Results And Discussion

Cloning and Expression of Nonstructural Proteins of HCoV-NL63 Eight out of 13 cDNAs encoding nonstructural proteins have been cloned into the entry vector pENTR D-TOPO. Using bacteriophage lambda recombination technology, seven out of them could be recombined into the expression vector pEXPl-DEST. Until now, 6 genes encoding nonstructural proteins of HCoV-NL63 have been expressed successfully. Figure 1 displays the expression of Nsp3, Nsp7, Nsp8, Nsp9, and Nsp10, while the expression of...

Nucleocapsid Protein Expression Facilitates Coronavirus Replication

Barbara Schelle, Nadja Karl, Burkhard Ludewig, Stuart G. Siddell, and Volker Thiel* The coronavirus nucleocapsid (N) protein has been implicated in a number of functions. As a structural protein, it forms a ribonucleoprotein complex with genomic RNA. However, it has also been described as an RNA-binding protein1 that might be involved in coronavirus RNA synthesis.2 Here, we used a reverse genetic approach to elucidate N protein function(s) in coronavirus replication and transcription. We could...

Rnaprocessing Enzymes

A detailed sequence analysis by Gorbalenya, Snijder and colleagues of the SARS-CoV genome revealed that the enzymology of coronavirus RNA synthesis may be significantly more complex than previously thought.8 In this seminal study published in 2003, as many as five novel enzymatic activities were identified in the genomes of coronaviruses and, to a varying extent, other nidoviruses (Fig. 1). The putative activities included (i) a 3'-to-5' exonuclease (ExoN) associated with nsp14, (ii) an...

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Arteriviridae, grouped into the order Nidovirales, together with the Coronaviridae and the Roniviridae '2 In vivo PRRSV has a predilection for porcine macrophages that express porcine sialoadhesin.3,4 In vitro porcine alveolar macrophages (PAM), some cultivated peripheral blood monocytes and the non-macrophage African green monkey kidney cell line...

Equine Viral Arteritis

Balasuriya* 1. INTRODUCTION Equine viral arteritis (EVA) is a contagious disease of horses caused by equine arteritis virus (EAV). EVA was first described perhaps 200 years or more ago. Horsemen also recognized long ago that otherwise healthy stallions could transmit the disease to susceptible mares at breeding, and that these carrier stallions could be a source of infection for many years. EAV was first isolated by Doll et al. during an outbreak of respiratory...

Ace2

Human ACE2, 209, 219, 229, 261, 277, 281, 285, 295, 481 SARS-CoV, 209, 211, 229, 261, 294, 324, 479 Mustela vison, 507 NL63, 219, 223, 281, 285 Rat ACE2, 232 Antisense morpholino oligomers, 567 APN equine arteritis virus, 429 LDHV, 439 Minnesota isolate, PRRS,73 N protein, 145 Nsp2, 73 pathogenesis, 439 quasispecies, 435 replication, 145 vaccine, 449 Astrocytes, 409 Autoantibodies morphogenesis, 175 Bioinformatics, 395 Cathepsins, 237, 335 CD209L, 261, 265 Cholesterol role in entry, 305...

Contributors

Adair, The Scripps Research Institute, La Jolla, California Fernando Almaz n, Centro Nacional de Biotecnolog a, CSIC, Madrid, Spain Sara Alonso, Centro Nacional de Biotecnolog a, CSIC, Madrid, Spain Enrique lvarez, Centro Nacional de Biotecnolog a, CSIC, Madrid, Spain Yasushi Ami, National Institute of Infectious Diseases, Tokyo, Japan Kanchan Anand, University of L beck, L beck, Germany William D. Arndt, Arizona State University, Tempe, Arizona Yasuko Asahi-Ozaki, National Institute...

Polymerase And Helicase Activities

The coronavirus RdRp has been classified as an outgroup of the RdRp superfamily 1.36 It resides in the ORF1b-encoded nonstructural proteins 12, as was predicted about 15 years ago6 and now demonstrated for a bacterially expressed form of the SARS-CoV nsp12.37 The RdRp catalytic domain occupies the C-terminal two-thirds of nsp12,6 but also the N-terminal part of nsp12 seems to contribute to activity.37 Based on MHV co-immunoprecipitation data, the N-terminal part of nsp12 has been suggested to...

Insights From Animal Ace2 And Animalderived Viral Isolates

The ability of the ACE2 proteins of mice, rats, and palm civets to support SARS-CoV infection has been compared with that of human ACE2.22, 51 Compared with cells expressing human receptor, SARS-CoV infection was less efficient in cells expressing murine ACE2. Infection was nearly absent in those expressing rat ACE2. Consistent with a role for palm civets in transmitting virus, palm civet ACE2 supported SARS-CoV infection as efficiently as human ACE2. These results correlated with affinity of...

Development Of Vaccines And Passive Immunotherapy Against Sars Coronavirus Using Mouse And Scidpblhu Mouse Models

Masaji Okada, Yuji Takemoto, Yoshinobu Okuno, Satomi Hashimoto, Yukari Fukunaga, Takao Tanaka, Yoko Kita, Sechiko Kuwayama, Yumiko Muraki, Noriko Kanamaru, Hiroko Takai, Chika Okada, Yayoi Sakaguchi, Izumi Furukawa, Kyoko Yamada, Miwa Izumiya, Shigeto Yoshida, Makoto Matsumoto, Tetsuo Kase, J. S. M. Peiris, Daphne E. deMello, Pei-Jer Chen, Naoki Yamamoto, Yoshiyuki Yoshinaka, Tatsuji Nomura, Isao Ishida, Shigeru Morikawa, Masato Tashiro, and Mitsunori Sakatani* *Masaji Okada, Yuji Takemoto,...

Discussion

SARS-CoV is a zoonotic pathogen, and several cases of likely animal to human transmission have been reported in China. In most cases, these infections were mild and did not spread beyond the index case suggesting that animal viruses require additional adaptation prior to evolving efficient usage of humans as hosts.3,4,7 After adapting to the human host, most severe disease manifestations occur within elderly populations likely compromised by waning innate immune and acquired immune responses to...

Materials And Methods

Transient expression of viral protein in HeLa cells Constructs containing plasmid DNA under the control of a T7 promoter were transiently expressed in mammalian cells using the recombinant vaccinia virus (vTF7-3) system as described before.5 In this study, the transfection reagent used was Effectene (Qiagen). *Nanyang Technological University, Singapore, 637551, and Institute of Molecular and Cell Biology, Proteos, Singapore, 1386731. Immunoprecipitation Transiently transfected HeLa cells in...

1

Detected in a considerable percentage of samples. HCoV-NL63 could be detected in 17.4 of these croup patients and was therefore the most frequently identified respiratory virus for croup. Since most of the samples tested were derived from the year 2000-2001, we cannot exclude that the high percentage of HCoV-NL63-positive samples is due to a strong viral activity in this particular year, and long-term studies are needed to determine whether HCoV-NL63 infections occur in cycles peaking every two...

Plasticity Of The Substratebinding Site

A special feature first discovered for the SARS-CoV M ro (but most probably present in all coronavirus main proteinases) is that in the monoclinic crystals grown at pH 6.0, the two monomers have different conformations around the S1 substrate-binding site, because the loop 138-145, in particular Phe140, as well as Glu166 undergo dramatic conformational rearrangements. As a result, one protomer exists in an active and the other in an inactive conformation.9 In the latter, the S1...

Conclusion

Coronaviruses mature by a budding process at the cis-Golgi network endoplasmic reticulum-Golgi intermediate compartment.9 The two coronavirus envelope proteins M and E are known to be intracellularly retained.1, 2 For the coronavirus S proteins, others found some surface expression by using efficient expression vectors like vaccinia virus or baculovirus.10, 11 12 Even with vaccinia virus, the majority of the S protein was found intracellularly. The weak surface expression can be explained by an...

Antiviral Ppmo Selection And Efficacy

PMO complementary to the genomic (positive-sense) strand, which were designed to bind regions identified by conservation or noted in the literature as critical for viral RNA synthesis, were synthesized (Fig. 1a). PMO were covalently linked to peptides NH2-RRRRRRRRRFFC-CONH2 or NH2-RRRRRFFRRRRC-CONH2 designated as R9F2 or R5F2R4, respectively. Both types of peptide-conjugated PMO are henceforth referred to as P-PMO, and were used interchangeably in these studies. Cells treated with < 20 M PMO...

And Animals

Southeast China has long been regarded as an epicenter for influenza viruses.1 Indeed, avian influenza H5N1 97, H9N2 99, H7N7 03, and other recent avian H5 strains have occasionally crossed the species barrier in the last few years.2 These events keep reminding us that new pandemic viruses may emerge in this geographical region. However, Nature caught us by surprise when the first pandemic in this millennium was caused not by influenza viruses, or any known pathogens, but by a previously...

Coronavirus Mhvjhmv In Mixed Neural Culture327

Keiko Nakagaki, Kazuhide Nakagaki, and Fumihiro Taguchi 4.24. RECEPTOR-INDEPENDENT INFECTION OF MOUSE HEPATITIS VIRUS ANALYSIS BY Rie Watanabe, Kazumitsu Suzuki, and Fumihiro Taguchi 4.25. SARS-CoV, BUT NOT HCoV-NL63, UTILIZES CATHEPSINS TO INFECT CELLS VIRAL I-Chueh Huang, Berend Jan Bosch, Wenhui Li, Michael Farzan, Peter M. Rottier, and Hyeryun Choe

Virucidal Effect Of A Newly Developed Nickel Alloy On Mouse Coronavirus

Norio Hirano and Takenori Nakayama* 1. INTRODUCTION A newly recognized disease, severe acute respiratory syndrome (SARS), was first reported in China in February 2003. A few months after the first outbreak of SARS, the disease was transmitted worldwide in more than 20 countries of Asia, Europe, and North America. A novel coronavirus (CoV) was detected in patients with SARS and identified as causative agent. Civet cats have been suspected as natural host of SARS CoV, which infects human beings...

C G Gc

Predicted 5' UTR secondary structures of MHV and SARS-CoV. Figure 2. (A) 1H-15N HSQC and (B) 2J HNN-COSY spectra acquired with uniformly 13C,15N-labeled SL2sa Figure 2. (A) 1H-15N HSQC and (B) 2J HNN-COSY spectra acquired with uniformly 13C,15N-labeled SL2sa (C) 2J HNN-COSY spectrum acquired for a uridine-only frequency 600 MHz, pH 6.0, 0.1 M KCl, 5 mM MgCl2, 10-25 C. 3C,15N- U -labeled sample. Conditions 1H Strand ( wmt-SiMiL RNA + Slnind mKNAi> - Srrarul mRNAfi Jh H h 4 h 4h Sil...

Sars Cov Why Susceptible To Monkeys

Despite rapid advances in our knowledge of SARS-Coronavirus (SARS-CoV), the regions of the virus spike glycoprotein and host receptor that are important for virus entry remain to be completely elucidated. The tropism of SARS-CoV for cells derived from many diverse species was analyzed by virus titration and by the use of a multiplex RT-PCR assay that differentiates input virus from virus that has entered a cell and initiated replication.1 Cells derived from monkey, human, and mink were shown to...

Results

Transgenic mice expressing GFP under control of the oligodendroglia-specific PLP promoter were infected to analyze genes associated with MHC presentation based on the easy distinction between CD45lc microglia and GFP+ CD45- oligodendroglia by flow cytometry. Resident glial cells expressed few if any MHC molecules in the normal adult murine CNS (Table 1). By day 5 p.i., expression of Class I was detected on the majority of microglia and a subset of oligodendroglia. The initial delay in...