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Homozygous L-SIGN (CLEC4M) plays a protective role in SARS coronavirus infection
Human genetic basis of coronavirus disease 2019
Hao Deng, Xue Yan & Lamei Yuan
From bedside to bench: regulation of host factors in SARS-CoV-2 infection
Samantha Y. Q. Ong, Ilya M. Flyamer, … Simon C. Biddie
Genome-wide analysis provides genetic evidence that ACE2 influences COVID-19 risk and yields risk scores associated with severe disease
Julie E. Horowitz, Jack A. Kosmicki, … Manuel A. R. Ferreira
Host genetic basis of COVID-19: from methodologies to genes
Kristina Zguro, Chiara Fallerini, … Alessandra Renieri
Allelic imbalance of HLA-B expression in human lung cells infected with coronavirus and other respiratory viruses
Yuanxu Zhang, Yisheng Sun, … Yanfeng Zhang
Common human genetic variants of APOE impact murine COVID-19 mortality
Benjamin N. Ostendorf, Mira A. Patel, … Sohail F. Tavazoie
SARS-CoV-2 pathogenesis
Mart M. Lamers & Bart L. Haagmans
Host genomics of SARS-CoV-2 infection
Claire Redin, Christian W. Thorball & Jacques Fellay
ELF5 is a potential respiratory epithelial cell-specific risk gene for severe COVID-19
Maik Pietzner, Robert Lorenz Chua, … Claudia Langenberg
Vera S F Chan1 na1,
Kelvin Y K Chan2,3 na1,
Yongxiong Chen1 na1,
Leo L M Poon4,
Annie N Y Cheung2,
Bojian Zheng4,
Kwok-Hung Chan4,
William Mak5,
Hextan Y S Ngan3,
Xiaoning Xu6,
Gavin Screaton7,
Paul K H Tam1,5,
Jonathan M Austyn8,
Li-Chong Chan2,
Shea-Ping Yip9,
Malik Peiris4,
Ui-Soon Khoo2 &
Chen-Lung S Lin1
Nature Genetics volume 38, pages 38–46 (2006)Cite this article
Severe acute respiratory syndrome (SARS) is caused by infection of a previously undescribed coronavirus (CoV). L-SIGN, encoded by CLEC4M (also known as CD209L), is a SARS-CoV binding receptor that has polymorphism in its extracellular neck region encoded by the tandem repeat domain in exon 4. Our genetic risk association study shows that individuals homozygous for CLEC4M tandem repeats are less susceptible to SARS infection. L-SIGN is expressed in both non-SARS and SARS-CoV–infected lung. Compared with cells heterozygous for L-SIGN, cells homozygous for L-SIGN show higher binding capacity for SARS-CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, homozygosity for L-SIGN plays a protective role during SARS infection.
SARS is an acute respiratory disease resulting from infection of a previously undescribed coronavirus (SARS-CoV) that spreads mainly through a respiratory route1,2,3. The spike (S) proteins of most coronaviruses are large type I membrane glycoproteins that associate with cellular receptors to mediate infection of target cells4,5. Angiotensin converting enzyme-2 (ACE2) is the only known functional receptor for SARS-CoV infection6. Sequence analysis has shown that the SARS-CoV spike proteins have multiple N-linked glycosylation sites and share great similarity in the sequence motifs with the HIV envelope proteins7, suggesting a similar binding pattern between these two viruses.
L-SIGN (for liver/lymph node-specific ICAM-3 grabbing non-integrin, encoded by CLEC4M), also known as DC-SIGNR, for DC-SIGN related, is a DC-SIGN (for dendritic cell-specific ICAM-3 grabbing non-integrin, encoded by CD209) homologue, with which it shares 77% amino acid identity8. Unlike DC-SIGN, which is expressed, for example, on dendritic cells, L-SIGN is expressed in the liver, lymph nodes and placenta9,10. The extracellular regions of both CLEC4M and CD209 contain tandem repeats of a highly conserved 23-amino acid sequence, followed by a C-terminal C-type carbohydrate recognition domain (CRD)11,12,13. In contrast to CD209, CLEC4M has considerable polymorphism in the tandem repeat domain of exon 4, which consists of three to nine repeats of a 69–base pair segment, with seven repeats being predominant (>50%) in the general population10. This tandem repeat segment encodes the extracellular neck region and has been suggested to be important for homo-oligomerization of L-SIGN on the cell surface, which brings the CRDs into proximity for high-affinity ligand binding10,11. It has been suggested that heterozygous expression of polymorphic variants of L-SIGN, in which neck lengths differ, may prevent the formation of hetero-oligomers and may thus lead to a reduced ligand-binding affinity8.
L-SIGN and DC-SIGN share the ability to bind high-mannose oligosaccharides through their CRDs, and L-SIGN serves as a receptor for many viruses, such as HIV, hepatitis C and Ebola, as well as for Mycobacterium tuberculosis14,15,16,17. Recently, it has been shown that pseudotyped lentiviral particles presenting SARS-CoV spike protein can bind to both DC-SIGN18 and L-SIGN19. Recent data have further demonstrated that L-SIGN can bind SARS-CoV20. It has also been shown that individual susceptibility to SARS infection may depend on genetic factors of the host: for example, HLA class I polymorphism21,22. As L-SIGN can bind SARS-CoV and its homo- or heterozygosity may determine the ligand-binding capacity, we hypothesized that CLEC4M homo- or heterozygosity might affect individual susceptibility to SARS infection. We therefore performed a genetic risk association study and a series of in vitro experiments to examine the biological role of L-SIGN in SARS infection.
CLEC4M genotypes in studied cohorts
We genotyped 285 confirmed SARS patients infected during the outbreak in 2003, as well as three groups of controls that included (i) 'random controls' consisting of 380 healthy blood donors randomly recruited before the outbreak; (ii) 'outpatient controls' consisting of 290 individuals randomly recruited from the general outpatient clinics at least 2 months after the SARS outbreak with no clinical history, signs or symptoms of inflammation or infection; and (iii) 'health care worker controls' consisting of 172 health care workers who had worked in SARS wards but remained disease-free and were confirmed to be seronegative for SARS. For comparison with corresponding controls, and because at least one-fifth of SARS patients in Hong Kong and elsewhere were health care workers23, as also reflected in our series, we further subclassified our SARS patients into two groups: (i) 67 who were health care workers (hereafter called 'health care workers with SARS') infected in hospitals during the course of duty and (ii) the remaining 218 who were recruited from the community ('community SARS'; Table 1). The CLEC4M 69-nucleotide tandem repeats in exon 4 were genotyped by PCR followed by gel electrophoresis, and results were further verified by DNA blotting analysis in selective cases of representative genotypes (data not shown).
Table 1 Summary of the CLEC4M genotypes in study groups
All groups except the health care worker controls were in Hardy-Weinberg equilibrium (HWE; Table 1). As a high frequency of homozygous 5/5 genotype was observed in the health care worker controls and may have thus contributed to the Hardy-Weinberg disequilibrium, DNA blot analysis was repeated and confirmed all samples with 5/5 genotype detected by PCR from all five groups (data not shown). There was no statistically significant difference in the genotype frequencies (P = 0.737, CLUMP method) or in the L-SIGN homo- or heterozygosity (P = 0.755, χ2 test) between the outpatient controls and random controls (Table 1).
CLEC4M homozygotes show lower risk of SARS infection
When all SARS patients (health care workers with SARS + community SARS) were compared with random controls, homozygous individuals had a significantly lower risk association for SARS infection with an odds ratio of 0.706 (95% confidence interval (c.i.) = 0.519–0.961, P = 0.027, Table 2), with heterozygotes as the reference group. To analyze whether the status of being a health care worker may have an impact on susceptibility, we performed a similar analysis separately in the non–health care worker population and health care worker populations. In the non–health care worker population, community SARS versus outpatient controls gave an odds ratio of 0.698 (95% c.i. = 0.490–0.993, P = 0.045; Table 2). In the health care worker population, health care workers with SARS versus health care worker controls demonstrated an odds ratio of 0.536 for homozygotes (95% c.i. = 0.303–0.950, P = 0.031; Table 2). In the stratified analysis between these two populations, the test of heterogeneity comparing the reduced odds ratios in the non–health care worker and the health care worker populations (odds ratio = 0.698 and 0.536, respectively) did not demonstrate a significant difference (P = 0.442, Table 2). This subsequently allowed us to perform a test for overall association based on the non–health care worker and health care worker populations between the SARS patients and control subjects. Results again demonstrated an overall odds ratio of 0.649 (95% c.i. = 0.481–0.876, P = 0.005, Table 2), further confirming that a reduced risk was associated with homozygotes, irrespective of whether an individual was a health care worker. Furthermore, as the outpatient controls and the random controls did not differ from each other in their individual genotype frequencies or homo- or heterozygote frequencies (P = 0.737 and 0.755, respectively, Table 1), these two controls were grouped together as 'combined controls'. Analysis of all SARS patients versus combined controls again gave an odds ratio of 0.691 for homozygotes (95% c.i. = 0.523–0.913, P = 0.009). On the other hand, analysis of the allele and genotype frequencies showed that the effect of differences in allele or genotype frequencies was either absent or inconsistent (Table 3), indicating that the homo- or heterozygosity per se, but not any specific allele or genotype that constitutes the homo- or heterozygosity, influences the susceptibility to SARS infection.
Table 2 Statistical analysis of CLEC4M homo- or heterozygosity
Table 3 Statistical analysis of CLEC4M allele and genotype frequencies
L-SIGN is expressed in SARS lung and small bowel
We performed in situ hybridization for L-SIGN on paraffin-embedded lung tissues with an L-SIGN–specific antisense RNA (cRNA) probe, which can differentiate expression of DC-SIGN and L-SIGN on transfected cell lines (data not shown). We performed fluorescence in situ hybridization (FISH) for ACE2 at the same time and detected L-SIGN in some of the cells at the alveolar surface (Fig. 1a) in all nonlesional samples from eight patients who received lung resection for lung cancer and who had no SARS infection. We also detected L-SIGN in cells that seemed to be scattered within the alveolar space (Fig. 1a). The sense control for L-SIGN in situ hybridization was negative (Fig. 1a). Furthermore, the FISH study showed that ACE2 was also expressed by cells at the alveolar surface and scattered within the alveolar space of nonlesional lung samples in four of eight lung cancer patients (Fig. 1a). When compared with L-SIGN expression, L-SIGNbright cells were either ACE2dim/− or ACE2bright (Fig. 1a), where 'bright' represents strong signal intensity, indicating a positive result, and 'dim' or 'dim/−' represent a weak signal or no signal, indicating a negative result. The sense control for ACE2 FISH gave a negative result (Fig. 1a). We did not detect an ACE2 signal in nonlesional lung tissues from the other four lung cancer patients.
Figure 1: L-SIGN is expressed in non-SARS lung and also in the lung and small bowel of patients fatally infected with SARS.
(a) In situ hybridization for L-SIGN in paraffin-embedded non-SARS lung tissues was performed with an L-SIGN specific antisense RNA (cRNA) probe (dark purple, upper left panel). Fluorescence in situ hybridization (FISH) for ACE2 was also performed on the same section (shown in green, upper right panel). L-SIGNbright cells either do (white arrows, upper left panel versus red arrows, upper right panel) or do not (sky blue arrows, upper left panel) co-express ACE2. Sense controls are shown in lower panels respectively. (b) Paraffin-embedded SARS lung samples were first stained with monoclonal antibody against the SARS-CoV nucleocapsid antigen and anti-mouse universal immunoalkalinephosphatase polymer, and the color was subsequently developed with fast red substrate system (shown in red, left panel). FISH for ACE2 (shown in green, middle panel) and in situ hybridization for L-SIGN (shown in dark purple, right panel) were also performed on the same section. SARS-CoV antigen was identified in L-SIGNbrightACE2bright cells (blue arrows, left panel versus red arrows, middle panel versus red arrows, right panel). No viral antigens were detected in L-SIGNbrightACE2dim/− cells (sky blue arrows, right panel). SARS-CoV antigen was identified in L-SIGNdim/−ACE2dim/− cells (left panel, white arrows). (c) Similar staining procedures were conducted on small bowel samples from SARS patients as described in b. SARS-CoV antigen could be identified in L-SIGNbrightACE2bright cells on luminal surface of small bowel of patients fatally infected with SARS (blue arrows, left panel versus red arrows, middle panel versus red arrows, right panel).
To examine the phenotype of cells infected by SARS-CoV in patients fatally infected with SARS, we first stained dewaxed paraffin sections with monoclonal antibody against the SARS-CoV nucleocapsid antigen (Fig. 1b). We performed FISH for ACE2 and in situ hybridization for L-SIGN (Fig. 1b) on the same sections. In lung samples, we identified SARS-CoV antigen in cells coexpressing ACE2 and L-SIGN (Fig. 1b). We did not detect viral antigens in L-SIGNbrightACE2dim/− cells (Fig. 1b). We also identified SARS-CoV antigen in L-SIGNdim/−ACE2dim/− cells (Fig. 1b). In small bowel samples, we detected SARS-CoV antigen in cells lining the luminal surface and coexpressing ACE2 and L-SIGN (Fig. 1c).
L-SIGN mediates proteasome-dependent viral degradation
CHO cells, which do not express ACE2 (data not shown), were transiently transfected with L-SIGN containing seven tandem repeats (N7 L-SIGN/CHO, where N denotes 'neck'), five repeats (N5), or both seven and five repeats (N7/N5 cotransfectants) in exon 4. These transfectants expressed high levels of L-SIGN on post-transfection day 2, and flow cytometry analysis confirmed that levels of surface expression were comparable for both the heterozygous and homozygous forms (data not shown). The transfectants (2.5 × 104 cells) were pulsed with 1 plaque forming unit (PFU) of SARS-CoV per cell for 1 h at 4 °C. After extensive washing, the cells were lysed and RNA was obtained for analysis of the copy number of ORF-1b sequence as described24. ORF-1b is a genomic RNA of SARS-CoV, and part of it encodes the viral polymerase25,26. Cultures of all three transfectants had a higher viral genomic copy number than the mock transfectants. Furthermore, homozygous N7 and N5 L-SIGN/CHO cells had a higher binding capacity than heterozygous N7/N5 transfectants (Fig. 2a). Binding of SARS-CoV to these transfectants was confirmed to be L-SIGN–dependent, as ORF-1b copy numbers were substantially reduced when blocking monoclonal antibodies against L-SIGN were included during the viral pulse and incubation (Fig. 2a).
Figure 2: L-SIGN is a binding receptor for SARS-CoV and mediates proteasome-dependent viral degradation.
(a) Viral ORF-1b copy number of N7, N5 and N7/5 L-SIGN transfected CHO cells (L-SIGN/CHO), and mock transfectants (CHO), pre-treated with anti-L-SIGN antibodies (clones 120526 and 120612, 10 μg/ml each) or isotype control antibody before being pulsed with 1 PFU/cell SARS-CoV and incubated for 1 h at 4 °C and washed. (b) Total viral ORF-1b copy number (cell lysates plus supernatants) of N7, N5 and N7/5 L-SIGN transfected CHO cells, and mock transfectants, pulsed with 1 PFU/cell SARS CoV for 1h, washed and subsequently incubated at 37 °C for 24 and 48h. (c) Total viral ORF-1b copy number of N7 L-SIGN transfected CHO cells, and mock transfectants, pulsed with 1 PFU/cell SARS CoV for 1 h at 37 °C, washed and subsequently incubated for 4 h in the presence or absence of 10 μM MG132 or proteasome inhibitor I (Pro. Inh. 1). *P < 0.05 in comparison to cells treated with DMSO solvent control. Data are expressed as mean ± s.d. from triplicate, and are representative of 3 experiments.
In separate experiments, transfectants were pulsed with virus for 1 h, washed, and incubated at 37 °C for 24 h and 48 h, after which the total ORF-1b copy numbers were determined for combined cell lysates and supernatants. After binding to L-SIGN, the total viral copy number decreased substantially by 24 h and 48 h in all three transfectants (Fig. 2b) with no obvious cytopathic effect (data not shown). Thus, there was no evidence of productive viral replication. Furthermore, the reduction of total viral copy number over at least 24 h was greater for N7 and N5 L-SIGN/CHO than for N7/N5 transfectants (Fig. 2b). In addition, the viral copy number in the supernatants at 24 h and 48 h was extremely low (Supplementary Fig. 1 online). We obtained similar results when the experiment was repeated using N7 L-SIGN–transfected A549 cells, a type II pneumocyte cell line27 that does not express ACE2 or L-SIGN (Supplementary Fig. 2 online). These results suggested that after binding to L-SIGN, there was little or no viral dissociation and that SARS-CoV was actually degraded over time.
To investigate the mechanism(s) for viral degradation, N7 L-SIGN/CHO cells were pulsed with SARS-CoV as above and incubated in the presence of 10 μM proteasome inhibitor MG132 or proteasome inhibitor I. After 4 h, the total viral ORF-1b copy number in wells treated with proteasome inhibitors was significantly higher than those treated with solvent control (Fig. 2c). Treatment with proteasome inhibitors for 4 h did not lead to excessive cell death, but a substantial cytotoxicity was observed after treatment for 24 h, thus precluding studies at a later time point (data not shown).
L-SIGN facilitates trans but not cis infection of SARS-CoV
To examine whether L-SIGN can mediate trans infection of permissive cells by SARS-CoV, each of the L-SIGN–transfected CHO cell lines was pulsed with 1 PFU/cell or 0.01 PFU/cell SARS-CoV for 1 h at 37 °C. After extensive washing, cells were subsequently cultured for 24 h or 48 h with fresh Vero E6 cells, which express ACE2 (data not shown) and have been used for in vitro propagation for SARS-CoV in our laboratory. Quantification of the viral genomic ORF-1b transcripts showed that higher final copy numbers were noted in cultures with N7/N5 cells than with homozygous N7 or N5 cells after pulsing with either 1 PFU/cell or 0.01 PFU/cell (Fig. 3a), although this difference was not observed at 48 h (Supplementary Fig. 3 online). Moreover, after initial viral pulse with 0.01 PFU/cell and incubation for 24 h, total viral copy number significantly increased in cultures of fresh Vero E6 cells with virus-pulsed L-SIGN/CHO cells, but not with virus-pulsed mock transfectants (CHO; Fig. 3a). These results imply that L-SIGN can deliver SARS-CoV to permissive cells in trans, and that 24 h after exposure to SARS-CoV, homozygous N7 and N5 L-SIGN/CHO cells have a lower ability for trans infection than heterozygous N7/N5 transfectants.
Figure 3: L-SIGN facilitates trans, but not cis, infection of SARS-CoV.
(a) N7, N5 and N7/5 L-SIGN/CHO and mock transfected CHO cells at 1 × 104/well in 96-well plates were pulsed with 1 PFU/cell (left panel) or 0.01 PFU/cell (right panel) SARS-CoV for 1 h at 37 °C. After wash, cells were cultured with 1 × 104 Vero E6 cells for 24 h and the total copy number of ORF-1b transcripts was determined. *P < 0.05 in comparison with homozygous N7 or N5 L-SIGN/CHO (left panel). *P < 0.05 and **P < 0.01 in comparison with homozygous N7 or N5 L-SIGN/CHO (right panel). (b) N7 and N7/N5 L-SIGN transfected Vero E6 cells and mock transfected Vero E6 cells were exposed to 0.01 PFU/cell SARS-CoV for 1 h at 37 °C. After wash, cells were incubated for 24 h and 48 h and the total copy number of viral ORF-1b (left panel) and nucleocapsid (right panel) transcripts were determined.
Because ACE2+L-SIGN+ cells that contained SARS-CoV antigen were identified in our SARS lung and small bowel samples (Fig. 1b,c), it is possible that L-SIGN may also be able to facilitate cis infection of SARS-CoV. To examine this possibility, Vero E6 cells transfected with N7 or N7/N5 L-SIGN were infected with 0.01 PFU/cell SARS CoV. At least 60% of the cells expressed surface L-SIGN on day 2 post-transfection as determined by flow cytometry (data not shown). After 1 h at 37 °C, we washed the cells and further incubated them for 24 h and 48 h and subsequently measured total ORF-1b and nucleocapsid copy number. Expression of N7 or N7/N5 L-SIGN on transfected Vero E6 cells ('L-SIGN/Vero') did not result in higher viral ORF-1b (Fig. 3b, left) or nucleocapsid (Fig. 3b, right) copy numbers. Infection of Vero E6 cells with 1 PFU/cell induced substantial cell death after 24 h and 48 h culture owing to the cytopathic effect of the virus (data not shown), precluding studies using this higher titer. Because the majority of nucleocapsid gene sequence was derived from subgenomic mRNA produced upon active replication28,29, the results indicate that the virus replicates with the same efficiency in these cells. We obtained similar results in additional experiments using CHO and A549 cell lines that had been transfected with ACE2 alone, or with both ACE2 and N7 L-SIGN (Supplementary Fig. 4 online).
L-SIGN on Vero E6 cells captures/adsorbs released viruses
We next examined the distribution of viral copy number in cell lysates compared with culture supernatants of SARS-CoV–infected L-SIGN–transfected Vero E6 cells. After cells were infected with 0.01 PFU/cell SARS-CoV and incubated for 48 h, the viral ORF-1b copy number was significantly higher in the cell lysates of N7 L-SIGN/Vero cultures than in N7/N5 L-SIGN–transfected and mock-transfected Vero E6 cultures (Fig. 4a). Furthermore, viral copy number was significantly lower in the supernatants of N7 L-SIGN/Vero cultures than in N7/N5 L-SIGN/Vero cultures and mock-transfected Vero E6 cultures (Fig. 4a). Infection of fresh Vero E6 cells for 48 h with serially titrated supernatants harvested from the 48 h culture of infected N7 L-SIGN/Vero E6 cells indeed resulted in a substantially lower final viral genomic copy number than did infection with supernatants from infected N7/N5 L-SIGN/Vero cells and mock transfectants (Supplementary Fig. 5 online). To exclude the possibility that expression of N7 L-SIGN in permissive cells might interfere with virus budding or release, N7 L-SIGN/Vero cultures were treated with antibodies against L-SIGN before and during the viral pulse and subsequent 48 h incubation. Notably, blockade of surface L-SIGN resulted in an increase of the number of viral genomic copies in the supernatants, with a reciprocal decrease in cell lysates (Fig. 4b). In separate experiments, we also treated N7 L-SIGN/Vero cells with 5 mM EGTA for an additional 1 h at the end of 48 h culture before harvest. Treatment with EGTA, a calcium-chelating reagent that can inhibit and reverse calcium-dependent binding such as that to L-SIGN, resulted in a decrease of viral copies in the cellular lysates of N7 L-SIGN/Vero cells with a reciprocal increase in the supernatants (Fig. 4c).
Figure 4: L-SIGN expressed on permissive Vero E6 cells captured/adsorbed infectious viruses released into the supernatant.
(a) N7, N7/N5 L-SIGN transfected Vero E6 cells and mock transfectant Vero E6 cells were infected with 0.01 PFU/cell SARS-CoV for 1 h at 37 °C. After wash, cells were incubated for 24 h and 48 h before cells were harvested separately from supernatant for the quantification of the viral ORF-1b copy number in cell lysates and supernatant. *P < 0.05; **P < 0.01 in comparison to heterozygous N7/5 L-SIGN/Vero E6 and mock transfected Vero E6. (b) N7 L-SIGN transfected Vero E6 cells were pre-treated with anti-L-SIGN antibodies or isotype control antibody, followed by infection with 0.01 PFU/cell SARS-CoV for 1 h at 37 °C. After wash, cells were further incubated for 48 h in the presence of anti-L-SIGN antibodies or isotype control antibody before determination of the viral ORF-1b transcripts in cell lysates and supernatant. (c) N7 L-SIGN transfected Vero E6 cells and mock transfectants were infected with 0.01 PFU/cell SARS-CoV for 1 h at 37 °C. After wash, cells were incubated for 48 h. The N7 L-SIGN/Vero E6 cells were further treated with PBS or 5 mM EGTA for 1 h before harvest for quantification of ORF-1b copy number in cell lysates and supernatant. *P < 0.05 in comparison to N7 L-SIGN/Vero treated with EGTA; **P < 0.01 in comparison to N7 L-SIGN/Vero treated with PBS. Data are expressed as mean ± s.d. of triplicates, and are representative of three experiments.
Homozygous L-SIGN reduces viral titers in a closed system
To determine the collective role of L-SIGN, stable transfectants of N5 and N7/N5 L-SIGN-transfected CHO cells were selected by limiting dilution assay and were confirmed to express high levels of L-SIGN (Supplementary Fig. 6 online). Vero E6 cells (1 × 104 cells) were cultured with 1 × 104 or 1 × 103 N5 or N7/N5 L-SIGN/CHO cells, respectively, and were subsequently pulsed with 0.01 PFU SARS-CoV per Vero E6 cell. Vero E6 cells were also cultured with the same number of parental CHO cells as controls. We did not conduct any washes in this experiment in order to maintain a constant viral titer for infection (that is, a closed system). There was no difference in the total number of viral ORF-1b copies (in both cellular lysates and supernatant) at 24 h of culture (data not shown), but there was a significant reduction in the viral genomic copy number at 48 h in 1:1 Vero:N5 L-SIGN/CHO cultures than in 1:1 Vero:N7/N5 CHO cultures (Fig. 5a). The total number of nucleocapsid transcripts was also significantly lower in cultures of homozygous N5 L-SIGN transfectants than in cultures of heterozygous N7/N5 (Fig. 5b). In addition, a stable clone of N2 L-SIGN/CHO was also obtained after prolonged culture of a stable N7 clone that subsequently lost five tandem repeats. When these N2 L-SIGN/CHO cells were cultured with Vero E6 cells and subsequently exposed to 0.01 PFU/cell SARS-CoV in a closed infection system, total ORF-1b copy number was lower than in cultures with parental CHO cells (Supplementary Fig. 6).
Figure 5: Homozygous, but not heterozygous, L-SIGN reduced the final total viral genomic and subgenomic copy number in a closed infection system.
(a) 1 × 104 Vero E6 cells were cultured with 1 × 104 (ratio 1:1) or 1 × 103 (ratio 1:0.1) stable transfectants of N5 or N7/5 L-SIGN transfected CHO cells, and subsequently infected with 0.01 PFU per Vero E6 cell. Without washing, cells were further incubated for 48 h before the determination of the total viral genomic ORF-1b copy number. (b) Cells were treated as in a for the determination of the total nucleocapsid copy number. Data are expressed as mean ± s.d. from triplicate and are representative of three experiments. *P < 0.05, **P < 0.01 in comparison to heterozygous N7/5 L-SIGN/CHO.
Among the five cohorts genotyped, we demonstrate Hardy-Weinberg equilibrium (HWE) in all except the health care worker controls (Table 1). As 22% of all SARS cases in Hong Kong were health care workers23, it is likely that the unaffected health care workers recruited for study may represent a preselected group with overrepresentation of homozygotic genotypes. Nevertheless, direct comparison of SARS-infected individuals and the corresponding controls, together with the stratified analysis, shows that CLEC4M homozygotes are significantly less susceptible to SARS infection, irrespective whether an individual is a health care worker (Table 2). Such a reduced risk is associated with L-SIGN homozygosity per se, rather than any specific allele or genotype (Table 3). In a separate study, we compared the genotypes of Chinese random controls with those of 58 unrelated individuals of European descent from the CEPH Reference Family. The genotypes of both Chinese and European cohorts were in HWE (Supplementary Table 1 online), and the allele frequencies of our European cohort were similar to those previously reported in other European populations10,30. Allele frequencies of CLEC4M tandem repeats in the Chinese population were significantly different from those in Europeans (P < 0.0001, Supplementary Table 1). There was also a significant difference in the genotype and homo- or heterozygote frequency in these two populations (P < 0.0001 and P = 0.002, respectively, Supplementary Table 1). It remains to be investigated whether these differences between Chinese and Europeans may result in different susceptibility for SARS infection.
It has been reported that the primary organ for SARS-CoV infection is lung31,32, and virus has also been detected in small bowel of patients fatally infected with SARS-CoV33. Using in situ hybridization and immunohistology, we have shown the presence of L-SIGN+ cells in nonlesional lung samples from lung cancer patients who had no SARS infection. These L-SIGN+ cells are either ACE2bright or ACE2dim/− (Fig. 1a), implying that L-SIGN may be expressed by at least two different subsets of cells. Indeed, in a separate study we found that L-SIGNbrightACE2dim/− cells expressed cytokeratin, indicating that they are epithelial cells, as also suggested in a recent report20. In contrast, L-SIGNbrightACE2bright cells do not express cytokeratin (data not shown). The nature of these cells is unknown and needs to be further characterized. Although L-SIGNbrightACE2dim/− cells are consistently found in lung tissue of all non-SARS samples, L-SIGNbrightACE2bright cells are found only in samples from half of the non-SARS individuals (four of eight); this needs further investigation. Nevertheless, the finding that L-SIGN is consistently expressed in cells that expressed both SARS-CoV antigen and ACE2 in the lung and small bowel of patients fatally infected with SARS (Fig. 1b,c) strongly suggests that L-SIGN may be involved in SARS-CoV infection. Notably, SARS antigen is found in L-SIGNdim/−ACE2dim/− cells in SARS-infected lung (Fig. 1b). The nature of these virus-containing cells that do not express the only known functional receptor, ACE2, is unclear, but they are not alveolar macrophages, because they do not express CD68 (Supplementary Fig. 7 online), a macrophage-specific marker34.
Our observation that homozygous L-SIGN cells have a higher SARS-CoV binding capacity than heterozygous cells (Fig. 2a) agrees with the suggestion that heterozygous expression of polymorphic variants of L-SIGN may result in reduced ligand-binding affinity8,10,11. After binding to L-SIGN, there is no or little viral dissociation, and viruses undergo degradation over time, with the homozygous cells more efficient than the heterozygous cells (Fig. 2b). It has been reported that levels of SARS-CoV subgenomic RNA were increased 24 h after infection of L-SIGN–expressing cells20. However, such an increase was transient, and SARS-CoV antigen was detected by immunofluorescence staining in fewer than 1% of the infected L-SIGN–expressing cells20, which argues against efficient and productive viral replication mediated by L-SIGN. Furthermore, L-SIGN–mediated viral degradation is, at least in part, proteasome-dependent, because it can be inhibited partially by proteasome inhibitors (Fig. 2c), although interference with proteasomes may also lead to indirect inhibition of upstream processes involved in the degradation of endocytosed SARS-CoV particles.
L-SIGN can also bind HIV and SIV and mediates infection of permissive cells in trans14. Expression of L-SIGN on permissive cells facilitates trans, but not cis, infection of SARS-CoV, at least when cells are initially infected with 0.01 PFU/cell (Fig. 3), a titer that may be more representative of the setting of primary infection than a higher one. The observation that N7 L-SIGN/CHO cells bind SARS-CoV more efficiently but mediate a poorer trans infection than N7/N5–expressing cells is interesting. It is likely that a higher binding capacity may lead to more efficient internalization and degradation, thereby resulting in a lower ability for trans delivery of the virus. In separate experiments in which the viral copy number was determined separately in cell lysates and supernatants, the cell lysates of SARS-infected homozygous N7 L-SIGN/Vero cells contained significantly higher viral copy number than those of heterozygous N7/N5 L-SIGN/Vero cells (Fig. 4a). This finding is consistent with a recent report that after infection with SARS-CoV spike protein–containing pseudotyped virus and incubation for 3 d, the luciferase activity in the cell lysates, which should correlate with the viral copy number, is higher in nonpermissive cells transfected with both ACE2 and N7 L-SIGN than in cells transfected with ACE2 alone19. Reciprocally, supernatants of SARS-infected homozygous N7 L-SIGN/Vero cells have a significantly lower viral copy number than supernatants of heterozygous N7/N5 L-SIGN/Vero cells (Fig. 4b). This implies that cells expressing N7 L-SIGN, but not heterozygous N7/N5, seem to retain a high proportion of viruses in a cell-associated form, leading to decreased levels of infectious virions in the supernatants. Taken together, we suggest that L-SIGN expressed on permissive cells (such as in L-SIGNbrightACE2bright cells; Fig. 1) may actually capture or sequester SARS viruses that have been liberated after replication, at least after 48 h incubation, when the viral copy numbers significantly increase. Capture, absorption and degradation of the released viruses via binding to L-SIGN would result in a lower amount of infectious virions in the local environment, which could limit subsequent viral spread to other permissive cells for further infection. In vivo, however, the outcome of SARS-CoV infection is likely to be a combination of L-SIGN–mediated capturing, internalization and degradation and/or trans delivery of virus to permissive cells. In a closed infection system in which no wash procedures were conducted in order to maintain a constant titer for infection, we demonstrated that cultures of permissive cells with homozygous, but not heterozygous, L-SIGN–expressing cells have significantly lower final viral genomic and subgenomic copy numbers (Fig. 5). This observation confirms that homozygous L-SIGN does have a protective role in SARS-CoV infection.
In summary, we have shown from a genetic risk-association study that CLEC4M homozygotes have a significantly lower risk for SARS-CoV infection. This observation is supported by our in vitro studies showing that homozygous L-SIGN plays a protective role by reducing the final total viral titers in cultures with permissive cells. In part this can be attributed to an increased binding capacity of homozygous L-SIGN which results in greater cell association of virions, increased proteasome-dependent viral degradation and a consequent lower capacity for trans infection. Our results show a novel role of L-SIGN in SARS infection. Whether our findings are also relevant to the pathogenesis and disease susceptibility of other L-SIGN-binding pathogens deserves further investigation.
Recruitment of subjects for case-control genetic association study.
Signed informed consent, with prior approval from the relevant Institutional Review Boards, was obtained from subjects donating blood. SARS patients were recruited from four major hospitals that treated SARS patients in Hong Kong during the 2003 outbreak: namely, Queen Mary Hospital, Pamela Youde Nethersole Hospital, Princess Margaret Hospital and United Christian Hospital. All 285 SARS patients selected for study were unrelated and confirmed by serology and/or quantitative RT-PCR assay. Of these, 67 were health care workers, and the remaining 218 were from the community. Controls included (i) random controls, consisting of 380 healthy blood donors randomly recruited before the outbreak by the Hong Kong Red Cross Blood Transfusion Service; (ii) 290 patients unaffected by SARS, attending general outpatient clinics and recruited at least two months after the outbreak; and (iii) 172 health care workers who had worked in SARS wards but remained disease-free and were confirmed to be seronegative for SARS. Serology for SARS was performed as previously described35. Genomic DNA was extracted from whole blood.
CLEC4M 69-nucleotide tandem repeats in exon 4.
As the sequence of the CD209 and CLEC4M genes are highly similar, special care was taken to design primers that specifically amplified exon 4 of CLEC4M: L-SIGN-Ex4-F and L-SIGN-Ex4-R (Supplementary Table 2 online). PCR was performed using High Fidelity Platinum Taq DNA Polymerase (Invitrogen), which facilitated amplification of long and short alleles with similar efficiency. The 20 μL PCR consisted of 1× PCR buffer, 1.5 mM MgCl2, 200 μM dNTP, 150 nM of each primer, 1 unit (U) High Fidelity Platinum Taq DNA Polymerase and 50 ng of genomic DNA. The reaction was carried out at 95 °C for 5 min, 40 cycles of 95 °C for 20 s, 59 °C for 30 s and 72 °C for 60 s, and final extension at 72 °C for 5 min. The amplified products were separated by 2% agarose gel electrophoresis of wide lane width (wells 6 mm wide, each loaded with 20 μL PCR products) to provide good visualization of alleles. The genotype of each case was checked (blind) by three separate individuals. Some representative cases were confirmed by DNA blot analysis as well as by direct sequencing. Genotyping was also performed in the 58 unrelated European individuals from CEPH Reference Families.
Genetic association for heterozygote and homozygote genotype comparison was assessed by two-tailed χ2 test using SPSS software (Version 11.0), and odds ratio and 95% c.i. were used to measure strength of association in genetic risk association study. Mantel-Haenzel test for stratified analysis was also carried out using SPSS. Comparison of allele frequencies or genotype frequencies was done using the CLUMP program. The Hardy-Weinberg Exact Test was calculated by the Markov chain method, taking P < 0.05 as the cutoff for assessing significance. (Markov chain parameters: dememorization number = 1,000, number of batches = 100, number of iterations per batch = 1,000). For all in vitro studies, statistical significance was calculated using Student's two-tailed t-test.
Preparation of anti-SARS-CoV nucleocapsid antibody.
BALB/c mice were immunized intraperitoneally with 0.1 ml of heat-killed SARS coronavirus HK39849 (107 TCID50 ml−1). Injections of similar doses were repeated weekly for 2 months. Four days after the final booster, 108 spleen cells from an immunized mouse were fused with 107 of NSI myeloma cells, with polyethylene glycol (PEG, M.W. 4,000, BDH, Poole) as the fusing agent. Hybridomas were screened for production of antibodies against SARS coronavirus (HK39849) by ELISA. Those that produced specific antibodies were cloned twice by limiting dilution. Purified hybridomas were then injected intraperitoneally into mineral oil–primed mice for the production of ascitic fluid. Monoclonal antibodies were enriched from the ascitic fluid by precipitation with 50% of ammonium sulfate. The antibodies reacted specifically with the nucleocapsid protein by protein blotting (data not shown).
Immunohistochemistry for SARS nucleocapsid antigen, in situ hybridization for L-SIGN and FISH for ACE2.
Immunohistochemistry was performed on 5 μm paraffin sections of formalin-fixed samples of lung tissues from eight adults having lung resection (lobectomy) for lung cancers. Parts of the nonlesional lung tissues were excised with approval by the Institutional Review Board of the University of Hong Kong. Sections of the lung and small bowel tissues taken after autopsy from five patients fatally infected with SARS were also retrieved for study. Samples were first deparaffinized and rehydrated. After microwave heating, mouse antibody against SARS-CoV nucleocapsid (1:2,000) was applied as a primary antibody at 4 °C overnight, and sections were then incubated with anti-mouse universal immunoalkalinephosphatase polymer (Nichirei Corporation, ready-to-use) for 2 h at room temperature. The color was subsequently developed with fast red substrate system (Sigma). FISH and in situ hybridization were performed as described previously36 on the same sections by using a mix of a FITC-labeled ACE2 cRNA probe, which corresponds to ACE2 full length cDNA and a digoxigenin-labeled L-SIGN cRNA probe which corresponds to nucleotides 109–168. Sections were then mounted with medium for fluorescence with DAPI (Vector Laboratories). Electronic images of the immunohistochemical staining and the FISH fluorescence visualized under a fluorescence microscope (Nikon eclipse E600) were saved to PC. Sections were then further incubated with sheep anti–digoxigenin alkaline phosphatase Fab fragment (1:200, Roche) for 2 h at room temperature. Finally, the color for L-SIGN in situ hybridization was developed with NBT/BCIP substrate system (Roche), and the sections were mounted with aqueous mounting medium (DakoCytomation). The same fields as in the immunohistochemistry and FISH fluorescence images were subsequently selected and visualized under the same microscope and also saved as electronic images for comparison and analysis of colocalization. Antisense and sense FITC-labeled ACE2 cRNA probe or digoxigenin-labeled L-SIGN–specific cRNA probe were generated by a nonradioactive RNA labeling kit with fluorescein-12-UTP or digoxigenin-11-UTP, respectively (Roche), according to the manufacturer's instructions.
N5 L-SIGN expression vector construction.
The entire exon 4 (fragment B) harboring five tandem repeats was amplified from genomic DNA from an N5 allele carrier using exon4-F and exon4-R (Supplementary Table 2). The 5′ end (exon 1 to partial exon 4 (fragment A)) and 3′ end (partial exon 4 to the stop codon in exon 7 (fragment C)) of the CLEC4M gene transcript were amplified from the pcDNA3-L-SIGN (obtained from the AIDS Research and Reference Reagent Program, US National Institutes of Health (NIH)) using two primer sets: exon1/BamHI-F and exon4/partial-R; and exon4/partial-F and exon7/stop-R (Supplementary Table 2). Fragments A and B, which overlapped for 62 bp of the sequence (5′ end of exon 4), were mixed and extended in the first ten thermal cycles reaction using the Expand High Fidelity Plus PCR System (Roche Diagnostics) without the primers. After initial extension, exon1/BamHI-F and exon4-R were added to the reaction to generate a complete fragment length of exon 1 to exon 4 in an additional 25 thermal cycles. The amplified product (fragment A/B) was then gel purified. Fragments A/B and C, containing 38 bp overlapping sequences (3′ end of exon 4), were mixed and a similar procedure was performed, using exon1/BamHI-F and exon7/stop-R to generate the full length of N5 L-SIGN which was subsequently cloned into the pGEM-T Easy vector (Promega). The plasmid was extracted and subjected to DNA sequencing to ensure that no error was introduced in the N5 L-SIGN sequence. The N5 L-SIGN in pGEM-T was then subcloned to BamHI- and NotI-digested pcDNA3.1/Hygro(+) vector (Invitrogen). The sequence and reading frame of N5 L-SIGN in the vector was subsequently confirmed by DNA sequencing.
Plasmids and cell lines.
pcDNA3-L-SIGN plasmid expressing L-SIGN with seven tandem repeats (N7) in exon 4 was obtained from the AIDS Research Program, NIH. The N5 L-SIGN was cloned as described above. CHO and Vero E6 cells were maintained in DMEM supplemented with 10% FBS, 10 units ml−1 penicillin and 10 μg ml−1 streptomycin, unless otherwise stated. N7 L-SIGN plasmid, N5 L-SIGN plasmid or a mixture of N7+N5 plasmids (ratio 1:1) were transfected into CHO cells or Vero E6 cells using Lipofectamine 2000 (Invitrogen) according to manufacturer's instruction. L-SIGN expression was verified by immunostaining with L-SIGN specific antibody (clone 120604, AIDS Research Program, NIH) 48 h after transfection, and the cells were used immediately for experiments. Stable clones of CHO cells expressing N5 or N7/5 L-SIGN variants were obtained by limiting dilution of L-SIGN–transfected CHO cells and selected with 1 mg ml−1 G418 and/or 0.5 mg ml−1 hygromycin B for 2 to 3 weeks. Homozygosity and heterozygosity of L-SIGN in the stable clones were verified by RT-PCR by amplifying the neck domain of the L-SIGN. The stable clones were maintained in complete media containing selection antibiotics.
RT-PCR for quantification of ORF-1b and nucleocapsid copy number.
Total RNA from cultured cells was extracted using RNeasy RNA Mini Kit (Qiagen) and viral RNA from culture supernatant was extracted separately using QIAmp Viral RNA Kit (Qiagen). RNA was resuspended in 30 μl RNAse-free water. The total viral RNA copy number was determined by quantitative RT-PCR amplifying the SARS-CoV ORF-1b sequence as previously described24. PCR for the SARS-CoV nucleocapsid (N) sequence was done using 250 nmol l−1 forward and reverse primer (Supplementary Table 2) with the following conditions: 5 min at 95 °C, followed by 50 cycles of 95 °C for 15 s, 57 °C for 15 s and 72 °C for 15 s. Reactions were performed using ABI PRISM 7700 Sequence Detector (Applied Biosystems). Fluorescence signals were analyzed using SDS 1.9.1 software (Applied Biosystems) and the total copy number was calculated against a standard with a known amount of plasmid containing the corresponding gene.
Binding assay for SARS-CoV on L-SIGN transfectants and treatment with proteasome inhibitors.
N7, N5 and N7/5 L-SIGN transfectants and mock transfected CHO cells were pulsed with 1 PFU/cell SARS-CoV in serum-free DMEM at post-transfection day 2. After 1 h incubation at 4 °C, cells were washed to remove unbound viruses and were immediately lysed to obtain RNA for analysis at time point 1 h. Some of the cells were further incubated in triplicate at 2.5 × 104 per well in a 96-well plate in complete media for 24 h and 48 h at 37 °C for determination of viral ORF-1b or nucleocapsid sequence as described in individual experiments. In experiments using proteasome inhibitors, N7 L-SIGN/CHO or mock-transfected CHO cells were pulsed with 1 PFU/cell SARS-CoV in serum-free DMEM for 1 h at 37 °C on post-transfection day 2. After wash, 2.5 × 104 cells were harvested at time point 1 h. The remainder of the cells were seeded at 2.5 × 104 cells per well in a 96-well plate in triplicate and were cultured in complete DMEM media in the presence of 10 μM MG-132 as described37 or proteasome inhibitor I (both from Calbiochem) for 4 h according to the manufacturer's instruction. DMSO (0.1%) was used as a vehicle control.
Trans infection of SARS-CoV by L-SIGN–transfected CHO cells.
N7, N5 and N7/5 L-SIGN transfected CHO and mock-transfected CHO cells were plated at 1 × 104 per well in a 96-well plate on post-transfection day 2 and were pulsed with 0.01 or 1 PFU/cell CoV at 37 °C in serum-free DMEM for 1 h. After wash, 1 × 104 Vero E6 cells were added to each well and further cultured for 24 h and 48 h. Total RNA was extracted from the wells and ORF-1b copy numbers were determined.
Cis infection of SARS-CoV in N7 and N7/N5 L-SIGN transfected Vero E6 cells.
Vero E6 cells were transfected with N7 or N7/N5 L-SIGN similarly to transfection of CHO cells, as described above. On post-transfection day 2, cells were exposed to 0.01 PFU/cell for 1 h at 37 °C. After washing, cells were further incubated at 1 × 104 cells per well in a 96-well plate in triplicate. Determination of the ORF-1b or nucleocapsid sequence after 24 h and 48 h was performed from cellular lysates or supernatants, or from both, as indicated in individual experiments. In some experiments, EGTA (Sigma) or PBS (as a control to EGTA) was added to each well at a final concentration of 5 mM at the end of 48 h culture and the cells were further incubated at 37 °C for 1 h. Supernatants and cellular RNA were then harvested separately and viral RNA copy numbers were determined.
Blocking experiments using antibodies against L-SIGN.
2.5 × 104 N7, N5 and N7/N5 L-SIGN transfected CHO cells were plated in 96-well plate and incubated with serum-free DMEM containing either a cocktail of antibodies to L-SIGN (a mixture of clones 120526 and 120612, 10 μg/ml each) or mouse IgG2a isotype control antibodies (20 μg/ml) at room temperature for 40 min. Subsequently, 1 PFU/cell SARS-CoV was added to the cultures, and cells were further incubated at 4 °C for 1 h in the continuous presence of the antibodies. Cells were then extensively washed and collected for quantification of ORF-1b transcripts. In separate experiments, 1 × 104 N7 L-SIGN–transfected Vero E6 cells were plated in 96-well plates and incubated with serum-free DMEM containing either a cocktail of antibodies against L-SIGN cocktail or mouse IgG2a isotype control antibodies (20 μg/ml) as described above. Then, 0.01 PFU/cell SARS-CoV was added to the cultures and cells were further incubated at 37 °C for 1 h in the continuous presence of the antibodies. Cells were then extensively washed and incubated in complete DMEM media for 24 h and 48 h, after replenishment of anti-L-SIGN antibodies and isotype control antibody. Determination of the ORF-1b sequence after 24 h and 48 h was performed from cellular lysates or supernatant, as indicated in individual experiments.
Infection of the cultures of Vero E6 and stable L-SIGN/CHO transfectants without wash in a closed system.
Vero E6 cells (1 × 105) were first homogenously mixed with 1 × 105 or 1 × 104 stable transfectants of N5, N7/N5 L-SIGN/CHO and parental CHO cells in 200 μl serum-free DMEM and subsequently infected with SARS-CoV at 0.01 PFU per Vero E6 cell in suspension at 37 °C for 1 h. Without washing off the virus, 20 μl of this infected cell suspension, which contained a suspension of 1 × 104 Vero E6 cells and 1 × 104 L-SIGN/CHO (a ratio of 1:1) or a suspension of 1 × 104 Vero E6 cells with 1 × 103 L-SIGN/CHO (a ratio of 1:0.1) was placed in a 96-well plate in triplicate. After addition of 180 μl complete media in each well to make a final volume of 200 μl, cells were subsequently incubated. After 24 h and 48 h, cells in the wells were lysed and collected, together with supernatant, for the measurement of the total ORF-1b and nucleocapsid copy numbers.
Accession codes.
GenBank: ACE2 full-length cDNA, AB046569; L-SIGN cRNA probe, nucleotides 109–168: AF290887.
URLs.
CLUMP program, http://www.mds.qmw.ac.uk/statgen/dcurtis/software.html; Hardy-Weinberg Exact Test, http://wbiomed.curtin.edu.au/genepop/.
Note: Supplementary information is available on the Nature Genetics website.
Accession codes
GenBank/EMBL/DDBJ
Peiris, J.S. et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319–1325 (2003).
Ksiazek, T.G. et al. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1953–1966 (2003).
Drosten, C. et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967–1976 (2003).
Bonavia, A., Zelus, B.D., Wentworth, D.E., Talbot, P.J. & Holmes, K.V. Identification of a receptor-binding domain of the spike glycoprotein of human coronavirus HCoV-229E. J. Virol. 77, 2530–2538 (2003).
Breslin, J.J. et al. Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C. J. Virol. 77, 4435–4438 (2003).
Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454 (2003).
Kliger, Y. & Levanon, E.Y. Cloaked similarity between HIV-1 and SARS-CoV suggests an anti-SARS strategy. BMC Microbiol. 3, 20 (2003).
Soilleux, E.J., Barten, R. & Trowsdale, J. DC-SIGN; a related gene, DC-SIGNR; and CD23 form a cluster on 19p13. J. Immunol. 165, 2937–2942 (2000).
Soilleux, E.J. DC-SIGN (dendritic cell-specific ICAM-grabbing non-integrin) and DC-SIGN-related (DC-SIGNR): friend or foe? Clin. Sci. (Lond.) 104, 437–446 (2003).
Bashirova, A.A. et al. A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J. Exp. Med. 193, 671–678 (2001).
Mitchell, D.A., Fadden, A.J. & Drickamer, K. A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J. Biol. Chem. 276, 28939–28945 (2001).
Feinberg, H., Mitchell, D.A., Drickamer, K. & Weis, W.I. Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294, 2163–2166 (2001).
Guo, Y. et al. Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR. Nat. Struct. Mol. Biol. 11, 591–598 (2004).
Pohlmann, S. et al. DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc. Natl. Acad. Sci. USA 98, 2670–2675 (2001).
Pohlmann, S. et al. Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J. Virol. 77, 4070–4080 (2003).
Lin, G. et al. Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR. J. Virol. 77, 1337–1346 (2003).
Koppel, E.A. et al. Identification of the mycobacterial carbohydrate structure that binds the C-type lectins DC-SIGN, L-SIGN and SIGNR1. Immunobiology 209, 117–127 (2004).
Yang, Z.Y. et al. pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J. Virol. 78, 5642–5650 (2004).
Marzi, A. et al. DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J. Virol. 78, 12090–12095 (2004).
Jeffers, S.A. et al. CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. USA 101, 15748–15753 (2004).
Lin, M. et al. Association of HLA class I with severe acute respiratory syndrome coronavirus infection. BMC Med. Genet. 4, 9 (2003).
Ng, M.H. et al. Association of human-leukocyte-antigen class I (B*0703) and class II (DRB1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome. J. Infect. Dis. 190, 515–518 (2004).
Chan-Yeung, M. Severe acute respiratory syndrome (SARS) and healthcare workers. Int. J. Occup. Environ. Health 10, 421–427 (2004).
Poon, L.L. et al. Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin. Chem. 49, 953–955 (2003).
Rota, P.A. et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394–1399 (2003).
Marra, M.A. et al. The Genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404 (2003).
Nardone, L.L. & Andrews, S.B. Cell line A549 as a model of the type II pneumocyte. Phospholipid biosynthesis from native and organometallic precursors. Biochim. Biophys. Acta 573, 276–295 (1979).
Snijder, E.J. et al. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 331, 991–1004 (2003).
Drosten, C. et al. Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus. J. Clin. Microbiol. 42, 2043–2047 (2004).
Lichterfeld, M. et al. The tandem-repeat polymorphism of the DC-SIGNR gene does not affect the susceptibility to HIV infection and the progression to AIDS. Clin. Immunol. 107, 55–59 (2003).
Lee, N. et al. A major outbreak of severe acute respiratory syndrome in Hong Kong. N. Engl. J. Med. 348, 1986–1994 (2003).
Tsang, K.W. et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong. N. Engl. J. Med. 348, 1977–1985 (2003).
To, K.F. et al. Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases. J. Pathol. 202, 157–163 (2004).
Soilleux, E.J. et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J. Leukoc. Biol. 71, 445–457 (2002).
Guan, M. et al. Evaluation and validation of an enzyme-linked immunosorbent assay and an immunochromatographic test for serological diagnosis of severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11, 699–703 (2004).
Xiong, H. et al. A novel gene encoding a TIG multiple domain protein is a positional candidate for autosomal recessive polycystic kidney disease. Genomics 80, 96–104 (2002).
Erler, J.T. et al. Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. Mol. Cell. Biol. 24, 2875–2889 (2004).
We are indebted to L.-C. Tsui and P. Sham for their advice and support. We thank K.F. To and P. Beh for providing autopsy materials of patients fatally infected with SARS. We thank L.Y.C. Yam, S.T. Lai, C.M. Chu, T.Y. Wong, E. Chow, E.S.K. Ma, E.Y.T. Chan, R.Y.L. Chen and M.M. Garcia-Barcelo for sample collection and C. Wong, J. Chan, W. Liu and W.C. Xue for their technical support. This project was funded by the SARS Research Fund from the University of Hong Kong, the European Research Project SARS-DTV, the US National Institute of Allergy and Infectious Diseases, the Research Fund for the Control of Infectious Diseases of Hong Kong and the Research Grant Council, HKSAR, China.
Vera S F Chan, Kelvin Y K Chan and Yongxiong Chen: These authors contributed equally to this work.
Department of Surgery, Hong Kong Jockey Club Clinical Research Center, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, SAR, China
Vera S F Chan, Yongxiong Chen, Paul K H Tam & Chen-Lung S Lin
Department of Pathology, Hong Kong Jockey Club Clinical Research Center, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, SAR, China
Kelvin Y K Chan, Annie N Y Cheung, Li-Chong Chan & Ui-Soon Khoo
Department of Obsterics and Gynecology, Hong Kong Jockey Club Clinical Research Center, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, SAR, China
Kelvin Y K Chan & Hextan Y S Ngan
Department of Microbiology, Hong Kong Jockey Club Clinical Research Center, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, SAR, China
Leo L M Poon, Bojian Zheng, Kwok-Hung Chan & Malik Peiris
Department of Genome Research Centre, Hong Kong Jockey Club Clinical Research Center, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, SAR, China
William Mak & Paul K H Tam
Human Immunology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
Xiaoning Xu
Division of Medicine, Imperial College, London, UK
Gavin Screaton
Nuffield Department of Surgery, University of Oxford, Oxford, UK
Jonathan M Austyn
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, SAR, China
Shea-Ping Yip
Vera S F Chan
Kelvin Y K Chan
Yongxiong Chen
Leo L M Poon
Annie N Y Cheung
Bojian Zheng
Kwok-Hung Chan
William Mak
Hextan Y S Ngan
Paul K H Tam
Li-Chong Chan
Malik Peiris
Ui-Soon Khoo
Chen-Lung S Lin
Correspondence to Ui-Soon Khoo or Chen-Lung S Lin.
Supplementary Fig. 1
Reduction of the total SARS-CoV ORF-1b copy numbers is not due to viral dissociation from L-SIGN. (PDF 90 kb)
A549 cells do not express ACE2 and L-SIGN, and SARS-CoV binding to L-SIGN-transfected A549 cells also underwent degradation over time. (PDF 179 kb)
L-SIGN delivered SARS-CoV to Vero E6 cells in trans, but no difference was noted among N7, N7/N5 and N5 L-SIGN transfectants after 48 h incubation. (PDF 101 kb)
L-SIGN expressed in ACE2-transfected CHO cells or in ACE2-trasnfected A549 cells did not facilitate cis infection of SARS-CoV. (PDF 153 kb)
48 h supernatant from SARS-CoV infected N7 L-SIGN/Vero E6 cells contained a lower number of infectious virions than that from the infected N7/N5 L-SIGN/Vero E6 cultures. (PDF 98 kb)
Co-cultures of stable clones of homozygous L-SIGN/CHO cells with Vero E6 cells in a closed infection system resulted in a reduced final SARS-CoV titer. (PDF 231 kb)
CD68bright cells did not express SARS-CoV mRNA in fatal SARS lung. (PDF 138 kb)
Supplementary Table 1
Genotype and allele frequencies of CLEC4M (L-SIGN) in Chinese random controls and Caucasians of European descent (CEPH). (PDF 90 kb)
List of primers used. (PDF 72 kb)
Chan, V., Chan, K., Chen, Y. et al. Homozygous L-SIGN (CLEC4M) plays a protective role in SARS coronavirus infection. Nat Genet 38, 38–46 (2006). https://doi.org/10.1038/ng1698
Issue Date: 01 January 2006
DOI: https://doi.org/10.1038/ng1698
SARS-CoV-2 S glycoprotein binding to multiple host receptors enables cell entry and infection
Irena Trbojević-Akmačić
Tea Petrović
Gordan Lauc
Glycoconjugate Journal (2021)
Genetic variants of the human host influencing the coronavirus-associated phenotypes (SARS, MERS and COVID-19): rapid systematic review and field synopsis
Emilio Di Maria
Andrea Latini
Giuseppe Novelli
Human Genomics (2020)
Modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice
Adam S. Cockrell
Sarah R. Leist
Ralph S. Baric
Mammalian Genome (2018)
Novel roles of DC-SIGNR in colon cancer cell adhesion, migration, invasion, and liver metastasis
Heya Na
Xiaoli Liu
Yunfei Zuo
Journal of Hematology & Oncology (2017)
Human lung ex vivo infection models
Andreas C. Hocke
Norbert Suttorp
Stefan Hippenstiel
Cell and Tissue Research (2017)
Nature Genetics (Nat Genet) ISSN 1546-1718 (online) ISSN 1061-4036 (print)


All Children are admitted full-time in the September following their fourth birthday. However, parents can request that their child attends part-time until the child reaches compulsory school age.
2. Children with a sibling* attending the school.
3. Children living within the priority admission area of Epping Upland C of E Primary School.
4. Children living within the priority admission areas of Coopersale and Theydon Garnon CE, Epping, Ivy Chimneys and Theydon Bois Primary Schools.
A 'looked after child' or a child who was previously looked after but immediately after being looked after became subject to an adoption, residence or special guardianship order will be given first priority in oversubscription criteria ahead of all other applicants in accordance with the Schools Admission Code 2012. A looked after child is a child who is (a) in the care of the local authority, or (b) being provided with accommodation by a local authority in the exercise of their social services functions (as defined in Section 22 (1) of the Children Act 1989).
For applications made in the normal admission round a relevant sibling is a child who has a brother, sister, adopted brother of sister or stepbrother or stepsister living in the same family unit in the same family household and address who attends the preferred school or partner school in any year group excluding the final year. Biological siblings who attend the preferred school in any year group excluding the final year will also be treated as siblings irrespective of place of residence. Children residing in the same household as part of an extended family, such as cousins, will not be treated as siblings.


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text:
Next ›12Next ›
105mm F1.4 Nikon lens
Vitaliy_Kiselev July 2016
Official specs:
14 elements in 9 groups, 3 ED elements
Min aperture F1.4, max - F16
9 rounded diaphragm blades
Minimum focusing distance is 1m
Ultrasonic ring focusing motor
$2199, £2049.99 or €2419 (yes, UK and EU are ripped again)
Available at : http://www.amazon.com/Nikon-NIKKOR-105mm-Focus-Cameras/dp/B01J4S9YRI/
27 Replies sorted by
Nikon Inc. is celebrating a momentous NIKKOR production milestone as well as the announcement of the AF-S NIKKOR 105mm f/1.4E ED, a premium lens for the most discerning photographers. This luxurious new medium-telephoto lens is engineered to help create flattering images with gorgeous bokeh, while delivering impressive sharpness and beautiful color rendition. As a testament to a lasting legacy of high-caliber lenses, Nikon has also announced that NIKKOR lens production has reached the landmark achievement of 100 million units produced worldwide.
"Surpassing the 100 million lenses produced milestone is a great honor and a testament to the photographers who both love and rely on NIKKOR glass to get the job done," said Kosuke Kawaura, Director of Marketing and Planning, Nikon Inc. "The AF-S NIKKOR 105mm f/1.4E ED represents the embodiment of the NIKKOR line of high-quality lenses, giving photographers a robust, well-balanced lens that provides intense sharpness, astounding image quality and background bokeh that is sure to be a favorite among portrait photographers."
Captivating Image Quality
The AF-S NIKKOR 105mm f/1.4E ED is a fast, full-frame (FX-format), medium-telephoto prime lens and is the world's first1 to strike the impeccable balance of 105mm focal length and large f/1.4 aperture for a truly outstanding optic. This new NIKKOR lens is ideal for professional and advanced enthusiast photographers, especially those capturing portraiture, from in-studio fashion to a golden hour engagement session on the beach. These users will love the precise sharpness from this fantastic glass, which provides a flattering compression to subjects with a sensational, sultry bokeh. Beyond the gorgeous image quality lies stellar resolving power, especially when used with Nikon DSLRs such as D5, D810 or D750.
The 105mm f/1.4E is a fast lens with endearing character, providing stellar optical performance, even in the peripherals. Sharp rendering is attained for shooting distant subjects throughout the aperture range – from maximum aperture to being stopped down by several stops. Gradual alteration from the focal plane helps render subjects in high-fidelity with a natural depth, giving colorful portraits and landscapes that little bit of extra "pop" to help create a truly distinct image. The AF-S NIKKOR 105mm f/1.4 is also an excellent choice for low-light photography, as its large maximum aperture allows photographers to shoot at the fastest possible shutter speeds. Additionally, this lens can reproduce point light sources faithfully without sagittal coma flare, even on the edges of the frame.
Masterful Lens Construction
Decades of precision, knowledge and experience are put forth into every NIKKOR lens, and the AF-S NIKKOR 105mm f/1.4E ED is an exceptional example of brilliant craftsmanship and Nikon technologies merging. Packed with the latest advanced optical innovations, the 105mm f/1.4 features Nikon's electromagnetic aperture control technology for consistent exposures, even during high speed continuous shooting.2
On the outside, solid build quality is well-balanced for all-day shooting, while the lens is sealed and gasketed to resist dust and moisture. Additional Fluorine coatings are used on the front and rear elements, so that smudges, dirt or moisture are easily removed. Inside the lens, the optical formula consists of 14 elements in nine groups, which include three ED glass elements. A 9-blade diaphragm further helps to create a circular, pleasing out of focus area. The lens also features Nikon's Nano Crystal Coat technology to significantly reduce instances of ghosting and flare.
The new AF-S NIKKOR 105mm f/1.4E ED joins other NIKKOR f/1.4 lenses in the Gold Ring Series, which include only premium primes with Nano Crystal Coat and pro-grade build quality. These other lenses include the AF-S NIKKOR 24mm f/1.4G, AF-S NIKKOR 35mm f/1.4G, AF-S NIKKOR 58mm f/1.4G, AF-S NIKKOR 85mm f/1.4G.
A Landmark in the NIKKOR Legacy
Nikon is also pleased to announce that the total production of NIKKOR lenses for Nikon interchangeable lens cameras reached the landmark 100 million milestone in mid-July 2016.
With a robust line-up of high-quality lenses for all types of photographers, the NIKKOR name has become synonymous with a rich history of superior lenses. Nikon continues to introduce new NIKKOR lenses that further optical technology, utilizing cutting-edge techniques and knowledge from experience cultivated over its long history. Such revolutionary advancements include Nano Crystal Coat, which acts as a powerful anti-reflection coating, and significantly reduces instances of ghosting and flare. More recently, NIKKOR lens technologies such as the use of Phase Fresnel (PF) and Fluorite elements have helped to create smaller and lighter lenses with superb balance and handling. Many NIKKOR lenses now also feature a Fluorine coat that helps repel a variety of contaminants such as dust, dirt, water, oils and grease. The introduction of an electromagnetic diaphragm has also helped to create lenses that provide consistently crisp exposures, even during high-speed shooting.
The AF-S NIKKOR 105MM f/1.4E ED will be available in late August for a suggested retail price (SRP) of $2,199.953.
http://www.nikon-image.com/products/lens/nikkor/af-s_nikkor_105mm_f14e_ed/sample.html
Vitaliy_Kiselev August 2016
https://www.flickr.com/photos/nikonrumors/with/28653864960/
img1125.jpg
More initial samples
https://www.facebook.com/photo.php?fbid=10155225934132715&set=a.10150840680827715.519372.616147714&type=3
https://taotzuchang.wordpress.com/2016/08/24/nikon105/
http://www.streetsilhouettes.com/home/2016/8/25/nikon-105mm-f14e
Vitaliy_Kiselev September 2016
Interview in Japanese about this lens
http://dc.watch.impress.co.jp/docs/news/interview/1017554.html
Second part of interview
DrDave September 2016
I've been looking at 100-105. I currently use the 1970s 105mm, a decently sharp lens but no coating. It will be interesting to see how this compares to the Canon 100mm F2 USM (not the macro) which is a fine lens and presumably less expensive. Obvious differences, of course.
Bokeh and very good contrast and sharpness across the full-frame sensor, even with the aperture opened to f1.4. This performance coupled with the build-quality of a professional Nikon lens and a reliable AF makes for a compelling package.
http://www.cameralabs.com/reviews/Nikon_Nikkor_AF-S_105mm_f1-4E_ED/
http://photoreview.com.au/reviews/lenses/full-frame/af-s-nikkor-105mm-f-1.4e-ed-lens
US - https://www.amazon.com/Nikon-NIKKOR-105mm-Focus-Cameras/dp/B01J4S9YRI/
UK - http://www.wexphotographic.com/buy-nikon-105mm-f1-4e-ed-af-s-lens/p1603567
https://www.dpreview.com/news/5370862528/nikon-105mm-f1-4e-ed-sample-images
Vitaliy_Kiselev October 2016
https://www.ephotozine.com/article/nikon-af-s-nikkor-105mm-f-1-4e-ed-review-30026
800 x 532 - 104K
http://www.imaging-resource.com/lenses/nikon/105mm-f1.4e-ed-af-s-nikkor/review/#shooting
Vitaliy_Kiselev November 2016
MTF measurement
https://www.lensrentals.com/blog/2016/11/nikon-105mm-f1-4-e-mtf-bench-tests/
Another review
http://petapixel.com/2016/11/18/nikon-105mm-f1-4-review-working-london-photographer/
Vitaliy_Kiselev December 2016
http://www.focus-numerique.com/test-3490/focale-fixe-nikon-af-s-nikkor-105-mm-f14-e-ed-test-8.html
http://dc.watch.impress.co.jp/docs/review/lens_review_2/1034431.html
Very good results
https://www.dxomark.com/Reviews/Nikon-AF-S-Nikkor-105mm-F1.4E-ED-review-The-best-performing-lens-in-the-lineup-below-200mm
tetakpatak December 2016
Wow, what a lens.... but I don't think Nikon will sell many units nevertheless.
http://www.intufisuri.ro/2016/12/review-nikon-10514e-ed.html
sample100.jpg
Vitaliy_Kiselev January 2017
Wide open at F1.4 the lens is almost tack-sharp across the frame. Stopping down to ƒ/2 actually introduces a slight amount of softness - we are talking micro-adjustments, here - but stopping down further to ƒ/2.8 slightly improves sharpness.
http://www.imaging-resource.com/lenses/nikon/105mm-f1.4e-ed-af-s-nikkor/review/
nikon 157
f1.4 21

redpajama_set_name: RedPajamaCommonCrawl


Lately the synchronization across devices with google drive doesn't work.
created in one device do not appear on the other device.
I am not sure when this started, possibly after the last updates.
I also tried to disconnect and reconnect to the google drive account but it did not solve the problem.
Since Enpass 6 is based on a new architecture, it will only sync with Enpass 6, and not with previous versions. So, to test the sync functionality, you'd need to install the Enpass 6 on your other devices as well.
How do i update my paid version on my Windows phone as it is not downloadable from Windows store?
Same architecture, different database structure. No backwards compatibility, forced upgrade, bug ridden application.
I think this is was a bad update strategy. If I am getting this correctly, I can only install v6,x from the MS Store if I have Win10 (Win7 machines are not able to download as they won't show up on the list of devices). Given that v6.x CAN sync to cloud, but v5.x (which is install-able on Win 7 machines) CANNOT sync to the cloud., then it seems like a big disconnect here. You've just made all of your users who are on Win7/8 (by choice) unable to install a version on their desktops and really be of any use. I run a mixture of Win 7 & Win 10 machines, but my favorite is definitely Win 7 and am going to keep Win 7 around for quite some time on them.
@blacktop, I think there is a misunderstanding. I am running 6.x on both Win 7 and Win 10 Desktops, and they sync back and forth as they should. If you want v6.x on Win 7, download it from the download page, it is called "Traditional Win32 (Windows 7 or later)". There is no need to stay on v5 with Win 7! Of course it is not in the MS Store, because the store is only for Windows 8 onwards.
There is no "big disconnect".
And the OP is talking about Win 10 PHONE (Mobile), not desktop.
Thank you so much for pointing that out. Lots of egg dripping off my face....I simply didn't scroll down the page far enough to see the download links for the "Traditional Win32". That just makes perfect sense....I was racking my brain trying to figure out why it was only going through the MS Store, and subsequently only available for Win10.


Amsterdam takes centre stage in the European GAA calendar this weekend as the 3rd round of the Benelux Mens Championship and 2nd round of the Benelux Ladies Championship takes place on Saturday May 10th.
For Amsterdam GAC, it's our first opportunity to show off our new clubhouse, which was completed last autumn by our long-time sporting partners Amstelveense Rugby Club. The new clubhouse is a top class facility, with 8 dressing rooms, a new bar & kitchen, and an impressive balcony extending from the bar providing spectators with an excellent vantage point of both football pitches where matches will be taking place throughout the day.
In the Mens Championship, Amsterdam will be looking to settle old scores with rivals Belgium who pipped them to the post in the final of Round 2 last month in Brussels. Dusseldorf GFC, Eindhoven Shamrocks, Luxembourg and newcomers to the Benelux Championship for the first time, Zurich Inneoin, will also be looking to walk away with the glory by Saturday evening.
In the Ladies Championship, Holland Ladies will make their season debut and take on Belgium, Dusseldorf, Luxembourg in what is always a close championship.
Matches will run from 9am to 6pm, and the club will host a BBQ from 5pm for players & spectators, whilst the bar will be open all day (and evening!!) for snacks.
Take Metro 51 from Centraal Station and leaving the tram at stop 'Sportlaan'. The pitch is just a few minutes walk away.
Buses 170, 171 and 172 from Central Station or Leidseplein to bus stop 'Sportlaan'.
Looking forward to a great day of sport and craic afterwards!


The Slub Stretch Twill Field Jacket offers a relaxed finish to casual outfits. The zip-up front and four zip pockets on the jacket combine with the form-skimming fit to create a work wear-inspired jacket perfect for everyday wear. Wear with chinos and a short-sleeved polo shirt for easy off-duty dressing.
Our model is 6ft3" and is wearing a US medium.


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text:
Differentiating Among Depression Delirium And Dementia In Elderly Patients 2020
June 18, 2019 by Wilma
DELIRIUM. DEPRESSION. DEMENTIA. DEFINITION Delirium is characterised by an acute fluctuating onset of confusion, disturbance in attention, disorganised thinking and/or decline in level of. the Older Person. Registerd Nurses Association Ontario (2003) Screening for Delirium, Dementia and Depression in Older Adults.
Older Adults (2010b) and Caregiving Strategies for Older Adults with Delirium, Dementia and Depression (2010a). It is to be used by. for the assessment and care of delirium and/or dementia and/or depression in older adults. The focus is. Identify and differentiate among signs and symptoms of delirium, dementia, and/or.
Distinguishing Between Depression and Dementia in the Elderly:. He claims that differentiation. tions of elderly patients tend to show marked.
depression. movement. disorders. psychosis. dementia. Depression with dementia ("pseudodementia"). Dementia with depression. PD with depression. Major Depression. Similar across lifespan but there may be some differences. Among older adults: Psychomotor disturbances more prominent (either agitation or.
• Differentiate between depression dementiaDifferentiate between depression, dementia, and delirium. Am J Geriatric Psychiatry. Symptoms Among Older Adults in.
Depression And Cognitive Complaints Following Mild Traumatic Brain Injury. A meta-analysis examining methodological variables and. depression after mild traumatic brain injury. cognitive complaints following mild traumatic. Traumatic Brain Injury (TBI), also known as intracranial injury, is commonly caused by vehicle accidents, bad falls and violence. A TBI occurs when there. Full-text (PDF) | Traumatic brain injury (TBI) is a common occurrence with multiple
Differentiating among Depression, Delirium, and Dementia in Elderly Patients. Published online. Journal Article. Full Text. Published version (via Digital Object.
Clarifying the confusion about confusion: Current practices. for delirium or differentiating it from dementia. in elderly patients with dementia.
Delirium and dementia are two causes of confusion in older adults. Delirium is an acute. The table below outlines some key differ- ences between delirium and dementia. Table 1. Differences Between Delirium and Dementia. Delirium. Dementia. nurse differentiate between delirium and dementia and help the patient.
bachelor's thesis animal assisted therapy among elderly patients with dementia – a systematic literature review ! janina vogt (s1010670) faculty of.
set of criteria among elderly patients, in diagnosing delirium in patients with or without dementia. Delirium is misdiagnosed as depression in up to
Over the course of 24 hours patients with dementia are. between depression, delirium and dementia in older. Differentiating among depression, delirium,
Free Depression Elderly. and other types of dementia and differentiating dementia from delirium and. Depression In Patients.
Aug 23, 2017. Delirium is mistaken for dementia or depression, especially when patients are quiet or withdrawn. However, by Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM5) criteria, dementia cannot be diagnosed with certainty when delirium is present. Health professionals can do Mini-Mental.
nosed as dementia or other psychiatric illness even though there are potential strat- egies (e.g. a more florid picture. Disorders of Sleep and Wakefulness. Delirium may first appear at night with fragmen- tation and reduction of sleep. Patients may wake from vivid dreams. among elderly patients, compared with DSM-III,
Screening for Delirium, Dementia and Depression in Older Adults. Establishing a Diagnosis of Depression in the Elderly…….. 75
The Folstein Mini-Mental State Exam is not useful in screening for delirium or differentiating it from dementia. As a subspecialist, I will often see patients with dementia and suspected depression, who then develop delirium as a result of failure to thrive.
Virtual Mentor American Medical. Differentiating among Depression, Delirium, and Dementia in Elderly Patients. delirium in elderly patients,
The following sections describe the signs and symptoms that are used to differentiate among the various types of dementia during a diagnostic evaluation. ALZHEIMER'S. It is important to evaluate and treat elderly patients for depression, because the symptoms of depression in older people often mimic dementia.
. in almost one half of elderly patients with delirium, can be useful in differentiating delirium from. of delirium, dementia, psychosis, and depression.
Differentiating among depression, delirium, however, the elderly patient with dementia sees only chaos and reacts directly to that chaos in an attempt to protect.
Depression in the elderly. depression Patients with dementia should still be screened. Differentiating delirium from dementia
There's no single test that can differentiate depression from dementia. Symptoms of Depression in the Elderly They're. Health.com may receive.
Find out more information about Understanding the role of delirium in dementia development.
Differentiating among Depression, Delirium, and Dementia in Elderly Patients A description and explanation of the distinguishing characteristics of depression.
DIFFERENTIATING DEMENTIA/DEPRESSION/DELIRIUM. dementia, depression, and delirium in older. Treat the patient,
Depression and delirium, particularly hypoactive delirium, may present with apathy, withdrawal and tearfulness. Delirium occurs suddenly (over a matter of hours or days) and the symptoms tend to fluctuate throughout the day; depression describes a negative change in mood that has persisted for at least two weeks; and the onset of dementia.
Dementia is a broad category of brain diseases that cause a long-term and often gradual decrease in the ability to think and remember that is great enough to affect a person's daily functioning. Other common symptoms include emotional problems, problems with language, and a decrease in motivation. A person's.
Table 1 Differentiating among dementia, delirium, and depression Patient Scenario Situational Features Scenario 1: A patient with dementia A 77-year-old woman was.
Gagliardi JP. Differentiating among depression, delirium, and dementia in elderly patients. Virtual Mentor. 2008;10(6):383-8. CrossRef PubMed Google Scholar
Compare the differences and similarities between dementia, depression, and delirium on several clinical dimensions. Introduction. Assessment of cognitive and behavioral disturbances is always complex, regardless of the age of the patient. However, older adults present unique challenges to assessment because they are.
Florida Heart CPR* Differentiating among Depression, Delirium, and Dementia in Elderly Patients. 1 hour. A description and explanation of the distinguishing.
Preventing Suicide in Older Adults, an issue brief from SAMHSA and the Administration for Community Living, assists health care and social service organizations in de¬veloping strategies to prevent suicide in older adults. The American Medical Association's Differentiating among Depression, Delirium, and Dementia in.
1 Care of the Hospitalized Patient with Delirium. % of elderly patients experience a delirium prior to. depression, delirium, and dementia can co.
Recognizing Dementia, Depression, and Delirium In Older Adults. Overview. Dementia, depression, and delirium are among the most common psychiatric disorders seen with older adults and speak to the complexity of the specialty of geriatrics. The overlap of symptoms prevalent in this triad is abundant.
Aug 1, 2007. The common secondary causes seen in most clinical practices are delirium, dementia, and depression. Among the dementing neurodegenerative disorders, psychotic symptoms are commonly seen in patients with Alzheimer's disease, dementia with Lewy bodies (DLB), and Parkinson's disease.
Differentiating among Depression, Delirium, and Dementia. delirium in frail elderly patients. Cognitive assessment and differentiating the 3 Ds (dementia,
Delirium in elderly patients has a. months later among patients with or without dementia. 89 These. Delirium among elderly persons.
Nursing Times Learning has launched a new unit on how to tell the difference between dementia, delirium and depression in older people, to ensure they receive the.
Another patient developed a delirium after the final. ECT session, associated with cognitive decline as evaluated by the MMSE. Discussion. This study investigates the usefulness of ECT for distinguishing between dementia and depression in elderly patients who suffer from resistant depression that mimics dementia.
differentiate between normal cognitive changes and delirium, independence, and yet changes in cognition are frequently unrecognized among older adults. Cognitive change in older adults can be observed due to age-related cognitive. Keywords: cognitive decline, delirium, depression, dementia, older adults, cognitive.
Delirium in the Elderly:. • History of prior delirium. Delirium in the Elderly. Differentiating between Delirium, Dementia and Depression
Delirium causes changes to the person's usual mental functioning. Its onset is rapid. In Intensive Care Units about 2 out of every 3 patients will get delirium. Although delirium. It is important for the medical team treating the person to differentiate between a diagnosis of delirium, dementia and depression. How is delirium.
Cognitive conditions like dementia, delirium, and depression represent. to differentiate depression from. among hospitalized older patients.
It can occur in up to 50 per cent of patients aged 65 years and over who have been admitted to hospital, and higher rates are seen among surgical patients and in intensive care. Dementia is a significant risk factor for the development of delirium. Being able to differentiate between dementia and delirium is essential for staff.
• Delirium • Depression. especially the elderly. People with depression. Differential Diagnosis of dementia Factsheet 2005.doc
Delirium in elderly patients and the risk of postdischarge mortality, institutionalization, and dementia: a meta-analysis
The common secondary causes seen in most clinical practices are delirium, dementia, and depression. 10% among elderly men. Hacked by 4Ri3 60ndr0n9.
Depression And Workplace Accomodations Depression And Cognitive Complaints Following Mild Traumatic Brain Injury. A meta-analysis examining methodological variables and. depression after mild traumatic brain injury. cognitive complaints following mild traumatic. Traumatic Brain Injury (TBI), also known as intracranial injury, is commonly caused by vehicle accidents, bad falls and violence. A TBI occurs when there. Full-text (PDF) | Traumatic brain
Gagliardi JP. Differentiating among depression, delirium, and dementia in elderly patients. Virtual Mentor 2008; 10, 6:383-388. Google Scholar
Pleasurable Activities And Depression: Nov 16, 2015. Positive strategies men regularly use to prevent and manage depression: a national survey of Australian men. The majority of these responses fell into three main categories of (i) specific pleasurable activities, (ii) relationships and/...
Depression Self Inflicted Injury: Learn About a Treatment for Adults with Depression. Get a Symptom Questionnaire. Under the federal health law and 2006 regulations, insurers can't deny medical coverage for an individual's injuries because they resulted from a medical condi...
Symptoms Of Depression In The Army: Depression army is an international growing movement centered around ending the stigma associated with mental illness and supporting those who have one. Back Depression Inventory Scoring Beck Depression Inventory II. Past Failure As I look back, I se...
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Signs Of Depression Cat: 6 Warning Signs That Your Cat Is Depressed. Depression may be a factor if your cat is sleeping more than normal and uninterested in the things that normally. HAMILTON — Johnny Football isn't intent on using the Hamilton Tiger-Cats as a springboard ba...
Studies On Depression And Exercise: Exercise is moderately more effective than a control intervention for reducing symptoms of depression, but analysis of methodologically robust trials only. Jun 14, 2016. Research has shown exercise to be an effective treatment for mild-to-moderate de...
Differentiating Among Depression Delirium And Dementia In Elderly Patients 2020 4.5 out of 5 based on 167 ratings.


Discussion in 'Emergencies / Diseases / Injuries and Cures' started by vantain, Mar 19, 2019.
Seems the issues with my hen are simply reproductive related, as she continues to have trouble with it.
The Baytril that "Eggsessive" recommended has come in, and the recommendation was for "Baytril dosage is 0.05 ml per pound orally twice a day for 5-6 days". When given orally, is that with a syringe, or can I add it to something she eats? What the best way to administer this? It's liquid. I think I need to start this ASAP.
She is lethargic today, all puffed up and not eating. She was fine this morning, running around, scratching and pecking in the run with her flock mates. I noticed this afternoon that she was standing off by herself. I brought her in and that is when she expelled the collapsed egg. She hasn't improved since doing that, and seems to be nodding off to sleep quite a lot.
ISA Brown, 11 months old.
She is lethargic today, and expelled an very shriveled soft shell, collapsed egg. It was bloodied.
3) How long has the bird been exhibiting symptoms?
4) Are other birds exhibiting the same symptoms?
5) Is there any bleeding, injury, broken bones or other sign of trauma.
Seems to be some internal bleeding, considering the collapsed soft shelled egg is reddish in color.
6) What happened, if anything that you know of, that may have caused the situation.
7) What has the bird been eating and drinking, if at all.
8) How does the poop look? Normal? Bloody? Runny? etc.
9) What has been the treatment you have administered so far?
Oxytretracycline for two days. Was waiting on the Baytril to arrive. Had her in the house for a few days, observing, and making sure she was getting food.
10 ) What is your intent as far as treatment? For example, do you want to treat completely yourself, or do you need help in stabilizing the bird til you can get to a vet?
11) If you have a picture of the wound or condition, please post it. It may help.
I would just give the Baytril (enrofloxacin) orally. It tastes bad according to others, and if put in food, she might not take it all. Sorry that she is not acting well. It sounds like her egg laying problems are continuing. Give her some calcium as well.
Thanks. It's unbelievably difficult to administer it orally. This bird fights. I managed to get it in there by dripping it on some scrambled egg that she ate from my hand.
In any case, about an hour after my last post from last night, our hen discharged a shell less egg. She perked up considerably after that, and even ate a 1/2 cup of egg and her normal layer crumbles.
I ended up putting her back out with the other hens for the night, and she had a clear crop again this morning. She's currently acting normal again.
I hope this issue clears up. I'll keep her on the Baytril twice a day for the next 10 days as suggested, and continue to give her a little extra calcium.
Also, because she is going to be on the antibiotics for a week or so, do I need to be concerned with feeding her other supplements due to the antibiotics ruining her normal gut bacteria?
I think the Baytril is only given for 5 days, maximum 6 days. You have to give it to her as best you can. If you can get her to take it in egg, that works. I use a little of Friskies canned pate cat food to hide meds from my animals. So glad that she laid, but I hope that she straightens up. You might want to check into suprelorin hormone implants to stop laying. They are too expensive for me to consider even if I found a vet to give them, but some with special pets will. Most of my birds with repro problems just seemed to stop laying eggs altogether.
I won't pay for any hormone implants. I will help keep her comfortable, and intervene as much as possible, but that is the extent of what I will do.
I would prefer if she did just stop laying permanently. I would prefer that to internal laying or EYP.
We'll see how things progress. Today she is happy and alert. When I went to check on eggs at lunchtime today, she was sitting on three eggs, trying to lay one of her own. I'm pretty certain the three already in there were from the other three hens. This makes me think she is either laying internally, or something else.
Poor girl...she's back in the house again, suffering yet another soft shelled egg issue.
I don't think I have heard of one having so many at one time. I suppose she is still getting calcium? Vitamin D 3 and phosphorus are all important in balance with calcium. Is she eating her chicken feed okay? Poultry nutridrench might be good for her today. I hope she feels better.
She was eating normal layer feed today..I think. I know she was eating from the feeder this morning, after I fed her her Baytril and a crushed calcium tablet with some scrambled egg.
Right now, she won't drink or eat, probably due to the pain of this soft shelled mess inside of her. She's clearly in pain.
I gave her the warm bath, and massages her a bit, but no luck on this one. She's definitely egg bound. Can't even get a finger into her vent, it is so swollen.
She's trying, but really in pain.


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Q: How to use a for each loop with a multi-dimensional array in C++ Okay, so first off you should note that I am using visual studio 2010.  Anyways, I am getting a very weird error in my for each loop that doesn't make any sense to me.
BYTE CPUKeys[][0x10] =
{
    {0x28,0x53,0x71,0xD9,0x7B,0x47,0xCC,0x50,0xAF,0x45,0x8D,0xB3,0xED,0x53,0x22,0x13},//Randy//encrypted
    {0x6E,0x38,0xB0,0xEF,0x6E,0x96,0x20,0x16,0xE5,0xCA,0x4B,0xE9,0x23,0x4E,0xC6,0xA5},//Josh//encrypted
    {0x95,0x2C,0x20,0x98,0xF9,0x99,0x28,0x0F,0xEE,0xA7,0x8F,0x48,0x58,0x01,0xB7,0x07},//Falcon//encrypted
    {0x64,0xDC,0x1E,0xFA,0xD2,0xAE,0x57,0x6C,0x0B,0xD0,0x39,0x6A,0x13,0x2C,0x91,0xE0},//Justin//encrypted
    {0x8D,0x82,0x6D,0x71,0x82,0xDC,0x83,0x8F,0x79,0x51,0xB8,0x7C,0x1F,0xC1,0xBF,0xD4},//Bypass//encrypted
    {0x27,0xF2,0xA7,0xF3,0xE2,0xDC,0x01,0x19,0x17,0xF4,0x11,0xFE,0xE9,0xB5,0x0C,0x2C},//Surge//encrypted
    {0x6D,0x7C,0x86,0x9A,0x6A,0xE1,0xD4,0x10,0x76,0x16,0x11,0x7C,0xED,0xB3,0x4D,0x56},//Noel//encrypted
    {0x3D,0x6C,0x88,0x8C,0x9D,0x3A,0xA5,0x40,0x64,0xDF,0xDF,0x8D,0x94,0xFD,0x28,0xF1},//XexRgh//encrypted
    {0x06,0x0D,0x8C,0xB6,0xB6,0x6D,0x29,0xD7,0x41,0xE1,0x13,0x35,0x84,0x80,0x68,0x31}//Zoobzy//encrypted
};

Alright so there is my multi-dimensional BYTE array.
for each(BYTE ID[0x10] in CPUKeys)
{

And there is my for each loop.  The weird thing that is happening with it is my error that I am getting on ID.  The error states: 'for each' iterator type "BYTE[16]" is not compatible with element type "BYTE[16]".  Any idea why it might say this?  What am I doing wrong, and how do I fix it?

A: Try this if it works:
for(auto& rows: CPUKeys)// Iterating over rows
{
    for(auto& elem: rows)
    {
        // iterate every row's column
    }
}

A: As far as i know there is not foeach loop in c++.
Use two for loops instead. Something like this
for (int i = 0; i < 9; ++i)
{
   for (int j = 0; j < 16; ++j)
   {
     BYTE value = CPUKeys[i][j];
   }
}

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