA sequencing, we studied the RNA virome of M. himalayana and discovered multiple novel viruses, especially picobirnaviruses (PBVs), which have a bi-segmented genome and belong to the family Picobirnaviridae. A total of 63% of the viral contigs corresponded to PBVs, comprising 274 segment 1 and 56 segment 2 sequences. Unexpectedly, four unsegmented PBV genomes were also detected and confirmed by PCR and resequencing. According to the phylogenetic analysis, the following nine PBV assortment types are proposed: C1:GI, C2:GIV, C4:GI, C4:GV, C5:GI, C7:GI, C8:GIV, C8:GV and C8:GII. We hypothesize a model of segmentation for the PBV genome, mediated by a 6-bp direct repeat sequence, GAAAGG. The model is supported by detection of the segmentation-associated sequence GAAAGG not only in the 5' untranslated regions of segment 1 (221 in 289) and segment 2 (57 in 80) of bi-segmented PBVs but also in the 5' untranslated regions and junction sequences between the capsid and RdRp genes of unsegmented PBVs. Therefore, with RNA sequencing, we found an unexpected biodiversity of PBVs in M. himalayana, indicating that M. himalayana is a special host for PBVs. We also proposed a putative model of how bi-segmented PBVs could be converted into unsegmented PBVs, which sheds new light on the processes of RNA virus genome evolution.

Hemorrhagic fever caused by a novel Bunyavirus in China: pathogenesis and correlates of fatal outcome.

BACKGROUND: Hemorrhagic fever-like illness caused by a novel Bunyavirus, Huaiyangshan virus (HYSV, also known as Severe Fever with Thrombocytopenia virus [SFTSV] and Fever, Thrombocytopenia and Leukopenia Syndrome [FTLS]), has recently been described in China. METHODS: Patients with laboratory-confirmed HYSV infection who were admitted to Union Hospital or Zhongnan Hospital between April 2010 and October 2010 were included in this study. Clinical and routine laboratory data were collected and blood, throat swab, urine, or feces were obtained when possible. Viral RNA was quantified by real-time reverse-transcriptase polymerase chain reaction. Blood levels of a range of cytokines, chemokines, and acute phase proteins were assayed. RESULTS: A total of 49 patients with hemorrhagic fever caused by HYSV were included; 8 (16.3%) patients died. A fatal outcome was associated with high viral RNA load in blood at admission, as well as higher serum liver transaminase levels, more pronounced coagulation disturbances (activated partial thromboplastin time, thrombin time), and higher levels of acute phase proteins (phospholipase A, fibrinogen, hepcidin), cytokines (interleukin [IL]-6, IL-10, interferon-γ), and chemokines (IL-8, monocyte chemotactic protein 1, macrophage inflammatory protein 1b). The levels of these host parameters correlated with viral RNA levels. Blood viral RNA levels gradually declined over 3-4 weeks after illness onset, accompanied by resolution of symptoms and laboratory abnormalities. Viral RNA was also detectable in throat, urine, and fecal specimens of a substantial proportion of patients, including all fatal cases assayed. CONCLUSIONS. Viral replication and host immune responses play an important role in determining the severity and clinical outcome in patients with infection by HYSV.

Analysis of synonymous codon usage in Shigella flexneri 2a strain 301 and other Shigella and Escherichia coli strains.

In this study, we analysed synonymous codon usage in Shigella flexneri 2a strain 301 (Sf301) and performed a comparative analysis of synonymous codon usage patterns in Sf301 and other strains of Shigella and Escherichia coli. Although there was a significant variety in codon usage bias among different Sf301 genes, there was a slight but observable codon usage bias that could primarily be attributable to mutational pressure and translational selection. In addition, the relative abundance of dinucleotides in Sf301 was observed to be independent of the overall base composition but was still caused by differential mutational pressure; this also shaped codon usage. By comparing the relative synonymous codon usage values across different Shigella and E. coli strains, we suggested that the synonymous codon usage pattern in the Shigella genomes was strain specific. This study represents a comprehensive analysis of Shigella codon usage patterns and provides a basic understanding of the mechanisms underlying codon usage bias.

Hemorrhagic fever caused by a novel tick-borne Bunyavirus in Huaiyangshan, China.

BACKGROUND: From April to July in 2009 and 2010, unexplained severe hemorrhagic fever-like illnesses occurred in farmers from the Huaiyangshan mountains range. METHODS: Clinical specimens (blood, urine, feces, and throat swabs) from suspected patients were obtained and stored. Mosquitoes and ticks in affected regions were collected. Virus was isolated from 2 patients and characterized by whole genome sequencing. Virus detection in additional patients and arthropods was done by virus-specific reverse transcription (RT) PCR. Clinical and epidemiological data of RT-PCR confirmed patients were analyzed. RESULTS: An unknown virus was isolated from blood of two patients and from Haemaphysalis ticks collected from dogs. Whole genome sequence analysis identified the virus as a novel member of the family Bunyaviridae, most closely related to the viruses of the genus Phlebovirus within which it forms a separate lineage. Subsequently, infection was confirmed by RT-PCR in 33 of 58 suspected patients. The illness in these patients was characterized by fever, severe malaise, nausea, vomiting, and diarrhea. Prominent laboratory findings included low white cell- and platelet counts, coagulation disturbances, and elevation of liver enzymes. Hemorrhagic complications were observed in 3 cases, 5 (15%) patients died. CONCLUSIONS: A novel tick-borne Bunyavirus causing life-threatening hemorrhagic fever in humans has emerged in the Huaiyangshan mountain areas of China. Further studies are needed to determine the epidemiology, geographic distribution and vertebrate animal ecology of this virus.

Hemorrhagic fever caused by a novel tick-borne Bunyavirus in Huaiyangshan,China / 中华流行病学杂志

Background:From April to July in 2009 and 2010,unexplained severe hemorrhagic fever-like illnesses occurred in farmers from the Huaiyangshan mountains range.Methods:Clinical specimens (blood,urine,feces,and throat swabs) from suspected patients were obtained and stored.Mosquitoes and ticks in affected regions were collected.Virus was isolated from 2 patients and characterized by whole genome sequencing.Virus detection in additional patients and arthropods was done by virus-specific reverse transcription (RT) PCR.Clinical and epidemiological data of RT-PCR confirmed patients were analyzed.Results:An unknown virus was isolated from blood of two patients and from Haemaphysalis ticks collected from dogs.Whole genome sequence analysis identified the virus as a novel member of the family Bunyaviridae,most closely related to the viruses of the genus Phlebovirus within which it forms a separate lineage.Subsequently,infection was confirmed by RT-PCR in 33 of 58 suspected patients.The illness in these patients was characterized by fever,severe malaise,nausea,vomiting,and diarrhea.Prominent laboratory findings included low white cell- and platelet counts,coagulation disturbances,and elevation of liver enzymes.Hemorrhagic complications were observed in 3 cases,5 (15%) patients died.Conclusions:A novel tick-borne Bunyavirus causing life-threatening hemorrhagic fever in humans has emerged in the Huaiyangshan mountain areas of China.Further studies are needed to determine the epidemiology,geographic distribution and vertebrate animal ecology of this virus.