Yan ; Feng

Sabine Santibanez

Hazel Appleton

Dr Yiyu Lu

Dr Jin Li
email: li.jin@hpa.org.uk

Application of new assays for rapid confirmation and 2 genotyping of isolates of rubella virus 3 4

Keywords: rubella virus isolates, PCR assays, genotyping rubella virus isolates, PCR assays, genotyping 22

Application of new assays for rapid confirmation and genotyping of isolates of rubella virus

INTRODUCTION 1

Rubella is a mild, sub-clinical disease with few complications, but infection 2 during early pregnancy can lead to severe birth defects known as congenital 3 rubella syndrome (CRS) [Best and Enders, 2007;WHO,2000]. Since the 4 introduction of rubella vaccination, the incidence of rubella has been reduced 5 greatly and CRS is very rare in countries, such as the United Kingdom and the 6 United States of America, which have established rubella vaccination 7 programmes [WHO, 2000]. In 2007, 975 suspected cases of CRS were reported 8 from 34 countries in the western hemisphere where CRS surveillance has been 9 established [WHO, 2008]. The World Health Organization (WHO) has estimated 10 that more than 100,000 cases of CRS occur in developing countries every year 11 [WHO, 2000]; this represents a considerable social and economic burden.

12 Rubella is regarded as another target for eradication in countries where 13 transmission of measles virus has already been interrupted [WHO, 2000]. Togaviridae, is a positive single stranded RNA virus [Chantler et al., 2001]. The 17 viral genome encodes five proteins, two non-structural proteins (P90 and P150) 18 and three structural proteins (E1, E2 and the capsid protein). Major neutralizing 19 and haemagglutinating epitopes and T cell recognized sites are located in the 20 structural proteins [Chantler et al., 2001;Cordoba et al., 2000]. The high G+C 21 content, of up to 65%, is the most significant characteristic of the viral genome,. weeks gestation [Enders and Jonatha, 1987].

6 7
Virus isolation is a conventional laboratory method, and important for monitoring 8 virus excretion in CRS infants. Although virus isolation is time-consuming, 9 labour-intensive and not particularly sensitive, it is still recommended by the 10 WHO for studying the aetiology and epidemiology of rubella [WHO, 2007]. RV 11 can be cultured in a variety of cell lines, such as RK13, SIRC, BHK and Vero 12 (Vero/SLAM) cells, but serial propagation can be established only in a limited 13 number of cell lines [Figueiredo et al., 2004]. Previous studies have shown that 14 specific CPE may appear in RK13 cells under controlled conditions, or in RK13 15 and SIRC cells after one or two passages in Vero cells [Figueiredo et al., 2000; 16 Best et al., 2005]. The immunofluorescence assay (IFA) is a conventional method for confirming 19 rubella growth, but it is time-consuming, relies on the operator's interpretation

Virus isolation and blind passages 12

Vero cells were seeded at a concentration of 5x10 5 cells/ml in glass tubes.

13

Growth medium (GM) was Dulbecco's MEM (DMEM) containing 5% foetal 14 bovine serum (FBS), 50µg/ml gentamicin and an additional 200mM L-glutamine.

15

Cells were incubated at 37°C. Once a monolayer of cells had formed, the GM 16 was removed and the cells inoculated with 100µl clinical specimen and left for 17 one hour at 37°C. 0.9 ml maintenance medium (MM) containing 2% FBS was 18 then added to the monolayer and the tubes were incubated stationary at 37°C.

19

The cells were observed daily. After seven days the tubes were frozen at -80°C 20 for one to two hours. After thawing, the cells were scraped into the culture 21 medium, thoroughly mixed and then centrifuged at 200 x g for 10min.

22 Supernatant and cells were harvested separately as the 1 st passage. 100µl cells Total RNA was extracted from 100µl tissue culture fluid supernatant/cells using 2 the MagNA Pure extractor (Roche, UK). In a 50µl reaction mixture, a 20µl 3 sample of RNA was reverse transcribed to cDNA using random primers as 4 described previously [Cooray et al., 2006;Vyse and Jin, 2002]. At the Robert Koch-Institut, total RNA was extracted from 140µl tissue culture 14 fluid using the QIAamp Viral RNA Mini kit (Qiagen, Germany) and was eluted 15 into 60µl RNase-free water. A 2.5µl sample RNA was used to generate cDNA 16 using the specific primer Ru1r. 12 The supernatant was discarded and the pellet re-suspended in distilled water.

13

The suspension was treated in an ultrasonic bath to break up clumps of cells. A with passage affecting about 30% cells in the 3 rd passage and 50% in the 5 th 13 passage.

14 15 RV particles were not observed by EM in the 2 nd passage, but small groups of 16 particles were seen in the 3 rd passage, and large clumps of virus particles were 17 found in the 4 th and 5 th passage (Figure 2). Rubella virus is usually strongly cell 18 associated and the viral particles observed were all attached to cell debris. In order to determine the genotypes of the six Chinese isolates, the E1-739nt 13 window was amplified using the new nested PCR-E860 assay. The amplicons 14 were purified and sequenced with primers Ru3r and Ru4f. These sequences, 15 together with reference strains recommended by the WHO, were used to 16 construct a phylogenetic tree (Figure 3). These results were in agreement with fourth step of incubating at 82°C for 5sec was added to increase the specificity. A 1 melting curve analysis for every test is strongly recommended as it is a 2 non-probe based real-time PCR assay [Robert et al., 2009]. Even though the 3 sensitivity was 100-fold lower than the nested PCR-E317, the Rtime-SGE317 is 4 one tenth of the cost and four times faster, and therefore suitable for rapid 5 detection of RV in large numbers of samples. The Rtime-SGE317 is less 6 sensitive compared to other real-time assays for rubella confirmation developed 7 previously in the UK or China [Jin and Thomas, 2007;Zhao et al., 2006]. This 8 could be due to its non-probe based method and the size of product. The 9 advantage of Rtime-SGE317 is that the product can be assessed quantitatively 10 and sequenced directly for confirmation and preliminary genetic classification.

11

Using plasmid DNA as standards, the viral load can be quantified based on copy 12 numbers. The copy numbers of viral load of two Sudanese isolates were higher 13 in cells than that in supernatant. Electron microscopy has confirmed that RV is 14 strongly cell associated. However, this does not mean that concentrated cells 15 are necessarily more suitable, as the remaining lysed cells may affect viral 16 propagation in fresh cells. The study results show that the Ct value is more reliable than viral genome copy Measles and Rubella. However, tissue culture fluid specimens have to be sent to 17 a national/regional laboratory for confirmation of RV growth because most 18 sub-national laboratories do not have a fluorescence microscope for IFA or the 19 reagents for the ICA [WHO, 2007;Zhu et al., 2007;Chen et al., 2007]. Results of

  RV), the sole member of the genus Rubivirus of the family 16

22 23

 22 Clinical diagnosis of rubella is unreliable and therefore laboratory confirmation is 24 necessary, particularly for the diagnosis of rubella-like illness during pregnancy. Serological diagnosis, by the detection of rubella-specific immunoglobulin M 1 (IgM) in a single serum or oral fluid sample, is the common method for 2 confirming rubella cases, and has proved a good indicator for demonstrating 3 recently acquired infection [Banatvala and Brown, 2004]. Prenatal diagnosis of 4 rubella infection based on rubella specific IgM alone is unreliable until at least 22 5

  17

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201

  and requires a fluorescence microscope. Recently, the reverse-transcriptase 21 polymerase chain reaction (RT-PCR) followed by hybridisation assays, the 22 replicons-based method and the indirect immunocolorimetric assay (ICA) have 23 been developed to detect rubella virus infected cells[Zhu, et al. 2007; Chen, et Genetic characterization of virus isolates has relied on nested RT-PCR assays to 2 generate the 739nt region of the E1 gene that is required for genotyping in 3 accordance with the recommendations of the WHO[WHO, 2005]. In this study, a 4 SYBR Green I-based real-time PCR assay (Rtime-SGE317) was developed, 5 initially at Health Protection Agency in the UK (WHO Rubella Strain Bank), and 6 applied subsequently to rapid confirmation of RV isolates by testing samples of 7 tissue culture fluid from Vero cells at the Centre for Disease Control and 8 Prevention of Zhejiang Province, China. Compared with other methods, namely 9 IFA and electron microscopy (EM), the Rtime-SGE317 can be carried out easily 10 at local public health laboratories. The nested PCR (PCR-E860), developed 11 initially at the Robert Koch-Institut, was used both at the Health Protection 12 Agency and the Centre for Disease Control and Prevention of Zhejiang Province 13 to generate the required E1-739nt region from eight confirmed RV isolates for 14 genotyping. By combining the two assays, RV isolates could be confirmed and 15 genotyped very quickly. These two tests offer the opportunity of extending the 16 molecular surveillance of rubella and contributing to the WHO goal of eradicating 17 were investigated in the study. Two (H1 and U6) were isolated 3 from nasal and throat swab specimens of two Sudanese infants with 4 serologically confirmed CRS [Omer et al., 2010]. These two isolates were also 5 used for comparing the PCR assays developed in this study with other detection 6 methods, EM and IFA (TableⅡ). The other six (ZJ1~ZJ6) were isolated originally 7 at the Centre for Disease Control and Prevention of Zhejiang Province, China 8 between 2005 and 2008 from throat swab specimens collected from patients 9 with acute rubella confirmed serologically.10 11

23

  and supernatant were passaged separately into fresh Vero cells to the 5 th 24 passage.

  RNA was extracted using the RNeasy Mini Kit (Qiagen, 7 Germany). 200µl tissue culture fluid mixture was extracted and eluted into 50µl 8 RNase-free water. cDNA was generated using M-MLV Reverse transcriptase 9 (Invitrogen or Promega) and either the specific primer Ru1r for the PCR-E860 or 10 random primers for both the nested PCR-E860 and Rtime-SGE317. The 10µl 11 reaction mixture contained 2.5µl sample RNA.

  17

7

  of E1.7 and E1.8, which is used routinely as a part of the nested 20 PCR-E317 assay at the Health Protection Agency and as described previously 21[Jin and Thomas, 2007], was adapted. The primer concentration was optimised 22 at a range of 0.5-1.0µM. The reaction with a final concentration 1.0µM gave the 23 best result for both sensitivity and specificity (results not shown). The assay was 24 performed using the QuantiTect SYBR Green PCR kit (Qiagen, Germany) on a Roche, Switzerland). Each 20µl reaction mixture contained 10µl 1 of 2x SYBR Green I buffer, 0.2µl of each primer (100pmol/µl), 7.6µl dH 2 O and 2 2µl cDNA or plasmid DNA. The Tm of the specific product amplified was 89°C 3 according to the melting curve, and therefore a fourth step of incubating at 82℃ 4 for 5 sec was added to eliminate the effect of non-specific amplification. The 5 optimised amplification conditions are shown in TableⅠ. 6 Sensitivity of the Rtime-SGE317 was evaluated using serial diluted plasmid DNA 8 of the E1 gene containing both the window 739nt and the target of the 9 Rtime-SGE317, and the random primer generated cDNA of the Thomas strain of 10 rubella (used routinely as a positive control at the Health Protection Agency).11The plasmid DNA used as standards for quantitation was diluted serially from10 6 12 copies/µl to 10 copies/µl. Standard curves were plotted with related copy 13 numbers, and the reproducibility of the assay for RV genome detection was 14 quantified in triplicate using tissue culture fluid samples of four RV isolates ZJ1-4 15 (TableⅡ).

  assay (PCR-E860) was developed initially at the Robert 19 Koch-Institut. The primers for the first round, Ru1r (5'-CTG CCG GGT CTC CGA 20 CAC AGC) and Ru2f (5'-GAC ACC GTG ATG AGC GTG TTC), and for the 21 nested round, Ru3r (5'-GTG GTG TGT GTG CCA TAC AC) and Ru4f (5'-CTT 22 GCT AGC TAC GTC CAG CAC) generate a 860nt fragment of RV E1 gene 23 (nt8687-9546, Genbank accession number L78917), covering the 739nt window 24 (E1 nt8731-9469) as recommended by the WHO for genotyping. In a total 25 per reaction, 2.5µl (Robert Koch-Institut, Centre for Disease 1 Control and Prevention of Zhejiang Province, China) or 10µl (Health Protection 2 Agency) cDNA was added as the template for the first round PCR. 2µL (Robert 3 Koch-Institut and Centre for Disease Control and Prevention of Zhejiang 4 Province, China) or 4µl (Health Protection Agency) of the first round amplicon 5 was used for the nested PCR-E860 assay. The PCR amplification conditions for 6 both first and second rounds are shown in TableⅠ.

9

  The products of Rtime-SGE317 and the nested PCR-E860 were purified using 10 the MinElute PCR Purification Kit (Qiagen, Germany) and then sequenced by 11 commercial companies (Invitrogen Com Ltd, Shanghai, China; MWG, Germany).12 The primers E1.7 and E1.8 for the amplicons of the Rtime-SGE317 and primers 13 Ru3r and Ru4f for the amplicons of the nested PCR-E860 were used for 14 sequencing. The phylogenetic trees were constructed using DNAStar software 15 on the basis of either the 280nt or 739nt sequences. removed from infected Vero cells and the cells were washed 19 gently with PBS. The PBS was removed and 0.25%Typsin-EDTA was added so 20 as to just cover the cell monolayer and left for 30 seconds. After a low speed spin, 21 the cells were re-suspended in PBS to the required cell density. The cells were 22 fixed on slides at -20℃ for 20 min with methanol and acetone (1:1), then 1:100 23 dilution mouse monoclonal antibodies containing anti-rubella capsid protein C 24 (provided by CDC, USA) and anti-structural glycoprotein E1(gifted by Dr. D , UK) was added and incubated at 37 o C for 20min. After 1 washing with PBS and deionised water, 1:20 diluted FITC-goat anti-mouse 2 IgG+M+A antibody (Invitrogen) was applied for 20min at 37°C. Negative and 3 positive control slides were prepared and observed at the same time according 4 to the WHO manual [WHO, 2007].

7

  Rubella is a fragile virus morphologically. Therefore, cultures were fixed prior to 8 processing, by adding Carson's buffered formaldehyde (Bios Europe Ltd) to give 9 a final concentration of 1.5% (v/v) formaldehyde in the culture medium, and 10 stored at 4oC until examined. Cells were scraped into the medium and the 11 mixture of cells and tissue culture fluid was centrifuged at 48000 x g for 45min.

2

  14 drop of suspension was stained with phosphotungstic acid (PTA) pH6.8 and 15 placed on a formvar-carbon coated EM grid. Grids were examined in a Philips 16 420 transmission electron microscope fitted with an AMT XR60 digital imaging The Rtime-SGE317 was developed to replace the probe-based real-time PCR 3 assay used previously [Jin and Thomas, 2007]. The new assay had the 4 advantage of being rapid and specific for confirmation of RV isolates. Even 5 though the primer pair gave consistent results with different RV strains, 6 specificity of the assay was evaluated further with measles virus and B19 virus, 7 which cause similar symptoms to rubella. Measles cDNA and B19 DNA were 8 tested by Rtime-SGE317 and all samples were negative with no cross-reactions 9 (results are not shown). The time taken to perform the Rtime-SGE317 assay was 10 2.5 hours less than for the previous assays (TableⅠ). Sensitivity of the 11 Rtime-SGE317 was also compared with nested PCR-E317 and nested 12 PCR-E860. The Rtime-SGE317 detected 200 copies/reaction of plasmid DNA 13 (TableⅠ).

3

  fluid samples from four Chinese strains (ZJ1-4) were tested in 16 triplicate to evaluate the reproducibility of the assay. Table Ⅱshowed that the Ct 17 values differed by less than one within each sample, whereas the deviation 18 based on the copy numbers ranged from 4.6 to 18.3 times, e.g. strain ZJ1 and 19 ZJ3 (indicated in italic in TableⅡ). Correlation between Ct values and copy 20 numbers was plotted using the data collected from all samples and RV plasmid 21DNA tested in this study. The samples with Ct value >/=40 were defined as 22 negative, though high copy numbers up to ~10,000 copies were estimated 23 based on the standards (Figure1). Figure1shows that distribution of copy 24 numbers was much wider when Ct values were >20 or copy numbers <10 6 , Ct value was more stable and reliable for estimation of viral 1 load in cell cultures. 2 Comparison of the methods for confirmation of RV isolates 4 The tissue culture fluids collected from the first to fifth passage of two RV 5 isolates from CRS patients (H1 and U6) were tested by three methods, IFA, EM 6 and the Rtime-SGE317 (TableⅡ). Clinical specimens from the two cases of CRS 7 were inoculated and passaged up to four rounds (the harvest from the last one 8 named as the 5 th passage, P5). Specimens were incubated in Vero cells for 9 7days each passage, but specific CPE was not clearly apparent. The infected 10 cells collected from the 2 nd to 5 th passage were tested by IFA and EM. Weak 11 fluorescence was observed in the 2 nd passage. Fluorescence became stronger 12

19

  and the 3 rd passage of sample H1. Results for both Ct value and copy number 23 are shown in TableⅡ. The copy numbers continually increased with each 24 subsequent passage and were higher for the cells than the supernatant. The 25 sample U6 was much greater than in sample H1 in every passage, 1 which corresponds with the EM observations (TableⅡ).

  obtained at the Centre for Disease Control and Prevention of 6 Zhejiang Province by inoculating throat swab specimens into Vero cells. Four 7 isolates were passaged four times (P1 to P5) and two twice only (P1 to P3). The 8 presence of RV in each passage was confirmed by detecting the RV genome 9 using the Rtime-SGE317. The six isolates were confirmed as the Ct values were 10 <35.0 after three blind passages.

  17those based on the 280nt products generated by Rtime-SGE317 (Results not 18 shown).

  739nt window of the E1 sequences (Figure 3), five isolates were 21 assigned to genotype 1E. Isolates RVi/Zhejiang.CHN/15.05 (ZJ-1) and 22 RVi/Zhejiang.CHN/17.05 (ZJ-2) originating from two outbreaks in Quzhou and 23 Lishui city within two weeks of each other in 2005 were 100% identical. 24 RVi/Zhejiang.CHN/15.07/1 (ZJ-3) and RVi/Zhejiang.CHN/15.07/2 (ZJ-4) outbreak in 2007 were also identical in sequence to each other, 1 but differed by 14nt from the 2005 isolates. RVi/Zhejiang.CHN/22.08 (ZJ-5) 2 isolated in Hangzhou city in 2008, was more closely related to the genotype 1E 3 strain detected in Hong Kong in 2008 (RVs/HongKong.CHN/27.08-1E, GenBank 4 accession no. FJ436377). The only 2B strain RVi/Zhejiang.CHN/23.08 (ZJ-6) 5 was isolated in Lishui city in 2008, and is close (4nt differences) to the Hong 6 Kong genotype 2B isolate, RVs/HongKong.CHN/49.08-2B (GenBank accession 7 no.FJ656218).

  in a variety of continuous cell lines. RK13 and SIRC cells 2 have been used because specific CPE is readily observed. However, these cells 3 do not generate high titre virus[Figueiredo, et al., 2000, Rajasundari, et al., 4 2008]. The Vero/SLAM cell line is currently recommended by the WHO for 5 measles and rubella isolation in the Global Measles/Rubella Laboratory Network 6[WHO,2007]. Viral CPE is not visible in Vero/SLAM cells in most cases, even 7 after several passages. A previous study has shown that RV can replicate rapidly 8 and reach higher titres in Vero cells because they do not produce interferon 9[Figueiredo, et al., 2000]. Therefore, the Vero cell line was used throughout this 10 study. Similar to Vero/SLAM cells, specific CPE was not visible in Vero cells 11 normally. However, it was visible when inoculated with a viral concentration of 12 10 5 TCID 50 or higher[Guido et al., 1989]; or when the tissue culture fluid 13 samples of each passage harvested in the first attempt were inoculated again 14 simultaneously into fresh cells and observed in parallel which was carried out in 15 the study (result not shown). It can be difficult still to differentiate between the 16 CPE produced in infected cells and normal dead cells. Determination of RV 17 growth after cell culture based on CPE observation alone is unreliable and 18 subjective. Therefore, a simple method for confirmation of RV presence is , the Rtime-SGE317 assay was shown to be simple, rapid and 22 specific for confirmation of RV isolation (TableⅠ). It has been reported 23 previously that the primer pair of E1.7 and E1.8 is specific and sensitive when 24 used in a block-based nested PCR (PCR-E317) [Jin and Thomas, 2007]. The25

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19

  number for estimation of viral load. The large deviation between copy numbers 20 may be caused by several factors, such as errors occurring during the manual 21 dilution and concentration measurement of the plasmid standards. In addition, 22 risk of contamination is greater using plasmid DNA. This suggests that plasmid 23 DNA as standard may not be necessary when RV isolates are confirmed by 24 Rtime-SGE317, so avoiding the risk of contamination and reducing the cost and local or sub-national laboratories. By testing a serial dilution of RV 1 cDNA, it was shown that confirmation of a rubella isolate could be based on the 2 viral load when the Ct value was <37 and the amplicon is large enough for 3 sequencing the 280nt of the E1 gene. If the Ct value is between 37 and 40, the 4 shape of the amplification curve should be carefully checked to ensure that the 5 sample is a real isolate. Samples with Ct value >40 could be passaged further,6but there is no point in continuing beyond the fifth passage as numbers of 7 defective virus particles may increase. Products with Ct value <30 may be 8 directly sequenced without purification.

9 10

 9 Two other methods, IFA and EM, were compared with Rtime-SGE317 using the 11 two CRS isolates. IFA is a routine assay used at the Health Protection Agency

12

  and is recommended by WHO. IFA indicates viable virus in cells by green 13 florescence. It is a classic assay, but requires fluorescence microscope and 14 experienced personnel to use it. The whole IFA process takes at least four hours 15 and a further PCR assay to generate the RV genome from confirmed strains for 16 genotyping. The recently developed indirect immunocolorimetric assay (ICA) 17 has the same sensitivity as IFA, and RV infected cells can be detected as 18 blue-staining spots on monolayers by the naked eye [Zhu et al., 2007; Chen et 19 al., 2007]. However, it was less sensitive than diagnostic PCR and standardised 20 high quality colorimetric reagents are not available commercially. Electron 21 microscopy requires highly experienced staff, is time consuming and is not 22 particularly sensitive. Although it can give an indication of the morphological 23 state of the viral particles, it is not suitable for rapid testing of large numbers of 24 samples. Results of phylogenetic analysis of the six Chinese RV strains supported the 1 previous suggestion that the 280nt of E1 region can be used for preliminary 2 genetic classification [WHO, 2005], especially when the 739nt window is not 3 detectable. Based on the E1-739nt window, five of the six Chinese RV strains 4 belonged to genotype1E and one to 2B, and the two strains isolated in 2008 5 (ZJ5-6) were close to two Hong Kong 1E and 2B isolates respectively. However, 6 there was no information available to link the viral transmissions. The results 7 indicated two genotypes of RV strains were co-circulating and genotype 1E 8 could have been the dominant genotype in Zhejiang province between 2005 and 9 2008. The preliminary identified RV isolates could finally be confirmed 10 definitively by sequencing the product of the nested PCR-E860 assay, the 11 window E1-739nt, as recommended by WHO for RV genotyping [WHO, 2005].

  rubella IgM and IgG tests, tissue culture for RV isolation is routinely 14 carried out in most of provincial laboratories and some municipal CDC 15 laboratories in China as well as in many other countries in the WHO LabNet for 16

20

  Figueiredo CA, Oliveira de MI, Afonso AM, Curti SP. 2004. Infectivity of wild-type

Figure 1 . 4 Figure. 2 .

 142 Figure 1. Correlation between Ct and copy number when detecting RV isolates

Figure 3 .

 3 Figure 3. Genetic relationship amongst the eight RV isolates (in bold) and the

  Figure 1

7

  Omer A, Abdel Rahim E.-H, Ali E.-E, Jin L. 2010. Primary Investigation of 31 8 infants with suspected congenital rubella syndrome in Sudan. Clin Micro and Rajasundari TA, Sundaresan P, Vijayalakshmi P, Brown DWG, Jin L. 2008. syndrome in the western hemisphere, 2003-2008. Wkly. Epidemiol Rec. Zhao LH, Ma Y, Wang H, Zhao SP, Zhao WM, et al. 2006. Establishment and Zhu Z, Xu W, Abernathy ES, Chen MH, Zheng Q, et. al. 2007. Comparison of

	1
		83:395-400.
	3
	4	Application of a TaqMan Real-Time Quantitative Reverse Transcription
	5	Polymerase Chain Reaction Assay for Rubella Virus RNA. Acta Biochimica et
		Biophysica Sinica 38:731-736.
	7
	9 8	Infect 16:678-682. four Methods using throat swabs to confirm rubella virus infection. J Clin F r o Microbiol 45: 2847-2852.
	11	P
	12	Laboratory confirmation of congenital rubella syndrome in infants: an eye e
	13	hospital based investigation. J. Med. Virol 80:536-546. e r
	14 15 16 17 18 19	Robert J, Pryor and Carl T. 2009. Real-Time Polymerase Chain Reaction and Melting Curve Analysis. In: Method in Molecular Biology Vol 336, Clinical R Application of PCR, 2 edition. Eds Y. M. Dennis Lo, Rossa W. K. Chiu & e K. C. Allen Chan 336:19-32. Vyse AJ, Jin L. 2002. PCR assay using oral fluid samples to detect rubella virus v i e genome for epidemiology surveillance. Mol Cell Probes 16:93-97. w
	20	WHO. 2000. Rubella vaccine. Wkly. Epidemiol Rec. 75:161-172.
	21	WHO. 2005. Standardization of the nomenclature for genetic characteristisation
	22	of wild-type rubella viruses. Weekly Epidemiol Rec. 80:126-132.
		WHO. 2007. Manual for the laboratory diagnosis of measles and rubella virus

10 2 6

the Rtime-SGE317, the Nested PCR-E860 and the Nested PCR-E317 assays

	Table Ⅰ Ⅰ Ⅰ Ⅰ Table Ⅱ Ⅱ Ⅱ Ⅱ
	Comparison among Method Confirmation	Rtime-SGE317	Nested PCR-E860	Nested PCR-E317
	Item
	Primer pair (100pmol/ul)	E1.7 and E1.8		Ru1r and Ru2f Ru3r and Ru4f	E1.5 and E1.6 E1.7 and E1.8
			50°C 2min; 95°C15min
			94°C 15sec
			55°C 20sec	45Cycles	96 o C 1 min
	Programme	72°C 20sec 82°C 5sec Single 95°C 0sec		94 o C 30 sec 55 o C 30 sec 68 o C 90 sec	35 Cycles	Nested PCR [Jin and Thomas, 2007]
			65°C 1min	Melting	68 o C 5 min
			95°C 0sec Cont
			40°C 30sec
		Plasmid DNA	200 copies/reaction	Not done	2 copies/reaction
	Sensitivity	Virus (Thomas) cDNA dilutions	10 -3		10 -2	10 -4
	Time for the process	1.5h		>4h	>4h
	Suggested application	Confirmation of RV isolates	Genotyping of RV isolates	Confirmation of clinical samples

and quantitative detection of RV isolates in Vero cell culture

	Sample (Type)	Method		1 st passage (inoculation)	2 nd passage	3 rd passage	4 th passage	5 th passage	Strain name GB accession No.
		IFA		Not tested	Pos (2 a )	Pos (30%)	Pos (40%)	Pos (60%)
	H1 (NS)	EM Rtime-SG E317	Sup	Not tested Neg	Not detected Neg	occasional single particles and small groups 4,000/35.2 24,400/36.6	some large groups Not tested 67,100/33.7	some large groups 17,900/32.4 80,800/33.1	RVi/Deweim.SD N/24.05[1E]CRS FJ775000
		(Copy no./Ct)	Cells	Neg	Neg 9,020/39.5	6,850/34.2 91,900/32.8	Not tested 180,000/30.8	43,200/30.7 199,000/30.5
		IFA		Not tested	Pos (4ª)	Pos (30%)	Pos (40%)	Pos (50%)
						Single particles,		Mix of more
		EM		Not tested	Not detected	pairs and small	several groups	large and few
						groups		small groups
			Sup	19,300/32.2 Not tested

John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology

Supernatant and cells of RV isolates were each tested quantitatively using the 21 Rtime-SGE317. RV genome was detected from the 1 st passage of sample U6


Dimitra ; Koukou

Ioanna Grivea

Eleftheria Roma

Heleni Tsioni

Georgios Trimis

Emmanuel Galanakis

Evagelia Farmaki

Elias Iosifidis

Athanassios Michos

Antigoni Siamopoulou-Mavridou

Maria Kalmanti

Heleni Papadopoulou

Emmanuel Roilides

Maria Theodoridou

George A Syrogiannopoulos

M.D. Vassiliki Syriopoulou

Greek Rotascore

"Frequency, clinical characteristics and genotype distribution of rotavirus gastroenteritis in Greece (2007-2008)"

provided the test kits of rotavirus antigen stool detection.

 (2007)(2008)in 5 Pediatric Hospitals to determine the prevalence, the clinical characteristics, and genotype distribution of rotavirus infection in Greece. Faecal samples were examined for the presence of group A rotavirus antigen by immunochromatography. Rotavirus strains were subjected to G and P genotyping by reverse-transcriptase polymerase chain reaction (PCR) and sequencing. A total of 393 children (216 boys) of median age 23 months, participated in the study. Rotavirus was the cause of acute gastroenteritis in 166 children, 42.3% (CI 95%, 37.4-47.1%) of non-hospitalized and 47.8% (CI 95%, 41.7-53.9%) of hospitalized patients. Rotavirus gastroenteritis occurred between December and April in 78.6% of the cases. Most children with RVG (77.8%) were between 3 months and 3 years old. The mean value of Clark severity score was 12.9±5.1 for RVG and 10.5±4.9 for non-RVG (p<0.01). Genotypes were determined in 117 strains and their distribution was as following: G1P[8], 49%; G2P[4], 31%; G4P[8], 10%; G9P[8], 9%; and G8P[14], 1%. Conclusion. Rotavirus is a frequent cause of acute gastroenteritis in Greece. The genotypes circulating are similar with those of other European countries.

KEY WORDS: epidemiological study; acute gastroenteritis; rotavirus; genotypes

INTRODUCTION

Rotavirus Gastroenteritis is the leading cause of acute gastroenteritis in children aged ≤ 5 years worldwide. Although the prevalence of the disease is similar in most geographic regions, the mortality is greater in developing countries due to malnutrition and poorer access to medical treatment [START_REF] Butz | Prevalence of rotavirus on high-risk fomites in day-care facilities[END_REF][START_REF] Cook | Global seasonality of rotavirus infections[END_REF][START_REF] Giaquinto | Clinical Consequences of Rotavirus Acute Gastroenteritis in Europe, 2004-2005: The REVEAL Study[END_REF]Kavaliotis et al., 2007;[START_REF] Leung | Rotavirus gastroenteritis[END_REF][START_REF] Parashar | Global illness and deaths caused by rotavirus disease in children[END_REF][START_REF] Raebel | Rotavirus disease and its prevention in infants and children[END_REF][START_REF] Soriano-Gabarró | Burden of rotavirus disease in European Union countries[END_REF]Van Damme et al., 2007a].

Rotaviruses are classified into G and P genotypes on the basis of the sequence diversities of the two outer layer proteins VP7 and VP4 respectively which are the two viral proteins that elicit neutralizing antibody responses [START_REF] Parashar | Global illness and deaths caused by rotavirus disease in children[END_REF][START_REF] Raebel | Rotavirus disease and its prevention in infants and children[END_REF]

. Although genotypes G1P[8], G2P[4], G3[P8], G4 [P8] and G9[P8]
are the predominant genotypes of the human strains circulating in most European countries, genotype variations have been observed among different geographic areas [START_REF] Iturriza-Gómara | Rotavirus surveillance in Europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data[END_REF]. The aim of the present study was to examine the epidemiology of rotavirus infection, the clinical characteristics and the genotype distribution in Greek children younger than 5 years of age.

MATERIAL AND METHODS

The present study was conducted in five Paediatric Hospitals located in different geographic areas of Greece (Athens, Thessaloniki, Larissa, Ioannina and Heraklion) between January 2007 and June 2008. These Hospitals cover nearly 65% of Greek pediatric population (Athens Hospital alone covers the 40%); however vast majority of mild to moderate and many severe cases of acute gastroenteritis are treated from primary care Pediatricians. Consecutive children aged ≤ 5 years who presented to the emergency room with symptoms of acute gastroenteritis were evaluated. Disease scoring system concerning the presence, intensity and duration of fever, vomiting, diarrhea, and changes in behaviour due to dehydration [START_REF] Rosenfeldt | Viral etiology and incidence of acute gastroenteritis in young children attending day-care centers[END_REF]. Stool examination was performed in children after parents gave a written informed consent to participate in the study. No parents refused. Ethics Committee provided its approval prior to study initiation.

A rapid stool immunochromatographic test for rotavirus antigen detection (test VIKIA, BioMérieux) was used. Fecal samples tested within 24 hours and those positive for Rotavirus Group A antigen were stored at 4ºC. Viral RNA was extracted from 10% faecal suspensions in balanced salt solution with the guanidinium isothiocyanate/silica method [START_REF] Boom | Rapid and simple method for the purification of nucleic acids[END_REF]. Complementary DNA was synthesized from the extracted viral RNA using reverse transcriptase enzyme in the presence of random hexamers [START_REF] Iturriza-Gómara | Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses[END_REF] and was further characterised by semi-nested multiplex polymerase chain reaction (PCR) using specific primers for the VP7 and VP4 genes to identify the G and P types respectively [START_REF] Gentsch | Identification of group A rotavirus gene 4 types by polymerase chain reaction[END_REF][START_REF] Gouvea | Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens[END_REF]Iturriza-Gómara et al., 2000a;[START_REF] Iturriza-Gómara | Rotavirus Genotyping: Keeping up with an evolving population of human rotaviruses[END_REF][START_REF] Simmonds | New oligonucleotide primers for P-typing of rotavirus strains: Strategies for typing previously untypeable strains[END_REF]. Amplification products were analysed by electrophoresis using a 2% agarose gel stained with ethidium bromide in TBE buffer. Specific segment sizes were visualised under ultraviolet light, and rotavirus genotypes were determined. For the strains which were not P typable by second round multiplex PCR, the first round PCR products were sequenced using the VP4 primers.

Sequencing was also carried out for PCR products of VP7 gene in one strain that displayed the G12 type. Sequencing was performed in a collaborating laboratory (Biogenomica, Athens, Greece) and the results were analyzed with the Bioedit Behavioral changes (irritability, hypotonia), signs of dehydration, weight loss, fever ≥38 0 C, vomiting, duration of diarrhea were more prevalent in rotavirus gastroenteritis than in non rotavirus gastroenteritis (p<0.01). Most children suffered from moderate (91.7% for rotavirus gastroenteritis vs 92.4% for non rotavirus acute gastroenteritis) or severe (7.1% for rotavirus gastroenteritis vs 6.1% for non rotavirus acute gastroenteritis) gastroenteritis (9-16 points and 17-24 points respectively). The mean value of severity was 12.9±5.1 for rotavirus gastroenteritis and 10.5±4.9 for non rotavirus acute gastroenteritis (p <0.01).

Children with rotavirus gastroenteritis as compared to those with non-rotavirus acute gastroenteritis were more likely to need clinical reevaluation during the course

DISCUSSION

The clinical burden of rotavirus gastroenteritis in Greece has a similar profile with that observed in other European countries. Rotavirus is the most common cause of acute gastroenteritis in children up to 5 years old in Greece (main risk period between 3 months and 3 years old), it has a greater clinical burden compared to non rotavirus acute gastroenteritis, and it occurs more frequently in winter months [START_REF] Parashar | Global illness and deaths caused by rotavirus disease in children[END_REF][START_REF] Soriano-Gabarró | Burden of rotavirus disease in European Union countries[END_REF]Van Damme et al., 2007a] In this report, the preliminary results of the first multicenter Hellenic study to determine the rotavirus genotypes circulating in Greece are presented. The predominant genotype was the G1P[8] which is the commonest worldwide, followed by G2P[4], G4P[8], G9P[8] that represent 99% of the circulating genotypes in this geographic region and are together with the G3P[8] the 5 commonest in Europe and worldwide [START_REF] Iturriza-Gómara | Rotavirus surveillance in Europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data[END_REF]Van Damme et al., 2007b]. Similarly with other countries [START_REF] Iturriza-Gómara | Rotavirus surveillance in Europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data[END_REF], some genotype variation was observed across the country; G2P[4] was the predominant genotype in southern and northern Greece (Thessaloniki, Heraklion) and G1P[8] in central Greece (Athens, Larissa).

Mismatches between the P[8] specific primer and its complementary region of the VP4 cDNA in the P[8] isolates would explain the failure of the typing RT-PCR of 15 strains. Of note, one strain was identified as P[14] and was closely related to human Rotavirus A P[14] strains isolated in Italy (PR/1300/04) and Hungary (Hun5) (maximum identity at nucleotide level 97% and 96% respectively) [START_REF] Medici | Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy[END_REF]. The VP7 gene of the same strain was identified as G8 and displayed high similarity to the Egyptian human Rotavirus A strain EGY1850 (maximum identity at nucleotide level 96%), while the sequence analysis of the VP6 gene identified it as subgroup I and displayed high nucleotide identity (97%) to the Slovenian Human Rotavirus A strain SI-R241/07 [START_REF] Holmes | Characterization of unusual G8 rotavirus strains isolated from Egyptian children[END_REF]. This uncommon strain G8P [14] was detected from a non-hospitalized child living in a rural region of Larisa who may have close contact with animals, and is possibly the result of reassortment between human and animal strains during coinfection [START_REF] Holmes | Characterization of unusual G8 rotavirus strains isolated from Egyptian children[END_REF][START_REF] Medici | Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy[END_REF][START_REF] Santos | Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine[END_REF]. There are few reports that associate P[14] with G types in humans, because P[14] is a rotavirus type that has been detected mostly in animals [START_REF] Holmes | Characterization of unusual G8 rotavirus strains isolated from Egyptian children[END_REF][START_REF] Matthijnssens | Are human P[14] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order Artiodactyla?[END_REF][START_REF] Medici | Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy[END_REF] , 2005]. G8 is a rotavirus type that can be found in cows at relatively high frequency and presents a great diversity of G-P combinations [START_REF] Santos | Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine[END_REF] In the previous decades human G8 was circulating mostly in tropical regions like Indonesia, Brazil and Africa and sporadically in some European countries like Finland, Italy and United Kingdom [Holmes et al. 1992]. Recently has reported mistyping of G8 human strains as G12 due to point mutations at the G8 specific primer binding site and the G8 and G12 primers were necessarily redesigned [START_REF] Aladin | Identification of G8 rotavirus strains determined as G12 by rotavirus genotyping PCR: updating the current genotyping methods[END_REF]. Interspecies transmission events and genome reassortment are frequently reported in the evolution history of rotavirus and highlight the role of animals as a source of rotavirus infection in humans [Matthijnssens et al., 2008a]. Sequencing of its genome and adopting a new classification system are recommended to generate reliable data and elucidate the rotavirus evolutionary patterns [Matthijnssens et al., 2008b].

Rotavirus strains circulating in Greece are well covered by current vaccines.

However, as there is seasonal fluctuation, next years results will help to form a more representative picture for rotavirus genotypes prevalence in Greece. Surveillance studies are necessary to monitor the changes in strain distribution during the pre-and post-vaccine era and to detect the emergence of antibody escape mutants that may occur after implementation of rotavirus vaccines [START_REF] Iturriza-Gómara | Rotavirus surveillance in Europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data[END_REF]Iturriza-Gómara et al., 2000b]. Although there is a limitation from the small sample size, results confirm that there is not a constant different distribution of rotavirus genotypes in non hospitalized compared to hospitalized patients with rotavirus gastroenteritis, either a different distribution in age groups, except neonates (not any case from this group in this study) [START_REF] Parashar | Global illness and deaths caused by rotavirus disease in children[END_REF]. [START_REF] Vesikari | European Society for Paediatric Gastroenterology, Hepatology and Nutrition/ European Society for Paediatric Infectious Diseases Evidence -based Recommendations for Rotavirus Vaccination in Europe[END_REF].

Rotavirus gastroenteritis has a major clinical burden in children between 3 months and 3 years old, a period where rotavirus vaccines have proven their efficacy against disease related to the serotypes that seem prevalent in Greece. This study supports the need for implementation of routine rotavirus vaccination program in Greece in order to reduce rotavirus gastroenteritis clinical burden and the associated cost for health care system.

  leading cause of acute gastroenteritis among young children worldwide. A prospective multi-center study was conducted

  by 24-point severity scale (Clark's scale), based on a clinical

  aligned with the BLAST nucleotide search [http://blast.ncbi.nlm.nih.gov/Blast.cgi, 2010]. RESULTS A total of 393 children of median age 23 months (range 2 months-5 years) participated in the study; 139 from Athens, 75 from Larissa, 71 from Ioannina, 57 from Heraklion, and 51 from Thessaloniki. Boys outnumbered girls by a ratio of 1.2 (216/180). The proportion of rotavirus gastroenteritis per geographic region is presented in Fig. 1. Rotavirus was the cause of acute gastroenteritis in 166 children, 42.3% (CI 95%, 37.4-47.1%) of non-hospitalized and 47.8% (CI 95%, 41.7-53.9%) of hospitalized patients. The 78.6% of rotavirus gastroenteritis occurred in winter months, between December and April. Most children with rotavirus gastroenteritis (77.8%) were between 3 months and 3 years old. The median age of rotavirus gastroenteritis diagnosis was 22 months (mean 20 ±12 months), while the median age of non-rotavirus acute gastroenteritis was 25 months (mean 22±11 months).

  (85.5 vs 74.9%, p<0.05), get hospitalized (72.9% vs 60.8%, p<0.05), have greater hospitalization time (median 4 vs 3 days, mean 4.2±2.7 vs 4±2.4 days) and more parents' days of absence from work (median 4 vs 3 days, mean 4.5±2.9 vs 3.8±2.6 days, p<0.05). No patient with rotavirus gastroenteritis had received any rotavirus vaccine. One hundred seventeen of the 166 rotavirus gastroenteritis positive samples (70%) were available for genotyping. The other 49 rotavirus gastroenteritis positive samples were not properly stored or the quantity was not adequate. All of the analyzed strains were successfully G typed by PCR. Sequence analysis of the VP4 gene was carried out for 15 strains that could not be P typed by PCR. Fourteen of those strains were classified as P[8] and one strain as P[14]. The last strain that displayed the unusual G12P[14] type was further analyzed by sequencing the VP7 and VP6 gene and was finally identified as G8P[14] group A rotavirus subgroup I. Two mutations were detected at nt 1 and 8 from the primes's 3' end of VP4 gene (C:A and T:G, respectively). Alignment of the VP7 sequencing demonstrated 3 base-pair mismatches at the positions nt 1, 10 and 15 from the primers' 3' end, which prevented G8 specific primer binding to the G8 primer binding site, whereas the great homology of the G12 primer with the corresponding binding site of the VP7 sequence resulted to the G12 mistype. One hundred sixteen of the 117 (99%) strains belonged to the most common genotypes that circulate in Europe during the last years [10,20], while 1 strain (1%) belonged to an uncommon genotype, G8P[14]. The distribution of the genotypes across the country (G1P[8] 49%, G2P[4] 31%, G4P[8] 10%, G9P[8] 9% and G8P[14]1%) and differences in genotype distribution by geographic region are shown in Fig.2

  B). No association was observed between genotypes and patients' age, severity of disease, and hospitalization rate.

  . Rotavirus gastroenteritis is responsible for nearly half of the cases visiting emergency clinics or requiring hospitalization due to acute gastroenteritis in Greek children up to 5 years of age (42.3% and 47.8% respectively); prevalence that is close to that reported in other studies conducted worldwide and in a previous study held in Greece (Kavaliotis et al., 2007; Soriano-Gabarró et al., 2006). However, Levidiotou et al, reported a lower prevalence (21.35%) of rotavirus infection in hospitalized children in Northwest Greece (Levidiotou et al., 2009). The different results obtained by the latter group may reflect differences in the screened population (different years and geographic regions) as well as in the detection method.Factors such as hospitalizations, emergency visits, increased mean hospitalization time (4.2 days), lost working days from parents (4.5 days as a mean) indicate social and economic cost, except from increased burden of Health System and suffering of children and their families. A limitation of the present study is that nosocomial infections, where rotavirus is the leading cause of acute gastroenteritis[START_REF] Parashar | Global illness and deaths caused by rotavirus disease in children[END_REF], cases visiting pediatric offices in community and domiciliary episodes are not included.

  diminished rotavirus activity in other countries; while, especially in USA, herd immunity has been demonstrated [CDC, 2008]. Medical societies, as ESPID and ESPGHAN, recommend introduction of routine infant rotavirus vaccination in European countries as a cost effective means of rotavirus gastroenteritis impact on society

ACKNOWLEDGEMENTS

We would like to thank all the members of the Greek Rotascore Extension Study Group, for their important contribution to the implementation of the epidemiological study.

We would like also to thank pharmaceutical companies VIANEX, Athens-Greece and Sanofi Pasteur MSD, Lyon-France for providing the test kits of rotavirus antigen stool detection.

John Wiley & Sons Journal of Medical Virology John Wiley & Sons Journal of Medical Virology


Angelina Plyusnina

Cvetko Lidija

Josip Krajinović

Jukka Margaletic

Kirill Niemimaa

Åke Nemirov

Alemka Lundkvist

Marica Miletić Marcotić

Tatjana Medved

Heikki Avšič-Županc

Henttonen

Åke ; Lundkvist

Kurkino38 Kurkino44

Croatia56 Greea

Kurkino44 Omskaa180 Omskaa189

Slovakia856 Sochiap1584

Lidija Cvetko Krajinovic

Josip Margaletic

Jukka Niemimaa

Kirill Nemirov

Alemka Markotic

Marica Miletic Medved

Tatjana Avšic Županc

Heikki Henttonen

Alexander Plyusnin
email: alexander.plyusnin@helsinki.fi

GENETIC EVIDENCE FOR THE PRESENCE OF TWO DISTINCT HANTAVIRUSES ASSOCIATED WITH APODEMUS MICE IN CROATIA AND ANALYSIS OF LOCAL STRAINS

Keywords: HFRS , hantavirus , Dobrava virus, Saaremaa virus HFRS, hantavirus, Dobrava virus, Saaremaa virus. 47

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three 29 hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice 30 trapped in [2003][2004] were screened for the presence of hantaviral N-Ag and 20 mice 31 (6.3%) were found either strongly positive or weak/suspected-positive. Partial 32 sequences of hantavirus M and S segments were recovered by RT-PCR from six mice 33 and subjected to (phylo)genetic analysis that revealed the presence of four novel 34 strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV 35 strains was found in A. agrarius mouse, i.e. not in the traditional host, A. flavicollis 36 mice, suggesting a spillover event. S segment sequences recovered previously from 37 HFRS cases (Markotic et al., 2002) were confirmed as DOBV sequences; one of which

64

[for a reviews, see [START_REF] Vapalahti | 457 Hantavirus infections in Europe (Review)[END_REF][START_REF] Schmaljohn | Hantaviruses: a global disease problem[END_REF]. Very little is 65 known about hantaviruses in Africa [Klempa et al., 2006[Klempa et al., , 2007] ] and nothing is 66 known about these viruses in Australia. [START_REF] Papa | Retrospective serologic and genetic 403 study of the distribution of hantaviruses in Greece[END_REF][START_REF] Avšic-Županc | 294 Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia-A 10-295 year survey[END_REF]Markotic et al., 2002;Jakab et al., 2007]. The 72 natural host for SAAV is the striped field mouse (Apodemus agrarius); the virus 73 causes mild HFRS in the Baltics, Central Europe and the European part of Russia 74 [Lundkvist et al., 1998;[START_REF] Plyusnin | 419 Dobrava hantavirus in Russia[END_REF]Golovljova et al., 2000;Sibold et al., 75 2001;Klempa et al., 2004Klempa et al., , 2008]]. It should be noted that SAAV was considered 76 initially a genetic variant of DOBV carried by A. agrarius [START_REF] Plyusnin | Dobrava 415 hantavirus in Estonia: does the virus exist throughout Europe?[END_REF]77 Nemirov et al., 1999] and for a while there was a controversy in the taxonomy and 78 terminology of SAAV. The virus is now recognized as a distinct species [for the 79 latest taxonomy, see www.ictvonline.org] but, in some publications, it is still named 80 "DOBV-Aa".

81

PUUV is carried by bank voles (Myodes glareolus) and causes HFRS, usualy 82 mild, in Northern and Central Europe, the Baltics, European part of Russia and 83 Alpe-Adria [START_REF] Settergren | Clinical characteristics 429 of nephropathia epidemica in Sweden: prospective study of 74 cases[END_REF], Mustonen et al., 1994;Tkachenko et al., 1998; 84 Golovljova et al., 2000Golovljova et al., , 2007;;Heyman et al., 2001;Markotic et al., 2002;Cvetko et al., 85 2005;[START_REF] Avšic-Županc | 303 Puumala hantavirus in Slovenia: analyses of S and M segment sequences 304 recovered from patients and rodents[END_REF]Hoffmann et al., 2008].

86

The whole of Croatia except costal region and islands is endemic for HFRS.

87

Serological and genetic findings in HFRS patients provided evidence that PUUV and 88 Dobrava virus (DOBV) have been two major pathogenic hantaviruses circulating in 89 Croatia and causing hantavirus infections in humans [Markotic et al. 2002, Cvetko et 90 al., 2005]. Apathogenic Tula virus was detected in wild rodents in Croatia as well 91 [START_REF] Scharninghausen | 433 Genetic evidence for tula virus in Microtus arvalis and Microtus agrestis populations 434 in Croatia[END_REF]. Croatia borders Slovenia to the northwest and has an 92 extended border with Bosnia and Herzegovina, one of the largest HFRS regions in 93 Europe [Markotic et al., 1995;Hukic et al., 2009]. Nowhere else is the co-existence of [Markotic et al., 2002;Cvetko et al., 2007;Lundkvist et al., 1997;[START_REF] Avšic-Županc | 294 Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia-A 10-295 year survey[END_REF].

96

During the HFRS outbreak in Croatia in 2002 when more than 400 patients were 97 serologically confirmed as HFRS positive, PUUV was shown to be dominant 98 pathogen [Miletic-Medved et al. 2002;Kuzman et al. 2003].

99

Phylogenetic analysis of wild-type (wt) PUUV strains from the 2002-outbreak 100 area showed that they clustered together with the strains from Slovenia and Austria 101 forming distinct Alpe-Adrian genetic lineage [START_REF] Cvetko | HFRS and Puumala virus in Croatia 308 during 2002 outbreak[END_REF]. Most recently,

102

Hungarian PUUV strains were added to this lineage [START_REF] Plyusnina | Co-circulation of three pathogenic hantaviruses: Puumala, 423 Dobrava and Saaremaa hantaviruses in Hungary[END_REF]. PUUV in Croatia, both in rodents and in association with human HFRS cases 245 [Markotic et al., 2002[START_REF] Cvetko | HFRS and Puumala virus in Croatia 308 during 2002 outbreak[END_REF], the data of (phylo)genetic analyses 246 suggested co-circulation of three pathogenic hantaviruses in this country (the 247 evidence of apathogenic Tula virus was earlier recorded as well [Scharninghausen et 248 al., 2002]). Thus, in that respect, Croatia resembles neighbouring Slovenia and 249 Hungary and also Slovakia where different hantaviruses have been found within a 250 relatively small territory [START_REF] Avšic-Županc | Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of 299 two distinct genetic lineages within the same natural focus[END_REF][START_REF] Avšic-Županc | 303 Puumala hantavirus in Slovenia: analyses of S and M segment sequences 304 recovered from patients and rodents[END_REF][START_REF] Plyusnina | Co-circulation of three pathogenic hantaviruses: Puumala, 423 Dobrava and Saaremaa hantaviruses in Hungary[END_REF] 251 [START_REF] Sibold | Short report: simultaneous occurrence of Dobrava, Puumala, 443 and Tula hantaviruses in Slovakia[END_REF][START_REF] Vapalahti | Isolation of Dobrava virus from A. flavicollis in Greece[END_REF]. So far, not a single SAAV genome sequence has been

38

  appeared particularly close to the prototype Slovenian DOBV isolate. Taken together 39 with earlier data on PUUV in Croatia, these results show a co-circulation of three 40 European hantavirus pathogens in this country. So far, not a single SAAV sequence 41 has been recovered from HFRS patients either in Croatia or neighbouring Slovenia 42 and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV 43 infection in humans in this part of Europe than for example in the Baltics. Hantavirus genus in the family Bunyaviridae 50 [Nichol et al., 2005]. They are enveloped viruses with tri-segmented RNA genome of 51 negative polarity. Small (S), Medium (M) and Large (L) RNA segments encode, 52 respectively, nucleocapsid (N) protein, a precursor for two surface glycoproteins Gn 53 and Gc, and the RNA-dependent RNA polymerase (L protein). In some 54 hantaviruses, the S segment encodes also the nonstructural protein NSs. The 55 function(s) of this protein in the virus replication cycle remain to be determined but 56 there is an evidence that it can counteract an interferon response [Jääskeläinen et al., 57 2007]. In nature, hantaviruses are maintained in rodents or insectivores infected 58 persistently. When transmitted to humans, usually via aerosolized excreta, some 59 rodent-borne hantaviruses can cause disease. In Europe, Dobrava-Belgrade (DOBV), 60 Saaremaa (SAAV) and Puumala (PUUV) viruses cause hemorrhagic fever with renal 61 syndrome (HFRS) of varying severity. In Asia, the main causative agents of HFRS 62 are Hantaan and Seoul (SEOV) viruses. In the Americas, Sin Nombre, Andes and 63 related viruses are associated with hantavirus (cardio)pulmonary syndrome (HCPS)

94

  PUUV and DOBV as obvious as in Croatia, Bosnia and Herzegovina and Slovenia

103Finnish

  The knowledge on DOBV and SAAV genetic variants in Croatia is still 104 restricted to partial S segment sequences recovered from three HFRS patients 105[Markotic et al., 2002]. To fill this gap, we attempted to recover and analyze DOBV 106 and SAAV sequences from carrier rodents, Apodenus mice, trapped during field 107 expeditions organized by the Faculty of Forestry, University of Zagreb and the 108 . Rodents were collected during two expeditions, in north-113 east Croatia in May 2003 and the south-west parts of Croatia in September, 2004 at 114 the following locations: Migalovci, Merolino, Loze, Nova Gradiška, Gratac/Mala 115 Kapela, and Delnice/Mala Kapela. Animals were trapped with snap traps, set in the 116 evening and checked early in the morning. Animals were placed in cooler box in the 117 field and kept subsequently in a refrigerator until dissected on the same day. Lung 118 tissue samples were taken from all animals and preserved in RNAlater reagent 119 (Ambion, Applied Biosystems, Carlsbad, CA, USA) for genetic analyses. Also blood 120 and serum samples were taken for further serological/epidemiological analyses by 121 pressing the heart on filter paper. 122 Screening of rodent samples. Lung tissue samples were screened first for 123 hantavirus nucleocapsid (N) protein antigen (N-Ag) by immuno(Western)blotting 124 (WB) as described earlier [Plyusnin et al., 1995]. In brief, approximately 100 mg of 125 tissue was placed into 400-500 µl of Laemmli sample buffer and homogenized by 126 sonication. Aliquots of 10 µl were separated by electrophoresis in 10% sodium 127 dodecyl sulphate-polyacrylamide gels and then immunoblotted with rabbit 128 polyclonal antibody raised against DOBV. Swine anti-rabbit antibodies conjugated 129 with the horse radish peroxidase (Dako, Glostrup, Denmark) were used as 130 secondary antibodies.

DOBV

  . In May, 2003, 362 rodents were trapped. Of those, 252 165 were Apodemus mice. In the field, 18 animals were classified preliminarily as A. 166 agrarius, 66 as A. flavicollis and 151 as A. sylvaticus. Of 245 small mammals trapped in 167 September 2004, 80 were classified as Apodemus mice. These included 75 yellow-168 necked mice (A. flavicollis) and five wood mice (A. sylvaticus). All 332 Apodemus mice 169 were subjected to the WB-screening for hantaviral N-Ag and 20 mice (6.3%) were 170 found either strongly positive or weak/suspected-positive. These mice, two A. 171 agrarius, six A. flavicollis, and 12 A. sylvaticus, were trapped at four localities: sequence identity to SAAV strains was substantially lower: 81-84%: 187 Consequently, the corresponding wild-type (wt) strains were designated as 188 was represented by a single sequence recovered from the 195 sample #12 of the May-2003 set. This sequence was more closely related to SAAV 196 sequences originated from Hungary, Slovakia, Central Russia and Estonia (identity 197 of 90-97%) than to DOBV sequences (81-84%). The corresponding wt strain was 198 designated as SAAV/Croatia_Migalovci/Aa12/2003, or SAAV/Croatia12 for short. 199 Phylogenetic analysis of the partial M segment sequences confirmed this 200 grouping (Fig. 1). All new Croatian DOBV strains clustered together with other 201 DOBV strains and shared with them a well-supported common ancestor. In 202 contrast, the new Croatian SAAV strain appeared monophyletic with other SAAV 203 strains. Within the SAAV cluster, the Croatian strain shared the most recent 204 common ancestor (TMRCA) with the Hungarian strain and, in turn, these two 205 shared a more anscient common ancestor with the Slovakian strain. Within the this study confirmed the circulation of DOBV in Croatia and also 241 presented the first evidence for the presence of SAAV in local A. agrarius mice. It 242 was clarified that the sequences described previously from Croatian human HFRS-243 cases belong to DOBV, not to SAAV. Taken together with the earlier reports on 244

252Fig. 1 .Fig. 2 .

 12 Fig. 1. Phylogenetic tree of hantaviruses based on partial M segment sequences (nt


M J Whelan

The development and early applications of the weak-beam technique

Keywords:

A short review is given of the way the weak-beam technique for observing dislocations at higher resolution than previously attained was developed, and some of the early applications.

1.

Introduction

The advance of scientific research is sometimes accompanied by what might be called 'blind spots', where some possible advance through available knowledge is overlooked until new minds are focussed on the subject. As an example consider the case of Dr. V.E. Cosslett, the leader of the electron microscope group in the Cavendish Laboratory, Cambridge. A graduate of Bristol University, in his early research career he led a somewhat peripatetic existence with fellowships at Bristol, Berlin and London, engaged in electron physics research and endeavouring to lay his hands on the newly developed instrument, the electron microscope, without success. During World War II he was evacuated from Birkbeck College, London, to the Electrical Laboratory of the Physics Department at Oxford, where he was engaged in teaching officer cadets, and where he remained until 1945, when he was advised by the Head of Physics (later Lord Cherwell) that there was no future for him in electron microscopy at Oxford (Mulvey [1]) -an Oxford blind spot! Thereupon he was recruited by Professor Bragg for the Cavendish Laboratory, where he remained for the rest of his career, becoming the father of electron microscopy and associated instrumentation in Cambridge University, and on whom our group in the Cavendish was heavily dependent when we embarked on electron microscope research in 1954.

The development of the weak-beam technique for the study of dislocation distributions at higher resolution than previously attained by conventional bright-field imaging attests to a further example. In 1960 a paper [2] was published calculating the averaged dark-field image profiles of a screw dislocation in a thin crystal using the so-called kinematical theory of diffraction, i.e. when the Bragg diffracted beam is weak (figure 1). At the time this was regarded as a poor approximation to the actual situation pertaining to dislocation images taken in bright-field under strong-beam (dynamical) diffraction conditions. Nevertheless it gave useful information on the visibility criterion depending on n (= g.b) , where g is the reciprocal lattice vector of the Bragg reflection and b is the Burgers vector of the dislocation. What is , g ξ being the extinction distance for the Bragg reflection g (for notation see reference [START_REF] Hirsch | Electron Microscopy of Thin Crystals[END_REF]). For n = g.b = 2 in figure 1, the half-width of the image is about / 1 π s and the peak is about 2 / 1 π s from the core of the dislocation. For s =2 x 10 -2 Å -1 , the peak is about 8Å from the core, with a half-width about 16Å. A strong-beam image is typically of width about 80Å. To observe such a weak-beam dark-field image in 1960 with the electron microscope available (Elmiskop 1) would have required a goniometer holder for tilting the specimen to an orientation to excite the weak beam, and reliable beam tilting facilities to align the weak beam on the microscope axis. The first was not available, and beam tilting could only be achieved with difficulty, using the mechanical controls designed to align the primary beam on the microscope axis. In addition the possibility of weak-beam imaging was not recognised -the blind spot! 2.

The development of the technique

Further advance required the presence of two brilliant research students, David Cockayne and Ian Ray, who joined the author in 1966 following the move of Peter Hirsch's group from Cambridge to Oxford. A problem was suggested to investigate the relation between lattice fringe images and the true position of lattice planes. The long-shot object in mind was to get information about dislocation core structure from fringe images, an object not achieved in a positive sense (see reference [START_REF] Cockayne | [END_REF]). Ray concentrated on experimental technique, while Cockayne carried out calculations on the Oxford KDF9 computer, although eventually he was involved in experimentation also, particularly in achieving the difficult beam tilting with the Elmiskop 1 to align the weak beam with the microscope axis. It is interesting to note that the development did not proceed by recognising the relevance of the kinematical profiles of figure 1. Cockayne carried out many-beam calculations, where the interest was in the lattice fringes at a dislocation formed by the strong beams, but as a by-product a weak beam was seen to have a narrow dislocation image profile. After this the relevance of the old kinematical theory was recognised.

The first report of this advance was published in 1969 [5], demonstrating weak-beam imaging of partial dislocations in Cu + 10 at .% Al alloy (figure 2), from which an estimate of stacking fault energy of 10.4 ± 1.8 erg cm -2 was derived. In figure 2 the imaging reflection is g = 0 2 2 , and the Burgers vector of the whole dislocation is 2 1 [ 0 1 1 ], which is dissociated into partial dislocations according to

2 1 [ 0 1 1 ]→ 6 1 [ 1 2 1 ] + 6 1 [ 1 1 2 ]
The products g.b for each of these in order are 2, 1 and 1. Many-beam calculations were carried out for this dislocation configuration. An example is shown in figure 3. For a number of computed images the average separations lay within 5 57 ± Å for Cockayne et al [5] proposed an approximate condition to determine the position of the weak-beam peak relative to the dislocation core, namely s g + (d/dz)(g.R) = 0 at a maximum of dR/dz along a column of crystal, where R is the displacement vector of the dislocation. This corresponds to the maximum tilt of diffracting planes along the column being just sufficient to compensate for s, i.e. to tilt to the reflecting position. For a screw dislocation with b =

2 1 [ 0 1 1 ]
and g = 0 2 2 , this leads to a position of the peak at / 1 π s. The kinematical theory curve of figure 1 for n = g.b = 2 has a peak at about 2 / 1 π s. Evidently the kinematical theory leads to a peak where the tilt over-compensates for s. A detailed discussion has been given by Cockayne [6], where many-beam calculations show that the actual peak may lie between these two values depending on the depth of the dislocation. on the microscope axis. Getting the correct photographic plate exposure, with long exposure times, and minimising specimen stage drift were all difficulties to be overcome. It was a considerable achievement on the part of both of them.

Applications

Those illustrated are necessarily selective, and micrographs are invariably at 100 keV beam potential.

Silicon

An early application was to the improvement of dislocation structure revealed by going from strong-beam imaging to weak-beam imaging (Ray and Cockayne [7]). Figures 5(a dislocations might be made visible when they are dissociated into stacking fault ribbons. The idea was just a hunch, with details of the stacking fault contrast remaining to be worked out.

Nevertheless, it was a crucial idea. At that time metallurgists and electron microscopists did not hold out any hope of seeing dislocations directly with the electron microscope. Their contrast ideas were focussed on density changes, which they thought would be too small to cause any effect. The contrast due to the phase 2 π g.R has been called Bragg diffraction phase contrast.

Peter Hirsch was a member of the Crystallographic Laboratory of the Cavendish Laboratory, and such a phase effect was well known to X-ray crystallographers.

The first (recognised) observations of dislocations in aluminium [START_REF] Hirsch | [END_REF] showed that even an undissociated dislocation is made visible by diffraction contrast due to the continuous strain field of a dislocation. The stacking fault ribbon is not necessary. At the same time Bollmann [11,[START_REF] Bollmann | Report of First European Regional Conference on Electron Microscopy[END_REF] obtained images of both dislocations and stacking faults in stainless steel. But it is interesting to note that figure 6(c) illustrates Peter Hirsch's hunch of 1954. For more detail on the history of the subject see reference [START_REF] Whelan | [END_REF].

Further early applications (a)

Stacking fault energies of f.c.c. metals. The partial dislocation separations for silver and copper have been measured for various orientations between edge and screw dislocations (Cockayne, Jenkins and Ray [14]). Figure 7 shows results for silver, from which a stacking fault energy γ of 7 . 1 3 . 16 ± erg cm -2 was derived. Similar measurements for copper gave a value for γ of 9 41 ± erg cm -2 . Carter and Ray [15] made similar studies for copper alloys (Cu + Al, Cu + Ge, Cu + Si, Cu + Zn) both from partial dislocation separations and (for some) from node shapes. This and various data from other sources led to a plot of γ versus the e/a ratio.

(b)

Observation of constrictions on dissociated dislocations. Carter and Ray [15] studied copper 7 at .% silicon alloy. Interesting weak-beam micrographs were obtained, of which figure 8 is typical showing constrictions presumably at jogs on dissociated dislocations. The motion of the constrictions was studied using a high-temperature specimen stage at ~300 o C.

(d)

The weak-beam technique has also been applied to the study of small interstitial Frank loops in silicon formed by irradiation with P + ions (Jenkins, Cockayne and Whelan [20]). Loops in the 100Å diameter size range were well imaged, but smaller loops (< 50 Å diameter) were not so clearly imaged. Small Guinier-Preston zones in aluminium-copper alloys have also been studied with the weak-beam technique (Yoshida, Cockayne and Whelan [21]).

Weak-beam contrast asymmetry for stacking faults

This topic has led to some considerable investigation, experimental and theoretical, since the first reported unusual (i.e. unexpected) contrast effects for weak-beam images of stacking faults in germanium and silicon of Cullis and Booker [START_REF] Cullis | Proceedings of the Fifth European Congress on Electron Microscopy[END_REF], followed by similar reports of others. Intrinsic and extrinsic nodes in Ge showed a reversal of the averaged contrast with reversal of s for fixed g, while intrinsic stacking faults in Si showed a reversal of the averaged contrast with reversal of g, s remaining the same in sign. The kinematical theory of stacking fault images of Whelan and Hirsch [START_REF] Whelan | [END_REF] leads to no background intensity asymmetry for the averaged contrast of an intrinsic fault by reversal of g or s, i.e. change of sign of (g.R)s. A similar result holds for an extrinsic fault provided its thickness can be neglected. But an extrinsic fault has a finite thickness because it is equivalent to two intrinsic faults on adjacent {111} planes, and for weak-beam conditions the depth periodicity of the intensity of the beam tends to 1/s. For s sufficiently large, the periodicity is comparable with the thickness of the fault, and this needs to be taken into account in the kinematical theory. A treatment so based was given by Föll, Carter and Wilkens [24], which suggested that an extrinsic fault would exhibit contrast asymmetry with change of sign of (g.R)s. This was confirmed by a quantitative study of Cockayne et al [25]. The situation for an intrinsic fault is not so clear. The kinematical theory predicts no asymmetry for an intrinsic fault with reversal of the sign of (g.R)s, contrary to what was observed [START_REF] Cullis | Proceedings of the Fifth European Congress on Electron Microscopy[END_REF]24,25]. Even for an intrinsic fault, electronic bond redistribution at the fault is to be expected, so that the fault region might appear to have a thickness. Calculations using multislice diffraction theory [26] have revealed a contrast asymmetry for an intrinsic fault. The situation was described by Cockayne [START_REF] Cockayne | Topics in Electron Diffraction and Microscopy[END_REF] as remaining to be fully exploited.

4

Concluding remarks

From what has been mentioned in this short review, it is clear that the weak-beam technique pioneered by David Cockayne and Ian Ray has led to significant advances in the study of materials.

I wish to thank Professor Cockayne for many stimulating discussions over more than forty years. Weak-beam diffraction geometries. In (a) the reflection + g is strongly excited and the image is formed with the weak systematic reflection -g. In (b) 3g is strongly excited and the image is formed with the weak beam g. In (c) no strong reflection is excited. From Ray and Cockayne [7]. Courtesy of the Royal Society. Constrictions along a dissociated dislocation in Cu + 7 at .% Si alloy after annealing at 320 o C in a heating specimen holder. The weak-beam image, taken with a 0 2 2 reflection, for which g and b for the various dislocations are indicated.

From Carter and Ray [15]. Courtesy of the Philosophical Magazine. Weak-beam image of a dislocation in Fe-26 at.% Al alloy with DO 3 -type long range order, showing four-fold dissociation into partials. Anisotropic elasticity theory shows that there is an instability orientation range from 105 o to 156 o (measured anticlockwise from [111]), which is circumvented by the partials adopting a zig-zag configuration.

From Crawford et al. [18]. Courtesy of the Philosophical Magazine.

3 1

 3 1 is that for larger s (the distance of the reciprocal lattice point of the reflection from the Ewald sphere) the dislocation image profile becomes sharper in x (distance on the image). The weak-beam image is sharper than the strong-beam image with the Bragg condition satisfied, where the width is about g ξ

1 -

 1 and 100kV electrons, which gave the estimate of stacking fault energy above.

Figure 4

 4 Figure 4 illustrates schematically some geometries which have been used for weak-beam imaging. In figure4(a) the reciprocal lattice point g is on the Ewald sphere, and the weak-beam image (broken line) is taken in -g, as in figure2where g = 202 . However, it can be shown from symmetry considerations that the same weak-beam image results if only the incident beam (and not the specimen) is tilted, so that 3 g is strongly excited (figure 4(b)). But in practice it is best to try to achieve a situation where no diffracted beam is strongly excited as shown schematically in figure4(c), a situation approximating to kinematical diffraction from one incident beam.As regards the realisation of the technique, once theory had pointed it out, Cockayne andRay worked together on the Elmiskop 1 to put it into practice. It should be remembered that at that time electron microscopes had been designed with the biological user's needs primarily in mind, with bright-field axial beam imaging, and with only single axis specimen tilt for stereo imaging. A Valdrè double-tilt specimen holder had become available, but considerable effort and ingenuity were required to achieve results. Ray was involved in specimen preparation, and both worked together to tilt the incident beam to the orientation required to excite a weak-beam

1 -

 1 ) and (b) are a pair of strong-beam and weak-beam images of a silicon specimen taken with a strong 220 type Bragg reflection (figure5(a)) and with the corresponding weak-beam condition (figure 5(b), ). The weak-beam image shows a great increase in detail visible, including dissociation into partial dislocations. Dissociation of three-fold nodes into extended intrinsic and extrinsic nodes was also observed. Such dissociation disproved an earlier suggestion of Booker and Brown[8] that dislocations in silicon were unextended. Figure6is a further example of the improvement for a dislocation line in silicon, showing: (a) a strong-beam 220 type image; (b) undissociated and dissociated segments taken with the 220 type reflection weak; (c) an image taken with a 111 type reflection such that g.b = 0 for the undissociated segments, which are therefore invisible, while the dissociated segments are made visible by the contrast due to the strip of stacking fault; (d) an image taken with a 220 type reflection which renders one of the partial dislocations bounding the dissociated segments invisible. An extended 3-fold node is also visible. The configuration is most likely due to a dislocation climbing at a shallow angle to its glide plane with dissociated segments in the glide plane and undissociated segments climbing out of the glide plane.

Figure 6 (

 6 Figure 6(c) is interesting from an historical point of view. The interest in our group inCambridge for entering the electron microscope field was generated by some early electron micrographs of beaten gold foil taken with a Metropolitan-Vickers EM3 electron microscope[START_REF] Hirsch | Proceedings of the Third International Conference on Electron Microscopy[END_REF]. Streaks on selected area electron diffraction patterns indicated the presence of stacking faults, while electron micrographs showed areas with dark bands parallel to the traces of {111} planes. Peter Hirsch had the idea that such bands might be due to inclined stacking faults cutting through the foil causing a difference in phase of 2 π g.R between electron waves scattered from opposite sides of the fault. Peter Hirsch pointed this out to the author when he joined his group as a research student in 1954, and said that because of this phase difference,

Figure 8 (

 8 at 20 o C) shows that one partial of the dissociated dislocation is more flexible at the constriction than the other partial. The more flexible partial is near edge orientation, while the other less flexible partial is closer to screw orientation. This difference in flexibility is well known from theory(Brown [16]), where the line tension of a dislocation is given by E(θ) + E'' (θ), E(θ) being the line energy as a function of orientation θ. The movement of constrictions along a dislocation at high temperature was observed with a high-temperature specimen stage.

  Superlattice dislocations in ordered Fe Al alloys.Ray, Crawford and Cockayne[17. 18] applied the weak-beam technique to ordered Fe Al alloys, where two types of ordering referred to as B2 and DO3 can occur. The B2 (Fe Al) ordering leads to a unit cell with Fe atoms occupying corner sites of the bcc Fe lattice, with Al atoms at the body-centre sites, while DO3 (Fe3 Al) has a unit cell eight times the volume of that of bcc Fe. Two-fold dissociation of dislocations into partials with anti-phase boundaries in B2 was observed, while four-fold dissociation in DO3, with appropriate anti-phase boundaries separating the partials, was observed with the weak-beam technique. Figure9is an interesting example in the DO3 structure showing zig-zag shapes of the partials near edge orientation due to the instability predicted byHead [19]. Anti-phase boundary energies were derived for orientations between edge and screw character.

Figure 1 .ξ

 1 Figure 1. Intensity profiles of images of a screw dislocation for various values of n = g.b. The actual image intensity for unit incident beam intensity is the ordinate multiplied by ( s g ξ 2 ) -2 , where

Figure 2

 2 Figure 2. 0 2 2 weak-beam image of a dislocation near edge orientation (b 2 1 = [ 0 1 1 ]) in

Figure 3 From

 3 Figure 3. Computed 0 2 2 image profile for a dissociated edge dislocation in Cu, for a separation of partial dislocations of

Figure 4 .

 4 Figure 4.Weak-beam diffraction geometries. In (a) the reflection + g is strongly excited and the image is formed with the weak systematic reflection -g. In (b) 3g is strongly excited and the image is formed with the weak beam g. In (c) no strong reflection is excited.

Figure 5

 5 Figure 5 (a), (b). (a) 0 2 2 strong beam image of dislocations in deformed silicon. (b) Corresponding weak-beam 0 2 2 image of the same area showing greatly improved resolution of detail. The diffraction patterns inset show the imaging beams encircled.

Figure 6

 6 Figure 6(a)-(d). Constricted and dissociated segments along a dislocation line in silicon imaged for various diffraction conditions. (a) 0 2 2 strong-beam image, where the dislocation structure is poorly resolved. (b) 0 2 2 weak-beam image showing partial dislocations at dissociated segments. (c) 111weak-beam image with g.b = 0 for the undissociated segments, which are therefore invisible. For the extended node at the left and the dissociated segments the contrast is due to the stacking fault. (d) 2 2 0 weak-beam image for which one of the partials with Burgers vector 6 1 [ 1 1 2 ] 07/09/2009 is out of contrast.

Figure 7 .

 7 Figure 7. Experimental measurement of partial dislocation separation ∆ as a function of dislocation line orientation θ in silver. The curves are computed using anisotropic elasticity theory. The dashed lines indicate the separations derived from a Peierls mode of the dislocation core. Stacking fault energies γ are indicated.

Figure 8 .

 8 Figure 8.Constrictions along a dissociated dislocation in Cu + 7 at .% Si alloy after annealing at 320 o C in a heating specimen holder. The weak-beam image, taken with a 0 2 2 reflection, for which g and b for the various dislocations are indicated.

Figure 9 .

 9 Figure 9.Weak-beam image of a dislocation in Fe-26 at.% Al alloy with DO 3 -type long range order, showing four-fold dissociation into partials. Anisotropic elasticity theory shows that there is an instability orientation range from 105 o to 156 o (measured anticlockwise from [111]), which is circumvented by the partials adopting a zig-zag configuration.

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