Roujian Lu

Etiology and Clinical Characterization of Respiratory Virus Infections in Adult Patients Attending an Emergency Department in Beijing

Background Acute respiratory tract infections (ARTIs) represent a serious global health burden. To date, few reports have addressed the prevalence of respiratory viruses (RVs) in adults with ARTIs attending an emergency department (ED). Therefore, the potential impact of respiratory virus infections on such patients remains unknown. Methodology/Principal Findings To determine the epidemiological and clinical profiles of common and recently discovered respiratory viruses in adults with ARTIs attending an ED in Beijing, a 1-year consecutive study was conducted from May, 2010, to April, 2011. Nose and throat swab samples from 416 ARTI patients were checked for 13 respiratory viruses using multiple reverse transcription polymerase chain reaction(RT-PCR) assays for common respiratory viruses, including influenza viruses (Flu) A, B, and adenoviruses (ADVs), picornaviruses (PICs), respiratory syncytial virus (RSV), parainfluenza viruses (PIVs) 1–3, combined with real-time RT-PCR for human metapneumovirus (HMPV) and human coronaviruses (HCoVs, -OC43, -229E, -NL63, and -HKU1). Viral pathogens were detected in 52.88% (220/416) of patient samples, and 7.21% (30/416) of patients tested positive for more than one virus. PICs (17.79%) were the dominant agents detected, followed by FluA (16.11%), HCoVs (11.78%), and ADV (11.30%). HMPV, PIVs, and FluB were also detected (<3%), but not RSV. The total prevalence and the dominant virus infections detected differed significantly between ours and a previous report. Co-infection rates were high for HCoV-229E (12/39, 30.76%), PIC (22/74, 29.73%), ADV (12/47, 25.53%) and FluA (15/67, 22.39%). Different patterns of clinical symptoms were associated with different respiratory viruses. Conclusions The pattern of RV involvement in adults with ARTIs attending an ED in China differs from that previously reported. The high prevalence of viruses (PIC, FluA, HCoVs and ADV) reported here strongly highlight the need for the development of safe and effective therapeutic approaches for these viruses.

Characterization of human coronavirus etiology in Chinese adults with acute upper respiratory tract infection by real-time RT-PCR assays.

Background In addition to SARS associated coronaviruses, 4 non-SARS related human coronaviruses (HCoVs) are recognized as common respiratory pathogens. The etiology and clinical impact of HCoVs in Chinese adults with acute upper respiratory tract infection (URTI) needs to be characterized systematically by molecular detection with excellent sensitivity. Methodology/Principal Findings In this study, we detected 4 non-SARS related HCoV species by real-time RT-PCR in 981 nasopharyngeal swabs collected from March 2009 to February 2011. All specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hMPV) and human bocavirus (HBoV). 157 of the 981 (16.0%) nasopharyngeal swabs were positive for HCoVs. The species detected were 229E (96 cases, 9.8%), OC43 (42 cases, 4.3%), HKU1 (16 cases, 1.6%) and NL63 (11 cases, 1.1%). HCoV-229E was circulated in 21 of the 24 months of surveillance. The detection rates for both OC43 and NL63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (P<0.001 and P = 0.003), and there was a higher detection frequency of HKU1 in patients aged over 60 years (P = 0.03). 48 of 157(30.57%) HCoV positive patients were co-infected. Undifferentiated human rhinoviruses and influenza (Flu) A were the most common viruses detected (more than 35%) in HCoV co-infections. Respiratory syncytial virus (RSV), human parainfluenza virus (PIV) and HBoV were detected in very low rate (less than 1%) among adult patients with URTI. Conclusions/Significance All 4 non-SARS-associated HCoVs were more frequently detected by real-time RT-PCR assay in adults with URTI in Beijing and HCoV-229E led to the most prevalent infection. Our study also suggested that all non-SARS-associated HCoVs contribute significantly to URTI in adult patients in China.

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged α2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α2AAR gene locus. Exploiting this mouse line, we evaluated α2AAR trafficking and α2AAR-mediated inhibition of Ca2+ currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the α2AAR subtype. We discovered a more rapid desensitization of Ca2+ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α2AAR evoked by clonidine than by guanfacine. Clonidine-induced α2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α2AAR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-α2AAR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

Enhanced Hypotensive, Bradycardic, and Hypnotic Responses to α2-Adrenergic Agonists in Spinophilin-Null Mice Are Accompanied by Increased G Protein Coupling to the α2A-Adrenergic Receptor

We previously identified spinophilin as a regulator of α2 adrenergic receptor (α2AR) trafficking and signaling in vitro and in vivo (Science 304:1940–1944, 2004). To assess the generalized role of spinophilin in regulating α2AR functions in vivo, the present study examined the impact of eliminating spinophilin on α2AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the α2AAR subtype, after systemic administration of the α2-agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in spinophilin-null mice. Mice lacking spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to α2AR stimulation. Whereas these changes in sensitivity to α2AR agonists occur independent of any changes in α2AAR density or intrinsic affinity for agonist in the brains of spinophilin-null mice compared with wild-type control mice, the coupling of the α2AAR to cognate G proteins is enhanced in spinophilin-null mice. Thus, brain preparations from spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of α2AAR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.

Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the First Imported MERS-CoV Case in China

ABSTRACT On 26 May 2015, an imported Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in Guangdong Province, China, and found to be closely related to the MERS-CoV strain prevalent in South Korea. The full genome of the ChinaGD01 strain was sequenced and analyzed to investigate the epidemiology and evolution of MERS-CoV circulating in South Korea and China.

Protein Kinase A Phosphorylation of Spinophilin Modulates Its Interaction with the α2A-Adrenergic Receptor (AR) and Alters Temporal Properties of α2AAR Internalization

Spinophilin plays critical roles in regulating trafficking and signaling of the α2-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940–1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-α2AAR interaction to regulate α2AAR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the α2AAR, and this event can be blocked by Ser → Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser → Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the α2AAR binding region of spinophilin, is sufficient to block the spinophilin-α2AAR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the α2AAR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the α2AAR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating α2AAR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.

Enhanced Hypotensive, Bradycardia and Hypnotic Responses to α2-adrenergic Agonists in Spinophilin Null Mice Are Accompanied by Increased G Protein Coupling to the α2AAR

We previously identified spinophilin as a regulator of α2 adrenergic receptor (α2AR) trafficking and signaling in vitro and in vivo (Science 304:1940–1944, 2004). To assess the generalized role of spinophilin in regulating α2AR functions in vivo, the present study examined the impact of eliminating spinophilin on α2AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the α2AAR subtype, after systemic administration of the α2-agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in spinophilin-null mice. Mice lacking spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to α2AR stimulation. Whereas these changes in sensitivity to α2AR agonists occur independent of any changes in α2AAR density or intrinsic affinity for agonist in the brains of spinophilin-null mice compared with wild-type control mice, the coupling of the α2AAR to cognate G proteins is enhanced in spinophilin-null mice. Thus, brain preparations from spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of α2AAR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.

Human Coronaviruses HCoV-NL63 and HCoV-HKU1 in Hospitalized Children with Acute Respiratory Infections in Beijing, China

The human coronaviruses (HCoVs) HCoV-NL63 and HCoV-HKU1 are two recently discovered coronaviruses that circulate widely and are associated with acute respiratory infections (ARI). We detected HCoV-NL63 and HCoV-HKU1 in specimens collected from May 2008 to March 2010 from patients with ARI aged <7.75 years of age attending the Beijing Childrens Hospital. Thirty-two (8.4%) and 57 (14.9%) of 382 specimens tested positive for HCoV-NL63 and HCoV-HKU1, respectively, by real-time RT-PCR. Use of a Luminex xTAG RVP Fast kit showed that coinfection with respiratory syncytial virus and parainfluenza 3 virus was common among patients infected with either virus type. In HCoV-HKU1-infected patients, the predominant clinical symptoms were cough, fever, and expectoration. In HCoV-NL63-infected patients they were cough, fever, and rhinorrhea. Phylogenetic studies showed that the HCoV-HKU1 nucleoprotein gene was relatively conserved compared to NCBI reference sequences, while the 1ab gene of HCoV-NL63 showed more variation.

Prevalence and Genetic Diversity Analysis of Human Coronavirus OC43 among Adult Patients with Acute Respiratory Infections in Beijing, 2012

To determine the prevalence, epidemiology and genetic diversity of human coronavirus OC43 (HCoV-OC43) among adult patients with acute respiratory infections (ARI) in Beijing,five hundred and fifty-nine nasopharyngeal swab samples were collected from adult patients with ARI in Beijing. The prevalence of HCoV-OC43 infection among these patients was assessed using two different OneStep reverse transcription polymerase chain reaction (RT-PCR) assays. The epidemiological profiles of the patients with HCoV-OC43 infection were described. Partial S and N genes of HCoV-OC43 circulating strains were sequenced followed by phylogenetic analysis and amino acid alignment. Our results showed that the prevalence of HCoV-OC43 infection was 12.52% (95% CI: 9.78–15.26%), and the epidemic peak occurred in autumn. Fifty partial S and 40 partial N fragments were obtained from these patients. Phylogenetic analysis based on neighbour-joining method showed that at least three distinct clusters (A, B, C/D) of HCoV-OC43 strains were circulating among adult patients with ARI in Beijing. In addition, some novel unique clusters (UNT) of HCoV-OC43 were found in the S- and N-based phylogenetic trees. Furthermore, consensus amino acids substitutes for each cluster were also found after alignment of partial S or N sequence coding region in this study. In conclusion, we herein describe the prevalence of HCoV-OC43 among adult patients and provide substantial evidence for the genetic diversity of HCoV-OC43 circulating in Beijing.

Cross-talk from β adrenergic receptors modulates α2A adrenergic receptor endocytosis in sympathetic neurons via protein kinase A and spinophilin

Background: Cross-talk between GPCRs is an important but undercharacterized mechanism regulating receptor responsiveness. Results: Co-activation of β and α2ARs accelerates α2AAR endocytosis in a PKA- and spinophilin-dependent fashion. Conclusion: βAR-mediated signaling modulates α2AAR endocytosis via PKA-dependent disruption of α2AAR/spinophilin interaction. Significance: Cross-talk from β to α2ARs may have important implications in basal adrenergic tone and the pharmacology of commonly used adrenergic therapeutics. Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α2ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α2AAR endocytosis and that α2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α2AARs. Importantly, we show that α2AR agonist-mediated α2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α2ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α2AARs and accelerating α2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α2AARs.