{'fraction_non_alphanumeric': 0.032764190124596214, 'fraction_numerical': 0.026995846792801106, 'mean_word_length': 4.850202429149798, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Identification of Ferret ACE2 and its Receptor Function for Sars-Coronavirus

Severe acute respiratory syndrome associated coronavirus (SARS-CoV) was the causative agent of SARS, which occurred as an emerging pneumonic disease in 2002. The epidemiological investigations showed that several wild animals such as Himalayan palm civet and raccoon dog had been infected by SARS-CoV. Experimental infection to several laboratory animal species such as mouse, hamster, ferret, cat, and monkey revealed the susceptibility of those animal species to SARS-CoV infection, however, 8


INTRODUCTION
Severe acute respiratory syndrome associated coronavirus (SARS-CoV) was the causative agent of SARS, which occurred as an emerging pneumonic disease in 2002. [1][2][3] The epidemiological investigations showed that several wild animals such as Himalayan palm civet and raccoon dog had been infected by SARS-CoV. 4 Experimental infection to several laboratory animal species such as mouse, hamster, ferret, cat, and monkey revealed the susceptibility of those animal species to SARS-CoV infection, 5-7 however, 8 to be a functional receptor for SARS-CoV. 9 To see whether ACE2 function as receptor is attributed to the disease manifestation, we cloned a ferret ACE2 (feACE2) gene and compared the feACE2 with that of human or mouse ACE2 in terms of receptor functionality for SARS-CoV.

MATERIALS AND METHODS
Amplification of partial ACE2 gene by RT-PCR: RNAs were extracted from heart, lung, kidney, and small intestine of a ferret. To select a suitable organ to amplify a full-length ACE2 gene, we amplified a partial gene of ACE2 (952 bp in size) by RT-PCR. As a control, all RNA specimens were subjected to RT-PCR with ß-actin specific primers.
Determination of nucleotide sequence of ferret ACE2 (feACE2): Two overlapping regions of feACE2 genes were amplified by RT-PCR from kidney RNA. One contained the region 5' to the initiation codon of feACE2 gene, while the other was located 3' to a National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan.

RESULTS
The partial feACE2 gene was amplified from RNAs isolated from the lung, heart, kidney, and small intestine by RT-PCR. The amount of amplicon varied among the organs (Fig. 1). An entire feACE2 gene was successfully amplified by RT-PCR from RNA isolated from the kidney. Its sequencing analysis showed that feACE2 consisted of 805 amino acid residues. There were 6 predicted N-glycosylation sites in the feACE2 (Table  1), and 4 of them (aa 53, 322, 546, 690) were conserved between ferret and human ACE2. Amino acid identities of ACE2 between ferret and human or mouse were 82.6% and 81.5%, respectively ( Table 2).
We selected 3 HeLa cell clones stably expressing feACE2 by IFA (Fig. 2) and Western blot (data not shown). HeLa cells expressing feACE2 supported SARS-CoV replication to the same extent as those expressing human ACE2 (Fig. 3). Replication of SARS-CoV

DISCUSSION AND CONCLUSIONS
We have determined the complete nucleotide sequence of feACE2 gene and established HeLa cell lines that stably express feACE2. Studies using these cells as well as cells expressing human or mouse ACE2 revealed that feACE2 functions as a SARS-CoV receptor as efficiently as human ACE2, while mouse ACE2 does less et al., who reported that human ACE2 is more efficient than mouse ACE2 in binding to the SARS-CoV S protein. 10 520 sequencing and was deposited in GenBank under accession number AB208708. Putative signal peptide, zinc binding, transmembrane and N-glycosylation sites were searched by PROSITE. We aligned entire ACE2 sequences of ferret, human (BC039902) and mouse (BC026801), and determined the identities between them by use of Multiple Alignment program in the CLUSTALW.
From these observations, it is postulated that animal species whose ACE2 functions as an efficient SARS-CoV receptor would be a susceptible in feACE2 expressing HeLa cells was over 10 times more efficient than that in mouse efficiently. Our findings shown in this study are in agreement with the observations by Li

IDENTIFICATION OF FERRET ACE2
host species for this virus. To establish a good animal model for SARS, a ferret model would be a good candidate. Furthermore, transgenic mouse expressing ferret or human ACE2 may serve a useful animal model for SARS-CoV infection.