Biomedical subjects

Yi Huang

Publications and source records attributed to Yi Huang.

At least 37 records · Page 2Linked to original sources

PubMed2006

Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1.

We sequenced and compared the complete genomes of 22 strains of coronavirus HKU1 (CoV HKU1) obtained from nasopharyngeal aspirates of patients with respiratory tract infections over a 2-year period. Phylogenetic analysis of 24 putative proteins and polypeptides showed that the 22 CoV HKU1 strains fell into three clusters (genotype A, 13 strains; genotype B, 3 strains and genotype C, 6 strains). However, different phylogenetic relationships among the three clusters were observed in different regions of their genomes. From nsp4 to nsp6, the genotype A strains were clustered with the genotype B strains. For nsp7 and nsp8 and from nsp10 to nsp16, the genotype A strains were clustered with the genotype C strains. From hemagglutinin esterase (HE) to nucleocapsid (N), the genotype B strains were clustered closely with the genotype C strains. Bootscan analysis showed possible recombination between genotypes B and C from nucleotide positions 11,500 to 13,000, corresponding to the nsp6-nsp7 junction, giving rise to genotype A, and between genotypes A and B from nucleotide positions 21,500 to 22,500, corresponding to the nsp16-HE junction, giving rise to genotype C. Multiple alignments further narrowed the sites of crossover to a 143-bp region between nucleotide positions 11,750 and 11,892 and a 29-bp region between nucleotide positions 21,502 and 21,530. Genome analysis also revealed various numbers of tandem copies of a perfect 30-base acidic tandem repeat (ATR) which encodes NDDEDVVTGD and various numbers and sequences of imperfect repeats in the N terminus of nsp3 inside the acidic domain upstream of papain-like protease 1 among the 22 genomes. All 10 CoV HKU1 strains with incomplete imperfect repeats (1.4 and 4.4) belonged to genotype A. The present study represents the first evidence for natural recombination in coronavirus associated with human infection. Analysis of a single gene is not sufficient for the genotyping of CoV HKU1 strains but requires amplification and sequencing of at least two gene loci, one from nsp10 to nsp16 (e.g., pol or helicase) and another from HE to N (e.g., spike or N). Further studies will delineate whether the ATR is useful for the molecular typing of CoV HKU1.

Adult↗

PubMed2006

[Amiodarone pneumonitis with Hypermastigote lung infection: a case report and review of the literatures].

OBJECTIVE: To describe the clinical features of amiodarone pneumonitis with Hypermastigote lung infection. METHODS: Case report and review of the related literatures. The clinical symptoms, laboratory tests, radiographic patterns, diagnosis, and therapeutic management of amiodarone pneumonitis with Hypermastigote lung infection were described. RESULTS: A 58 year old male patient presented dyspnea after exertion. Pulmonary function showed decrease of the diffusing capacity, and CT showed interstitial changes and alveolar exudation. Foamy cells and Hypermastigotes were found in the bronchoalveolar lavage fluid. After cessation of amiodarone and the start of anti-parasite therapy, the symptoms relieved. CONCLUSIONS: Amiodarone pneumonitis with Hypermastigote lung infection is very rare. The infection may be due to decrease of local immunity caused by amiodarone pneumonitis.

Amiodarone↗

PubMed2006

[Pb-tolerance of ectomycorrhizal fungi from polluted and unpolluted sites under different P levels].

By the method of liquid culture, this paper studied the effects of different P and Pb doses on the biomass production of three strains of ectomycorrhizal fungi, and the P and Pb concentrations in their mycelium. The results showed that under the application of P and Pb, the biomass production of strains ZC1 and ZC2, collected from mining area was significantly higher than that of strain GV collected from non-polluted site. At low P level (0.16 mmol x L(-1)), the growth of these three ectomycorrhizal fungi was inhibited by the increasing dose of Pb; while at higher P levels (0.81and 2.42 mmol x L(-1)), increasing dose of Pb inhibited the growth of GV significantly, but had little effects on ZC1 and ZC2. In all treatments, the Pb concentration in GV mycelia was significantly higher than that in the mycelia of ZC1 and ZC2. Under the same dose of Pb, the P concentration in GV mycelia was higher at low P level, but lower at higher P levels than that in ZC1 and ZC2 mycelia. The increase of Pb dose decreased the P concentration in ZC1 and ZC2, mycelia, but had little effect on that in GV mycelia. In comparing with GV, ZC1 and ZC2 had the characteristics of fast growth and low Pb and high P accumulation in their mycelia, indicating that the strains of ecomycorrhizal fungi collected from polluted area had a higher tolerance to environmental stress.

Ecosystem↗

PubMed2006

[Influence of copper, cadmium on growth and cation exchange capacity of two kinds of ectomycorrhizal funguses].

Ectomycorrhizal fungus has the ability to enhance the growth of higher plants in the contaminated area, especially ruined by heavy metals. And much attention was focused on how the fungus could enhance the resistance of higher plants. We focused on the resistance of ectomycorrhizal fungus in vitro to heavy metals. In the first experiment, the mycelium biomasses of two ectomycorhizal funguses growing in the Kottke media treated with different concentrations of Cu and Cd were measured after growth as well as the pH value of the medium. The results indicated that heavy metals could reduce the biomasses of the two funguses. Gomplhidius viscidus has higher tolerance to Cu but less Cd than that of Boletus edulis. With development of fungal mycila, the pH value of medium dropped significantly, and this effect might play an important role in enhancing its tolerance. In addition, the higher pH value change per biomass indicated that the fungus treated with heavy metals had the ability to adjust environment of pH more significantly. In the second experiment, the cation exchange capacity (CEC) of the cell walls of the fungus treated with heavy metals was measured according to Marschner's. The results indicated that with the increasing of the concentrations of Cu or Cd, the CEC of Gomphidius viscidus increased, but the CEC of Boletus edulis dropped.

Biomass↗

PubMed2006

[Construction of urban green space ecosystem by using corridor network: a case study in west urban area of Dongying City, Shandong Province].

This paper discussed the ecological significance of urban green corridors network in urban green space ecosystem, analyzed the present status of green space ecosystem in the west urban area of Dongying in Shangdong Province, and figured out the ways of constructing urban green corridors network in this area to strengthen the linkage between its fragmented greenbelts, and between these greenbelts and rural natural environment. Through the construction of this network, the greenbelt area in the west urban area of Dongying would increase 1400 hm2, greenbelt area per capita would increase to 66 m2, and urban and rural greenbelts would be integrated into a whole system to serve the whole city, giving a powerful support to enhance the life quality of local people and the stability of urban ecosystem.

China↗

PubMed2005

Clinical and molecular epidemiological features of coronavirus HKU1-associated community-acquired pneumonia.

BACKGROUND: Recently, we described the discovery of a novel group 2 coronavirus, coronavirus HKU1 (CoV-HKU1), from a patient with pneumonia. However, the clinical and molecular epidemiological features of CoV-HKU1-associated pneumonia are unknown. METHODS: Prospectively collected (during a 12-month period) nasopharyngeal aspirates (NPAs) from patients with community-acquired pneumonia from 4 hospitals were subjected to reverse-transcription polymerase chain reaction, for detection of CoV-HKU1. The epidemiological, clinical, and laboratory characteristics of patients with CoV-HKU1-associated pneumonia were analyzed. The pol, spike (S), and nucleocapsid (N) genes were also sequenced. RESULTS: NPAs from 10 (2.4%) of 418 patients with community-acquired pneumonia were found to be positive for CoV-HKU1. All 10 cases occurred in spring and winter. Nine of these patients were adults, and 4 had underlying diseases of the respiratory tract. In the 6 patients from whom serum samples were available, all had a 4-fold change in immunoglobulin (Ig) G titer and/or presence of IgM against CoV-HKU1. The 2 patients who died had significantly lower hemoglobin levels, monocyte counts, albumin levels, and oxygen saturation levels on admission and had more-extensive involvement visible on chest radiographs. Sequence analysis of the pol, S, and N genes revealed 2 genotypes of CoV-HKU1. CONCLUSIONS: CoV-HKU1 accounts for 2.4% of community-acquired pneumonia, with 2 genotypes in the study population. Without performance of diagnostic tests, the illness was clinically indistinguishable from other community-acquired pneumonia illnesses.

Adolescent↗

PubMed2005

Spermine oxidase SMO(PAOh1), Not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine analogue-treated human breast cancer cell lines.

The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H2O2. However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N1,N1-bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N1-acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H2O2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.

Acetyltransferases↗

PubMed2005

Baseline total and specific differential white blood cell counts and 5-year all-cause mortality in community-dwelling older women.

Increasing evidence demonstrates that inflammation is associated with many pathophysiologic processes and mortality in older adults. Increase in total white blood cell (WBC) counts is recognized as an important cellular marker of systemic inflammation. However, relationships of total WBC and individual differential counts with mortality in older adults, particularly in older women, have not been adequately evaluated. To address this important question, we obtained baseline total WBC and differential counts and 5-year all-cause mortality of 624 community-dwelling women age 65-101 in the Women's Health and Aging Study cohort, excluding those with WBC counts above the normal range. Using Kaplan-Meier survival and Cox proportional hazard regression analyses, and adjusting for age, race, body mass index, smoking, and education, we identified that baseline higher total WBC, higher neutrophil, or lower lymphocyte counts were independently associated with increased mortality. No significant associations of eosinophil, monocyte, or basophil counts with mortality were observed. These results suggest that beyond acute bacterial infection, changes in counts of baseline total WBC and its specific subpopulations predict increased mortality in older women. They provide a basis for further investigation into the role of leukocytes in age-related inflammation and its associated adverse outcomes in older adults.

Aged↗

PubMed2005

Role of p53/p21(Waf1/Cip1) in the regulation of polyamine analogue-induced growth inhibition and cell death in human breast cancer cells.

Intracellular polyamines are absolutely required for cell proliferation and many tumors have abnormal requirements for polyamines. Therefore, the polyamine metabolic pathway represents a rational target for antineoplastic intervention. A number of polyamine analogues act as potent modulators of cellular polyamine metabolism and exhibit encouraging effects against tumor growth in both cell culture and animal studies. In this study we demonstrate that specific polyamine analogues exhibit differential inhibitory action against growth of human breast cancer MCF-7 cells. Treatment of MCF-7 cells with oligoamine analogues and the symmetrically substituted bis(alkyl)-substituted analogue, BENSpm, produced a G1 cell cycle arrest, while the unsymmetrically substituted bis(alkyl)-substituted analogue, CHENSpm, induced a G2/M cell cycle arrest. All four compounds significantly upregulated p53 and p21 expression in MCF-7 cells. Stable transfection of small interfering RNA (siRNA) targeting p53 blocked the expression of p21 induced by the polyamine analogues and significantly reduced polyamine analogue-induced growth inhibition and apoptosis, suggesting that polyamine analogue-induced p21 expression occurs through p53-dependent mechanisms. The effects of analogue exposure on cyclins and cyclin dependent kinases varied with the specific agent used. Expression of p53 siRNA reversed only BENSpm-modulated the cell cycle arrest, suggesting that regulation of cell cycle arrest by p53/p21 induced by polyamine analogues occurs through agent-specific mechanisms. Understanding the mechanism of p53-mediated cellular responses to polyamine analogue may help to improve the therapeutic efficacy of polyamine analogues in human breast cancer.

Apoptosis↗

PubMed2005

Axial nonuniformity of geometric and mechanical properties of mouse aorta is increased during postnatal growth.

The hemodynamic conditions of aorta are relatively uniform prenatally and become more heterogeneous postnatally. Our objective was to quantify the heterogeneity of geometry and mechanical properties during growth and development. To accomplish this objective, we obtained a systematic set of data on the geometry and mechanical properties along the length of mouse aorta during postnatal development. C57BL/6 mice of ages 1-33 days were studied. The ascending aorta was cannulated in situ and preconditioned with several cyclic changes in pressure. We investigated the axial variations of geometry (diameter and length) and mechanical properties (stress-stain relation, elastic modulus and compliance) of the mouse aorta from the aortic valve to the common iliac. Our results show that the arterial blood pressure of mice increased from approximately 30 to 80 mmHg during the first 2 wk of life. The stretch ratio, diameter, wall (intima-media) thickness, and total lumen volume of mouse aorta increased with age. The aorta was transformed from a cylindrical tube at birth to a tapered structure during growth. Furthermore, we found the mechanical properties were fairly uniform along the length of the aorta at birth and become more nonuniform with age. We conclude that the rapid change of blood pressure and blood flow after birth alter the geometric and mechanical properties differentially along the length of the aorta. Hence, the axial nonuniformity of the aorta increases as the organ becomes more specialized during growth and development.

Aging↗

PubMed2005

Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats.

Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.

Amino Acid Sequence↗

PubMed2005

Oral bacteria in the occluded arteries of patients with Buerger disease.

OBJECTIVE: Recent studies have suggested that infectious organisms play a role in vascular diseases. In this study, to explore a possible link between oral infection and Buerger disease, we investigated whether oral (periodontal) bacteria were present in occluded arteries removed from patients with characteristic Buerger disease. METHODS: Fourteen male patients with a smoking history who had developed characteristics of Buerger disease before the age of 50 years were included in this study. Occluded arteries, including superficial femoral (n = 4), popliteal (n = 2), anterior tibial (n = 4), and posterior tibial (n = 4) arteries, were removed and studied. A periodontist performed a periodontal examination on each patient and collected dental plaque and saliva samples from them at the same time. The polymerase chain reaction method was applied to detect whether seven species of periodontal bacteria--Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedia , and Prevotella nigrescens--were present in the occluded arteries and oral samples. In addition, arterial specimens from seven control patients were examined by polymerase chain reaction analysis. RESULTS: DNA of oral bacteria was detected in 13 of 14 arterial samples and all oral samples of patients with Buerger disease. Treponema denticola was found in 12 arterial and all oral samples. Campylobacter rectus, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Prevotella nigrescens were found in 14% to 43% of the arterial samples and 71% to 100% of the oral samples. A pathologic examination revealed that arterial specimens showed the characteristics of an intermediate-chronic-stage or chronic-stage lesion of Buerger disease. All 14 patients with Buerger disease had moderate to severe periodontitis. None of the control arterial samples was positive for periodontal bacteria. CONCLUSIONS: This is the first study to identify oral microorganisms in the lesions of Buerger disease. Our findings suggest a possible etiologic link between Buerger disease and chronic infections such as oral bacterial infections.

Adult↗

PubMed2005

Total and differential white blood cell counts and their associations with circulating interleukin-6 levels in community-dwelling older women.

BACKGROUND: Interleukin-6 (IL-6) is an inflammatory biomediator, and age-related increases in IL-6 levels are associated with osteoporosis, sarcopenia, disability, and mortality in older adults. Although white blood cells (WBC), or leukocytes, are known to produce IL-6 in vitro, their in vivo relationship with circulating IL-6 levels is not well established. METHODS: In this cross-sectional analysis of data from the Women's Health and Aging Study I, the authors evaluated the relationships of total WBC and WBC differential (neutrophil, monocyte, lymphocyte, eosinophil, and basophil) counts to circulating IL-6 levels in 619 community-dwelling older women. Potential associations of age, race, and cigarette smoking with total and differential WBC counts and IL-6 levels were also assessed. RESULTS: Except for lymphocyte and basophil counts, significant associations of total WBC, neutrophil, monocyte, and eosinophil counts with IL-6 levels were identified. These associations remained highly significant after adjustment for age, race, and smoking status. Total WBC, neutrophil, monocyte, and eosinophil counts had significant stepwise increases in four escalating quartiles of IL-6 levels. In addition, age, race, and cigarette smoking were differentially associated with total and differential WBC counts and IL-6 levels. CONCLUSIONS: Positive in vivo associations of total WBC and its specific subpopulations were identified, including neutrophils, monocytes, and eosinophils, with circulating IL-6 levels in community-dwelling older women. These findings suggest significant contributions of WBC and its subpopulations to circulating IL-6 levels and potential effects from chronic elevation of IL-6 levels to the function of these circulating immune cells that warrant further investigation.

Age Factors↗

PubMed2005

Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes.

Amyloid beta protein (Abeta) is the principal component of neuritic plaques in Alzheimer's disease (AD). Abeta is derived from beta amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-site APP cleaving enzyme 1 (BACE1) has been identified as the major beta-secretase. BACE2 is the homolog of BACE1. The BACE2 gene is on chromosome 21 and has been implicated in the pathogenesis of AD. However, the function of BACE2 in Abeta generation is controversial. Some studies have shown that BACE2 cleaved APP at the beta-site whereas other studies showed it cleaved around the alpha-secretase site. To elucidate the involvement of BACE2 in AD pathogenesis, we compared BACE2 and BACE1 gene regulation and their functions in Abeta generation. We cloned and functionally characterized the human BACE2 promoter. The BACE2 gene is controlled by a TATA-less promoter. Though Sp1 can regulate both BACE1 and BACE2 genes, comparative sequence analysis and transcription factor prediction showed little similarity between the two promoters. BACE1 increased APP cleavage at the beta-site and Abeta production whereas BACE2 did not. Overexpression of BACE2 significantly increased sAPP levels in conditioned media but markedly reduced Abeta production. Knockdown of BACE2 resulted in increased APP C83. Our data indicate that despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation. BACE2 is not a beta-secretase, but processes APP within the Abeta domain at a site downstream of the alpha-secretase cleavage site. Our data argue against BACE2 being involved in the formation of neuritic plaques in AD.

Alzheimer Disease↗

PubMed2005

Molecular mechanisms of polyamine analogs in cancer cells.

The natural polyamines are aliphatic cations with multiple functions and are essential for cell growth. Soon after the critical requirement of polyamines for cell proliferation was recognized, the metabolism of polyamines was pursued as a target for antineoplastic therapy. Initially, much attention was focused on the development of inhibitors of polyamine biosynthesis as a means to inhibit tumor growth. The best-characterized inhibitor is alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. While compensatory mechanisms in polyamine metabolism reduce the effectiveness of DFMO as a single chemotherapeutic agent, it is currently undergoing extensive testing and clinical trials for chemoprevention and other diseases. There has been increasing interest over the last two decades in the cytotoxic response to agents that target the regulation of polyamine metabolism rather than directly inhibiting the metabolic enzymes in tumor cells. This interest resulted in the development of a number of polyamine analogs that exhibit effective cytotoxicity against tumor growth in preclinical models. The analogs enter cells through a selective polyamine transport system and can be either polyamine antimetabolites that deplete the intracellular polyamines or polyamine mimetics that displace the natural polyamines from binding sites, but do not substitute in terms of growth-promoting function. Synthesis of the first generation of symmetrically substituted bis(alkyl)polyamine analogs in the mid-1980s was based on the theory that polyamines may utilize feedback mechanisms to auto-regulate their synthesis. In the 1990s, unsymmetrically substituted bis(alkyl) polyamine analogs were developed. These compounds display structure-dependent and cell type-specific cellular effects and regulation on polyamine metabolism. More recently, a novel class of analogs has been synthesized, which include conformationally restricted, cyclic and long-chain oligoamine analogs. The development and use of these analogs have provided valuable information for understanding the molecular mechanisms of targeting the polyamine pathway as a means of cancer therapy.

Animals↗

PubMed2005

Prevotella multiformis sp. nov., isolated from human subgingival plaque.

Four bacterial strains isolated from the human oral cavity, PPPA19, PPPA21(T), PPPA28 and PPPA30, were characterized by determining phenotypic and biochemical features, cellular fatty acid profiles and the phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that each of the isolates was a member of the genus Prevotella. These strains were related to Prevotella denticola with about 95 % similarity. The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. However, the cells of these strains were often cocci (coccobacilli), depending on the cultivation time. Colonies of different sizes were detected on Eggerth Gagnon agar plates for these strains. The cells forming large colonies were cocci, whereas those forming small colonies were cocci and rods. However, 16S rRNA gene sequence comparison of colonies of different sizes revealed that only a single organism was present. Although these strains had phenotypic characteristics that were similar to those of P. denticola JCM 8528, they could be differentiated from P. denticola JCM 8528 by aesculin hydrolysis and d-cellobiose fermentation in API 20A tests. DNA-DNA hybridization experiments revealed the genomic distinction of these four strains with respect to P. denticola JCM 8528. On the basis of these data, a novel Prevotella species, Prevotella multiformis sp. nov., is proposed, with PPPA21(T) (=JCM 12541(T)=DSM 16608(T)) as the type strain.

Bacteroidaceae Infections↗

PubMed2005

In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU1 3C-like protease.

Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV-HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182-amino acid replicase polyprotein in the CoV-HKU1 genome showed that the replicase polyprotein of CoV-HKU1 is cleaved by its papain-like proteases and 3C-like protease (3CL(pro)) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CL(pro) revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CL(pro) are occupied by glutamine. This is also observed in CoV-HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV-HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV-HKU1 sequences from multiple specimens of different patients; the P2 and P1'-P12' positions of this cleavage site are consistent between CoV-HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C-terminal amino acid sequencing of the CoV-HKU1 helicase and trans-cleavage assays of the CoV-HKU1 3CL(pro) will confirm the presence of this novel cleavage site.

Amino Acid Sequence↗

PubMed2005

Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia.

Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 x 10(6) copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 x 10(6) copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.

Aged↗