Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability

ELSEVIER

Virus Research 35 (1995) 1-16

Virus Research

Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability Tetsuo Nakayama a,b,*, Takayuki Mori a, Shinya Yamaguchi a,b, Satomi Sonoda b Sinnji Asamura b, Ryoko Yamashita b, Yoshinao Takeuchi b Takashi U r a n o b a The Kitasato Institute, Department of Virology, 5-9-1, Shirokane, Minato-Ku, Tokyo 108, Japan b Keio University, School of Medicine, Department of Pediatrics, Tokyo 160, Japan

Received 2 June 1994; revision received 15 August 1994; accepted 16 August 1994

Abstract

A simple and sensitive method for the detection of measles virus genome was developed, amplifying the regions encoding the nucleocapsid (N) protein and hemagglutinin (H) protein of measles virus by reverse transcriptase-polymerase chain reaction (RT-PCR). We examined a variety of measles patients: 28 patients with natural infection, 4 with measles encephalitis and 1 with subacute sclerosing panencephalitis (SSPE). In 28 patients with natural measles infection a single step PCR amplifying the N region resulted in a high detection rate for all plasma samples (28/28) within 3 days of the onset of rash and 80% (20/25) even on day 7 of the onset of rash and later. Within 3 days of the onset of rash, 24/25 (96.0%) of nasopharyngeal secretions (NPS) and 27/28 (96.4%) of peripheral blood mononuclear cells (PBMC) were positive for the N region PCR and the positivity rate of PCR decreased in NPS and PBMC after 7 days of the rash. In acute measles infection, measles genome was detected in all cell fractions, CD4, CD8, B cells, and monocytes/ rnacrophages by the H gene nested PCR. Measles genome was also detected from cerebrospinal fluids (CSF) in patients with measles encephalitis, SSPE, and acute measles by the H gene nested PCR. PCR products of the N and H regions were sequenced and we confirmed the presence of measles genome. Based on the sequence data, chronological sequence differences were observed over the past 10 years. The sequences obtained from

* Corresponding author at address a. Fax: + 81 03 34446637. Elsevier Science B.V. SSDI 0168- 1702(94)00074- 3

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T. Nakayama et al./Virus Research 35 (1995) 1 16

the SSPE patient were closely related to those of the wild viruses that were circulating at the time when the patient initially acquired measles. RT-PCR for NPS, PBMC, CSF, and plasma provides a useful method for the diagnosis of measles and molecular epidemiological study in addition to virus isolation.

Keywords: Measles; Reverse transcriptase-polymerase chain reaction; Measles encephalitis; Subacute sclerosing panencephalitis; Sequence variability

1. Introduction

Virus isolation is a common laboratory diagnostic method using primary human or monkey cells (Enders et al., 1962). We used Vero cells, an established cell line of African green monkey kidney cells. However, the sensitivity of Vero cells was not sufficient for isolation of measles virus from clinical samples; at least 2 or 3 passages were required to produce clear CPE of measles virus (Nakagawa et al., 1985). Kobune et al. (1990) reported that Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line and derivative (B95a) cells showed a high susceptibility to measles virus infection and Ihara et al. (1992) reported that measles virus was isolated from children's peripheral blood mononuclear cells (PBMC) obtained between day - 1 and day 6 of the onset of rash, using B95a cells. Forthal et al. (1992) reported that measles virus was isolated from PBMC stimulated with phytohemagglutinin (PHA) obtained from adult measles patients, when co-cultivated with cord blood lymphocytes. Although these methods are sensitive for isolation of measles virus, measles virus is not recovered from PBMC on day 7 and later after the onset of rash. It is still difficult to isolate measles virus from plasma or cerebrospinal fluid (CSF), using these sensitive methods. Recently, nucleic acid amplification methods using polymerase chain reaction (PCR) have been used for detection of low concentrations of viral genome of fastidious pathogens in cell cultures. Measles virus is a single-stranded negative R N A virus, a member of the genus Morbillivirus in the family Paramyxoviridae. It has six structural proteins; nucleocapsid (N), phosphoprotein (P), m e m b r a n e / m a trix (M), hemagglutinin (H), fusion (F), and large (L) proteins (reviewed by Norrby and Oxrnan, 1990). Esolen et al. (1993) reported that measles genome was detected in PBMC by the F protein gene RT-PCR, especially in monocytes enriched adherent cells, but not in lymphocytes, up to 6 days after the appearance of the rash. And Shimizu et al. (1993) also reported M gene RT-PCR for the detection of measles genome from nasopharyngeal secretions (NPS). In this report, we detected measles virus genome from various kinds of clinical samples more sensitively than Esolen et al. (1993) or Shimizu et al. (1993) did. We obtained different results for the cell fractions infected with measles virus from those reported by Esolen et al. (1993) and we further sequenced the PCR products of the N and H regions. The sequence data demonstrated the chronological genetic variability of measles virus during the past 10 years in Japan.

T. Nakayama et al. / Virus Research 35 (1995) 1-16

3

2. Materials and methods 2.1. Patients a n d measles strains

The subjects of this study consisted of a variety of patients with different clinical degrees of measles virus infection: 28 patients aged 7 months to 22 years who had typical clinical features of measles with a serological response of HI antibodies to measles virus, 4 patients with measles encephalitis, and 1 patient with subacute sclerosing panencephalitis (SSPE) aged 9 years old. 11 strains of measles virus were used for the comparison of the sequences. The 87-K and 88-F strains were isolated using B95a cells in 1987 and 1988, respectively, and were further propagated in Vero cells. The 84-K, 84-S, 84-E, and 89-T strains were isolated in 1984, and in 1989 from PBMC using Vero cells. The 84-1 and 84-Y strains were isolated in 1984 by Dr. Kobune, Department of Virology, National Institutes of Health, Japan, and were kindly presented to us. SSPE 75 strain was isolated in 1975 using Vero cells co-cultured with brain tissues obtained during autopsy (Makino et al., 1977). The Edmonston strain was generously presented to us by Dr. Enders and was passaged to establish the AIK-C vaccine strain (Sasaki, 1974). 2.2. Virus isolation

B95a ceils generously supplied by Dr. Kobune were grown in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS). 0.1 ml of the samples was inoculated in B95a cell cultures maintained in minimum essential medium (MEM) supplemented with 5% FCS. After three passages, samples without CPE were discarded as negative for virus isolation. 2.3. Extraction o f total R N A

PBMC were separated from 3 ml of heparinized venous blood by ficoll-hypaque centrifugation. PBMC were washed more than 5 times with phosphate buffered saline (PBS). In 8 patients PBMC were fractionated into lymphocyte subsets. PBMC were incubated in a 4 well tissue culture plate at 37°C for 3 h and the adherent cells were obtained as monocytes/macrophages. Non-adherent ceils were adjusted at 3 - 1 0 6 / m l and further fractionated into cell subsets by means of a complement mediated T cell lysis with monoclonal antibodies, O K T l l , OKT8, OKT 4, and OKB7 (Ortho Diagnostics, Raritan, N J), as previously reported (Nakayama et al., 1987). NPS were obtained by direct suctioning. A Nelaton catheter (Fr. 8) connected to a disposable syringe was inserted into the nasal cavities and NPS were soaked into 1 ml of MEM supplemented with 5% calf serum (CS) and adequate antibiotics. Samples were stocked at -70° C until virus isolation and RNA extraction. Total RNA was extracted from 200 /zl of NPS, plasma, CSF and 2- 4- 106 PBMC, as reported by Chomczynski and Sacchi (1987) with minor modification. Briefly, 200 ~1 of lysis buffer containing solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M 2-

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T. Nakayama et al. / Virus Research 35 (1995) 1-16

mercaptoethanol, 0.5% Nonidet P-40) was added. RNA was extracted by means of a phenol chloroform mixture and was precipitated with one volume of isopropanol. The R N A pellet was resuspended in 20 /xl of sterile distilled water and 5 /xl of total R N A was applied for RT procedure.

2.4. Primer design and RT-PCR We synthesized the sets of linker-primers for the N and the H regions, referring to the sequence of the AIK-C strain, as previously reported (Mori et al., 1993). The genomic locations and the sequences of the primers are illustrated in Fig. 1. Measles genomic RNA was first converted to cDNA with NPB0 positive sense primer for the N region P e R and with MF1 positive sense primer for the H region P e R . We amplified the 169 bp of DNA segment of the N region using N P B I ( + ) and NPB2 (-) primers. As for the H region, the first P e R was done with a set of MH2( + ) and MH6(-) primers, amplifying the 635 bp of D N A fragment. A nested PCR experiment was done with a set of M H 3 ( + ) and MH4(-) primers, which yielded the 377 bp product. Viral genomic RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Life Technologies Inc., Gaithesburg, MD) at 37°C for 2 h. The reverse transcriptase activity was inactivated at 95°C for 5 min. 10 /~1 of

N I /I

P/C M I I "174~ 3402

NPBO 1279 NPB1

H I I 7247 I

L I ~ \9208 ,,

I

I 15894

\

NPB2

6 (. 1557

F I 4872

\ /

1725

I

",

I I~F1 7209 MH2

\

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8067 / P MH3

~" ",.

8701 MH4

77 Primer Sequence 51~ ~52 1279 NPB0 5' C A A G G C T T G T T T C A G A G A T T G C 3' 1557 NPB1 5' G A T C C G C A G G A C A G T C G A A G G T C 3' 1725 NPB2 5' A G G G T A G G C G G A T G T T G T T C T 3' 72~ MF1 5' G C T T C C C T C T G G C C G A A C A A T A T C G 3' 8067 MH2 5' G G G C T C C G G T G T T C C A T A T G 3' 8100 MH3 5' C A G T C A G T A A T G A T C T C A G C A A C T G 3' 9482 MH4 5' A T C C T T C A A T G G T G C C C A C T C G G G A 3' ~01 MH6 5' C ' I - r G A A T C T C G G T A T C C A C T C C A A T 3'

Fig. 1. Primer design for measles RT-PCR. The n u m b e r of the nucleotide is based on the genomic location of the AIK-C strain.

T. Nakayama et al./ Virus Research 35 (1995) 1-16

5

viral c D N A was amplified by PCR in a total volume of 100 ~1 mixture as recommended by the manufactures, using 2.5 units of Taq D N A polymerase (Perkin-Elmer Cetus, Norwalk, CT). Light mineral oil (Sigma Chemical Co., St. Louis) was layered onto the samples, which were then subjected to a D N A thermal cycler (Program Temp. Control System PC-700, Astec Co. Ltd. Fukuoka, Japan). The first three cycles were at 92°C for 2 min, 55°C for 3 rain, then 72°C for 2 rain. These were followed by 30 cycles (denaturation at 93°C for 1 min, reannealing at 58°C for 1 min, and extension at 72°C for 2 min) with final additional extension period of 5 min at 72°C. For the nested PCR experiments, 10/xl of the first PCR product diluted at 1 : 100 was mixed in a total of 100 ~1 PCR reaction mixture and the samples were subjected to PCR using the same thermal cycle program. PCR products were electrophoresed through 3% agarose gel (NuSieve 3:1, FMC Bioproducts Corp., Rockland, ME) and visualized by staining with ethidium bromide. R N A extracts from AIK-C strain of measles virus-infected and mock infected B95a cells were used as positive and negative controls. And the Sasazaki strain of mumps virus (106 TCIDs0) was also used as a negative control to examine the specificity. The negative control samples were interspersed and were tested in parallel with the test samples in each experiment to monitor for contamination as a source of false-positive.

2.5. Nucleotide sequences of PCR product 30 /~1 of PCR products was electrophoresed through low-melting-temperature 1% agarose gel and the specific bands were excised from the gel. The purified PCR product was directly sequenced by means of dye labeled primers using a Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems Japan Inc., Tokyo) and analyzed with an automated nucleotide analyzer, the 373A D N A sequencer (Applied Biosystems, Foster City, CA).

3. Results

3.1. Detection of measles genome in patients with natural measles R N A extracts from 105 TCIDs0 of measles virus were serially diluted and subjected to R T - P C R by the H gene nested PCR. The detection level of RT-PCR method was 10°-ITCIDs0 . No band was detected for the negative control of mumps virus to examine the specificity. R T - P C R was done in 28 natural measles patients and the results of the detection of measles virus genome from a series of samples obtained from a patient are shown in Fig. 2. We designated the day when patient had a typical measles rash as day 1 of the onset of rash. The measles genome of the N region was detected in PBMC, NPS, and plasma on day 1 and in plasma on day 5, 9 and 28. Through the first R T - P C R in the H region, the specific band was demonstrated in PBMC and NPS on day 1 and the nested PCR increased the detection rate in PBMC on day 5, 9, and 28. As for negative controls,

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T. Nakayama et aL / Virus Research 35 (1995) 1-16

Days after the onset of rash 1 5 9 28 I

I

r--I

r~l

Days after the onset of rash 1 5 9 28

r--i

MN L P L P L P

~

LPC

r--I

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Days after the onset of rash 1 5 9 28 r-'--I

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~1-169

489 ~

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H nested

PCR

Fig. 2. Difference of the sensitivity of RT-PCR in the N region and H region. M, DNA marker; N, NPS; L, PBMC; P, plasma; C, negative control.

no visible band was observed in the N gene and the H gene RT-PCR. The R T - P C R in the N region was sensitive to detect measles genome from plasma samples and the nested H gene P C R was suitable for the NPS and PBMC. The detection rates of measles genome in the N region from PBMC, NPS, and plasma are shown in Table 1. Measles genome was detected in 24 (96.0%) of 25 NPS, 27 (96.4%) of 28 PBMC, and in all of 28 plasma on day 1-3. On day 7 and later, the detection rate of measles genome decreased to 50% of NPS, 40% of PBMC, but 20 (80%) of 25 plasma samples were P C R positive.

3.2. Detection of measles genome from CSF We examined the detection of measles genome from CSF in 4 patients with measles encephalitis and 3 patients with acute measles without showing the symptoms related to measles encephalitis. The results are shown in Fig. 3. Patients No. 1, 2, and 3 were diagnosed as measles encephalitis in 1993 and patient No. 4 in 1988. Loss of consciousness was observed 3 - 9 days after the onset of rash and CSF and sera were obtained 3 - 1 0 days after the onset of rash. Pleocytosis was noted in one patient and the protein concentration in CSF slightly elevated in all. Glucose level in CSF showed within normal glucose range. The N gene R T - P C R was positive for plasma samples obtained from the patient No. 1 and 2 but was negative for CSF. Measles genome was detected from all CSF samples by the H

Table 1 Detection of measles virus genome by the N region PCR Samples NPS PBMC Plasma

Days after the onset of rash < 3 days

4-6 days

> 7 days

24/25 (96.0%) 27/28 (96.4%) 28/28 (100%)

7 / 8 (87.6%) 9/17 (52.9%) 14/17 (82.4%)

3 / 6 (50.0%) 10/25 (40.0%) 20/25 (80.0%)

T. Nakayama et al. / Virus Research 35 (1995) 1-16

Encephalitis

M

1

2

I---I

r--'l

NH PC

NH PC

7

Acute measles 3

4

1

~

HH CC

2 3

1"-3

H C

NHHH P C CCCoM ~1- 3 7 7 , ~ ~1-169 ~

603 310 194

Fig. 3. Detection of measles virus genome from patients with measles encephalitis and acute measles without symptoms of encephalitis. RT-PCR in the H region was done for the detection of measles genome from CSF in 4 patients with measles encephalitis and RT-PCR in the N region was done for the detection of measles genorne from plasma samples. In patient No.3, the second CSF sample was obtained 7 days after the initial sampling. C, CSF; P, plasma; M, DNA marker; Co, negative control.

gene nested PCR. In patient No. 3, initial sample was obtained 10 days after the onset of the rash, 3 days after the onset of encephalitis, and the second sample was obtained 7 days later. CSF sample of patient No. 4 was stocked for 5 years at - 2 0 ° C and measles virus specific PCR product was weakly visible. As for acute measles infection without showing the symptoms related to encephalitis, measles genome was detected from CSF samples obtained 3 days after the onset of rash by the H gene nested PCR and was detected from plasma in patient No. 1 by the N gene PCR. The results of R T - P C R in patient with SSPE are shown in Fig. 4. The specific band of 635 bp of the H gene first PCR was observed in PBMC and those

12341234CM

/

m 194 H 1st PCR nested PCR

Fig. 4. Detection of measles genome in patient with SSPE. Left half of the panel shows the H gene first step PCR and right half shows the H gene nested PCR. 1, PBMC; 2, CSF; 3, plasma; 4, PBMC stimulated with PHA; C, negative control; M, DNA marker.

T. Nakayama et al. / Virus Research 35 (1995) 1-16

~ o 2 °° ^ ~

~ o ~ °° ~..~, _

~1-489 ~1"267

16!

~ oo 1357 603 310 194

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41- 80 N 1st P C R

H 1st P C R

nested P C R

Fig. 5. Detection of measles genome from different lymphocytes fractions. CD4, T cells depleted of CD8 + cells, CD8, T cells depleted of CD4 + cells, N.C., negative control; M, DNA marker.

stimulated with phytohemagglutinin (PHA), while it was faintly visible from CSF, and plasma. By the nested PCR, the specific band became more clear. For the negative control, R N A extracts from mock infected B95a cells were examined and no band was visible after the H gene nested P C R procedure. No virus was isolated from CSF obtained from patients with SSPE and encephalitis.

3.3. Detection of measles virus genome from lymphocyte subsets In 8 patients, PBMC were fractionated into m o n o c y t e s / m a c r o p h a g e s and lymphocyte subsets. The results of the detection of measles genome from lymphocyte subsets obtained from one of them are shown in Fig. 5. When P C R was done in the N region, specific P C R product was visible in unfractionated lymphocytes, T, CD4 and CD8 cells. By the first P C R in the H region, measles genome was detected in PBMC, T, CD4, and B cells. When the nested P C R was done in the H region, measles genome was detected in all cell fractions including CD8 cells and m o n o c y t e s / m a c r o p h a g e s in which specific bands were not visible by the first H gene PCR. In the remaining 7 patients, measles genome was detected in all cell fractions similar to the results.

3.4. Nucleotide sequencing We determined the nucleotide sequence of 126 bp of the P C R products of the part of the N region amplified between the set of primers from plasma, NPS, and PBMC in comparison with the A I K - C vaccine strain as previously reported by Mori et al. (1993). Sequencing results of the N region are shown in Fig. 6. From the strains 89-U to 91-T, P C R products of the N gene were sequenced directly from clinical samples and 84-K, 84-S, 84-E, 84-Y and 84-I were clinical isolates. Nucleotide differences were observed at 4 sites between the A I K - C and the Edmonston strain (the parental strain of the A I K - C vaccine strain). Nucleotide

T. Nakayarna et al. / Virus Research 35 (1995) 1-16

AIK-C

Edm 84-K 84-S 84-E

1591 1601 1580 1581 8CTGACSCCC TICTTAIlaCT |CAAICCAT8 ..... ..... ..... .....

1611 8CAS|AATCT

1621 1631 1641 CiIAAGAACA AiSCTCASAC ACaSACACCC

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91 -T

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84-Y 84-1 89-U

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1691

84-$ 84-E 84-Y 84-1

...G ................ ...G ................ ...G ...............

6................................. G. . . . . . . . . . . . . . T. . . . . . . . . . . . . . . . . . G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...GG..

A. . . . . . . . . . .

6.................................

89-U 89-H 90-0

...GG..A ........... ...66..A ........... ...GG .............

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91 -T

...GG .............

T 6.................................

1701 G..

Fig. 6. Nucleotide sequence alignments of the N region.

differences at the genome positions of 1595, 1654, 1671 were conserved after the 1984 isolates. Reflecting the time of epidemics, several nucleotide differences were observed. Nucleotide alignment of the 84-1 strain isolated in 1984 was similar to that of the strains after 1989. We also examined the nucleotide sequences of 546 bp of the P C R products of the part of H region and the results are shown in Fig. 7 in comparison with me A I K - C vaccine strain. Two nucleotide differences were observed between the E d m o n s t o n and the A I K - C strains (8174 and 8282). G/A nucleotides at 8174 and C at 8282 were conserved in the wild strains and these two sites were characteristic of the A I K - C strain. G nucleotides at 8419 and 8641 were conserved from the 1984 isolates. C at 8131, T at 8156, C at 8167, G at 8206, C at 8285, T at 8311, G at 8383, A at 8516, and A at 8557 were conserved from the 1987 isolates. According to the conserved nucleotide changes and transient mutations, measles virus genomes were classified into three genotypes. Fig. 8 shows the nucleotide differences and predicted amino acid changes. We observed 6 - 1 2 nucleotide differences between the Edmonston strain and genotype A measles virus. 14-17 and 18-19 nucleotides differed from the Edmonston strain in genotype B and C measles viruses, respectively. Genotype A was common before 1985 and genotype B surged as a predominant lineage from 1985 to J990. In 1990 and later genotype C became ubiquitous strain, but this classification was not so definite. The 84-1 strain isolated in 1984 had a similar sequence alignment to genotype B and the 88-F strain isolated in 1988 was similar to the genome of 1990 and later. Patient No. 4 with measles encephalitis was diagnosed in 1988 and the sequence of this genome was similar to that of the 88-F genome alignment. SSPE 75 was isolated from a 14-year-old boy with SSPE in 1975 and was supposed to have originally infected the patient in early 1960's from his measles history. Based upon the sequence data of the SSPE 75 strain, 6 nucleotides differed from the Edmonston strain and 6-13

T. Nakayama et al. / Virus Research 35 (1995) 1-16

10

(a)

8131

8141

8151

AIK-C

TATGGTGKCT

TTKGGGGA6C

TCAAACTCGC AGCCCTT16T

Eda

...........................................

8161

8171

8181

CACCSGGAA6 ATTCTATCAC

8191

B201

8211

8221

AATTCCCTAT

CA6GKATCAG

6GAAAKSTGT

CAGCTTCCAG CTCGTCAAGC

SSPE 7 5

...................

84

K

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98

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A...............................

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gl

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k ............

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91

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k ...........

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93

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A. . . . . . . . . . . . . . . . . A............

T..........

k . . . . . . . . . . . . . . .. . . . .

C. . . . . .

8241

--8251

826"1 " T

AIK-¢

]AGGTSTCT6

8AAATCCCCA

ACCGACATSC A A ] C C T G G G ]

Edm

.........................................

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A...............................

8271

6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8281

8291

8301

8311

CACCTTATCA

ACGGATGATC C A S T G A X A 6 A C A K S C T T T A C

75

......................................

84

-

K

.........................................

C....................................................................

84

-

E

.............

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64

-

Y

.........................................

I

..............................

6 ..........

C..C .........................

-

K

..............................

G. . . . . . . . . .

C..C .........................

F

.........................................

T

..............................

H

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 86 69

-

89

8321

8331

8341""

CTCTCATCIC

ACASAGGTGT

TATCGCTGAC

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

$$PE

84-

T .....

6..................................

g3

SSPE 9 2

.

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I K

A......

.

1 ...........

84

k ............

.

T.........................................

87

A. . . . .

8231

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A..C

G. . . . . . . . . . . . . . . . . . . . . . . . . . .

....................................................................

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ] ....................................... T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C,.C .........................

] .......................................

G. . . . . . . . . .

C..C .........................

T.......................................

8 ..........

C..C .........................

T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90

+ 0

.........................................

C..C .........................

T.......................................

90

-

K

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C..C

T.......................................

91

-

Hk

.........................................

C..C .........................

. . . . . . . . . . . . . . . . . . . . . . . . .

T.......................................

91

-

H6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C..C

. . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93

-

KA

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C..C

. . . . . . . . . . . . . . . . . . . . . . . . .

T .......................................

93

-

K~

.........................................

C..C .........................

SSPE 92

.........................................

C....................................................................

ENCE

.........................................

C..C .........................

T.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C..C .........................

T.......................................

1

ENCE 4

(b)

8351

8361

8371

AATCAASCAA AAIGSGCTG]

AiK-C Edm SSPE 75

'illlll]i

illll

illl

8381

CCCSACAACA CGA4CkSAT6

i

ill]i

;]illlili~

G. . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8391

8401

6411

8421

8431

ACAASTTGCG

AAT6GASACA

188TTCCAAC

A66CGTGTAA

GGGTAAAAIC CAASCAC]CT

8441

GCSAGAATC¢

illlllllll

illlllllll

illlilli~i

illillilll

ilillllill

iillllllll

-

K

""

X.....................................

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

84

-

E

..............................

A .....................................

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

84

-

Y

..............................

A. . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-

K

87

I

88

-

F

89

-

T

89

-

H

90

~ 0

go

-

91

-

HA

91

-

HO

93 93

K

................................

G.

................................

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G.

G. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . .

C.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . .

C..........................

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...............................

-

KI 92

ENCE I £NCE

4

AIK-C

. . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . .

A..................... G.,A ..................... C......

G. . . . . . . . . . . . . . . . . . .

k ..................... A ........... . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . .

C..........................

6 ...................................

G. . . . . . . . . . . . . .

C..........................

G. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..............................

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..8 ...................................

................................

KA

SSP[

8451

illiiii211

84

84-

. .....................

T.......................

8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A.....................................

G.

8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .

G. . . . . . . . . . . . . . .

.

.

.

.

G. i l i i i i i i l l

8 . . . . . . . . . . . ... . . . . . . . . . . . . . . . . .

.

.

.

.

C.

. . . . . . . . . . . . . . . . . . . . . . . . .

C.

. . . . . . . . . . . . . . . . . . . . . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

.

iilc. ll . . . . . . . . . . . . . . . . . . . . . . .

.

.

.

G. . . . . . . . . . . . . . . . . . . . ~. L. . . . . . " . . . . . .. .. . . 656~ 8531 6541 S~51

8461

8471

6481

8491

8501

8511

8521

C6AGT6GSCA

CCATTGAAG6

A]AACAGGA]

]CCTTCATAC

6688]CTTST

CTSTISATC]

8AGICT6ACA

GT18ASCTTA

AAATCAAAAT

TSCIT¢GGGA

TTCSGG¢CAT

Edm SSPE

75 ....................................

84 84

E

84

Y

64

I

87

K

68

F

89

T

89

H

9O

0

90

K

91

HA

91

HO

93

KA

g3

KI

SSPE 92 ENCE 1 ENCE 4

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G. . . . . . . . . . . . . . . . . . . . . .

.... C. . . . . . . . . . . . . . . . . . . . . . . 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A ................ .......... ....... A . . . . . . . . . . . . . . . . G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • A ...................................

iiiiii~ii~

iii~iiiiii ili~iillll

,¢ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i~iiiillll

i

........

A........................................

A........................................

, . . . . .

A........................................

.............................................

..........

A. . . . . . . . . . . A. . . . . . . . . . . . .

~. . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . ............................

iiiillllll

Q. . . . . . . . . . . . . . . . . . . . . . G

T. . . . . . . . . . .

A... ^ ............. A.............

¢. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.............

C.......

C.................................

k ......................

C. . . . .

.............

C. . . . . . .

C...........

A ......................

¢ .................

A.............

.............

C.......

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A......................

C.................

A.............

.............

C.......

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A......................

C.................

.............

C.......

C.................................

A ......................

C.................

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. .............

C...... C......

G. . . . . . . . . . . . . . . . . . . . .

T...........

G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.C ................................. C

4 ...................... ..A

C

.......................................

Fig. 7. Nucleotide sequence alignments of the H region.

A. . . . . . . . . . . . .

A............. A............. G. . . . . . . . . . . . . . . . . . . . . . A A.............

T. Nakayama et al. / Virus Research 35 (1995) 1-16 (C) AIK-C

11

8571 8581 8591 8601 8611 8621 6631 9641 8651 8861 8671 TGATCACACACGGTTCAGG8 ATG6ACCTAT ACAAATCCAA CCACAACAAT 6T6TATTGGC TGACTATCCC ACCAATGAA8 AACCTAGCCT TAGGTETAAT CAACAC

Edli

SSPE 75

.......................................................................................................... ...................................................................... ................................................... C. . . . . . . . . . . . . . . . . .

84

'~

84 84

E Y

......................................................................

84 87

I K

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

88

F

89 89 90 90 91 91 93 93 SSPE ENCE ENCE

T H 0 K HA HO KA KI 92 1 4

.... T. . . . . . . . . . . . . . . . . . . ................................... ...................................

]]]]]]]]] ]]]2]]]i] ....................

]kll]]]]]i ]]]]]] 6. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.....................................................................

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . .

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................................... C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................ IC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................ C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C .....

8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...... .............................. ......... ......................................................................

G. . . . .

8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. . . . . . . . . . . . . . . . . . . . 8. . . . . . . . . . . . . . 1~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . li . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

" ....... C. . . . . . . . . . . . . . . . . . " .....

C. . . . .

Fig. 7 (continued).

d i f f e r e n c e s w e r e o b s e r v e d f r o m t h e 1984-85 wild strains. T h e S S P E 92 g e n o m e was d e t e c t e d f r o m t h e p a t i e n t w h o was d i a g n o s e d in 1992 a n d the p a t i e n t was s u p p o s e d to have a c q u i r e d m e a s l e s in 1984 o r 1985 a c c o r d i n g to the p a s t history. T h e s e q u e n c e results s u g g e s t e d t h a t the S S P E 92 was classified into g e n o t y p e A m e a s l e s virus, a p r e d o m i n a n t strain in t h e y e a r o f < 1985. N o s e q u e n c e d i f f e r e n c e was o b s e r v e d in this r e g i o n a m o n g g e n o m e s d e t e c t e d f r o m P B M C , p l a s m a , C S F , a n d P B M C s t i m u l a t e d with P H A . T h e results of c h r o n o l o g i c a l s e q u e n c e analysis w e r e c o i n c i d e n t with m e a s l e s history.

'?' ",~ AIK

- C

Edm

"FG T

1-rG

"t' GAG

Hpa II

,?, ,,,,

.lS~ ;1~ CTC

CTC

CTr

CCGG

Bst P I / / A v a

~2

CAA

II

0.~5

GGTCACC

.1;~,,,..

TrA

GAC

C

C

.G . . . . . . . . . . . .

C . . . . . . . .

C

.A ....

.G .....

C..

C

.A ....

CGA

. . . . . . . . . . .

~ 1985

. . . . . . . .

1 98 5..89

..C

. . . . . . . .

1990-

..C

..A

......

.......

C..

C ....

287

291

293

294

296

299

302

312

334

338

339

347

371

Cys

Leu

GlU

Leo

Leu

Leu

Arg

Gly

Gin

Thr

Leu

Asp

Arg

/r

~r

*

*

Phe

.

G~--rq

*

Arg

Pro

*

*

*

AIK

o C

WILD

........

GGA

.2:1

.G . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

A

0~

T

T .... T ....

G G

A..

C ....

T

..G

T

..G

Ava II

,/, AIK

- C

CAA

.3., . . ., . . . GGT

ATC

~,~.y..,6

1-rG GAT

TCA

G

.i~ GAT

. ,. . . . . .. . . ... . .

CTT AAA

TCG

GGACC

~,,

TCC ACT

CCA

~,2 TTA

Edm ~ 1985

..G

. . . . . . . . . . . . . .

1985,-,89

..G

. . . .

1990~

..G

..C

...

AIK WILD

- C

A . . . . . . . . . . C ....

C

...

. . . . . . . .

G

. . . . . . . . . . . . . . . .

A . . . . . . . . . .

A

.....

A ....

A

. . . . . . . . . .

C

.....

G

A . . . . . . . C

G ..G

..G ... ...

383

388

390

402

404

409

416

423

426

429

446

455

457

464

Gin

Gly

lie

Leu

Asp

Set A s p

Leu

Lys

Ser

Ser

Thr

Pro

Leu

*

*

Asn

*

*

*

*

*

Thr

*

*

*

*

Asn

Fig. 8. Nucleotide differences and predicted amino acid substitution differences among wild strains representative of the times of epidemics. Asterisks represent an identical amino acid with the AIK-C strain.

12

T. Nakayama et al. / Virus Research 35 (1995) 1-16

4. Discussion

Cell-associated viremia contributes to widespread dissemination to the tissues. The magnitude of measles viremia is considered to peak around the onset of the rash and thereafter decreases promptly when N T antibodies appear in the circulation. Forthal et al. (1992) and Ihara et al. (1992) reported that measles viremia lasted up to 7 days after the onset of rash according to their data using sensitive virus isolation methods. Godec et al. (1990) reported that the R T - P C R method for the detection of measles virus genome successfully amplified 400-500 bp from each of five genes in R N A extracts of wild measles virus cultured in Vero cells and in R N A extracted from SSPE brain tissue. Esolen et al. (1993) reported that 3 out of 7 children had detectable measles R N A by R T - P C R confirmed by Southern blotting within the first 2 weeks of the onset of measles rash and 4 out of 7 children exhibited measles R N A after long exposure. All monocyte enriched PBMC ( 5 / 5 ) from day 2 of the rash were positive, but only 25% ( 1 / 4 ) from day 3 was positive and none ( 0 / 2 ) from day 4 was positive in situ hybridization. They detected no viral R N A in plasma samples. They used a primer set designed for the detection of the fusion (F) gene. In this report we detected measles virus genome directly from the clinical samples, PBMC, NPS, plasma, and CSF. In our comparative study of virus isolation using B95a cells, measles virus was isolated in 16 (55.7%) out of 29 frozen NPS samples, while measles genome was detected in all samples. Our R T - P C R method was sufficiently sensitive to detect measles genome of 10 °-~ TCID50. Measles genome was detected at a high positivity rate of approximately 100% within day 3 of the onset of rash from PBMC, NPS, and plasma. Even after day 7 of the rash it was detected in 50% of NPS, 40% of PBMC, and 80% of plasma, probably up to 4 weeks and later after the onset of rash. The measles genome was found to be present in plasma and PBMC longer than expected, apart from the infectivity. The infectivity of measles virus was completely neutralized with a NT positive serum, but measles genome was detected even after the infectivity was lost (data not shown). The detection of measles virus genome did not reflect the presence of infectious particles. The different sensitivity of R T - P C R of the N and H region to plasma, PBMC, or NPS may depend upon the primer selection. We have difficulties in clinical diagnosis of measles in infants aged around 7 months or in cases with secondary vaccine failure because the typical symptoms are modified by low or undetectable levels of antibodies. In immuno-compromised patients they demonstrate questionable measles symptoms, with prolonged high fever and unusual rash. In these cases we could detect measles genome. In the patients with SSPE and encephalitis, measles genome was detected in PBMC, plasma, and CSF despite negative for virus isolation. Thus, our R T - P C R method has a definite clinical advantage for the detection of measles genome. Infectious measles virus was not recovered from the T cell subsets of the patients with natural measles. Hyypi~i et al. (1985) reported that replication of viral R N A was demonstrated in both T cells and B cells, and both OKT4 + and

T. Nakayama et al. / Virus Research 35 (1995) 1-16

13

OKT8 + depleted T cell subsets in vitro. They demonstrated the presence of measles virus R N A in PBMC obtained from 16 out of 26 patients. Most of their positive samples were obtained during the first week of illness, and one was obtained even 3 weeks after the onset of rash by spot hybridization. Fournier et al. (1985) reported that 7 0 - 9 0 % of PBMC from SSPE, 0 . 5 - 5 % of P B M C from healthy adult, and 10-15% of PBMC from young infants were found to contain measles virus R N A sequence using in situ hybridization. Later, however, measles virus specific sequences were detected only in patients with acute measles and one out of 16 healthy control donors (Schneider-Schaulies et al., 1991). In contrast, Esolen et al. (1993) reported that measles genome was found only in the monocytic fractions but not in the lymphocyte enriched fraction. However, in this report, we were able to detect the measles genome in T cells, B cells, and m o n o c y t e s / m a c r o phages. As for the T cell subsets, the measles genome was detected in both CD4 and CD8 cells. These different results for the cell fractions infected with measles virus are due to the sensitivity of the primer design for RT-PCR. We fractionated T cell into subsets by monoclonal antibody mediated T cell lysis and there may be some possibility that a small amount of contaminating cells could account for the positivity of nested P C R of the H gene. Measles genome was detected in T, CD4 + , and CD8 + cell fraction by the N gene PCR, less sensitive than the nested H gene PCR, and thus it would depend on the different proportions of the peripheral lymphocyte subsets. We confirmed that the presence of measles genomes by direct sequencing. Measles virus genomes were sequenced from the isolates before 1988 and directly from clinical samples, NPS, PBMC, plasma, and CSF after 1989 without virus isolation. Further sequence data also provided an important information on genetic variability. We sequenced 126 bp of P C R products of the N region and c o m p a r e d the sequencing results reported by Taylor et al. (1991) and Schulz et al. (1992). Rota et al. (1994) reported the alignment of the predicted amino acid sequences of the N and M genes of wild strains isolated from 1958 to 1989. The evolutionary patterns of the N and M genes suggested that the wild strains in the US in 1989 were more related to those in the U K from 1983 to 1988 than to those in the US. The wild strains isolated in J a p a n < 1985 differed from those isolated in the US in 1983 at 512 and 515 residues in comparison with the alignment of the deduced amino acid from the sequencing results in this report. We also sequenced the 546 bp of the H coding region of measles virus isolated from 1975 and more informative results were obtained. According to conserved nucleotide changes and transient changes, measles virus genomes were classified into three genotypes representative of the period before 1985, from 1985 to 1989, and after 1990. Rota et al. (1992) reported that the nucleotide replacement at 8516 from G to A reflected an amino acid change from Asp to Asn, predicting a new glycosylation site. The change at this site was noted in the 84-I strain isolated in Japan in 1984 and the genome after 1987 and later, as well as the McI strain isolated in the US in 1983 reported by Rota et al. (1992). In addition, the molecular size of the H protein of the recent isolates from 1988-89 in the US was larger than that of the Moraten strain (Rota et al., 1992). The same findings were reported by Saito et al.

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(1992) and Sakata et al. (1993). The 84-I type measles virus was not a majority strain before 1985. The 88-F genome was detected in 1988, but its sequence alignment was similar to that found from 1990 and later in the H regions. Thus, it appears that the measles virus of the next generation existed concurrently with the majority strain in epidemics. In comparison with the sequencing results of the recent isolates in the US from 1983 to 1989, reported by Rota et al. (1992) and those in the UK, in 1988, reported by Schulz et al. (1992), the conserved changes in the predicted amino acid (296, 302 and 416 residues) of the recent isolates in the US were common in the US, UK and Japan. But, in detail, a G nucleotide at 8206, T at 8311, and T at 8338 in Japanese strains after 1985 were A, C, and C in the UK strains, respectively. The measles viruses in Japan seem to be of a different lineage from those isolated in the U. K. and the US. It is suggested that our R T - P C R method and direct sequencing of the H region are useful method for molecular epidemiological study. It is still unclear how measles virus is persistently present in the brain of the patients with SSPE. Using this sensitive RT-PCR, we detected measles genome from CSF obtained from patients with SSPE, measles encephalitis, and acute measles infection without demonstrating symptoms related to encephalitis. No difference in the sequence alignment in the part of the H gene was observed among the genomes obtained from CSF, PBMC and plasma in the patient SSPE 92. Genome pattern was similar to that of < 1985 and it demonstrated the possibility that the measles virus had infected persistently in PBMC and the brain since acute measles episode. We propose the possibility that measles virus could be transported by infected PBMC to the CNS and could initiate a persistent infection. Mutations in the M gene of SSPE viruses were frequently observed and the same was reported in the H and F genes with less frequency (Cattaneo et al., 1989, Schmid et al., 1992). Baczko et al. (1993) analyzed the M gene sequences in different regions in one SSPE brain and indicated that different biased hypermutation events had occurred during the persistence of measles virus. We will now have to study the sequence alignments of the M gene in the brain and PBMC of SSPE and the chronological sequence changes of the M gene.

Acknowledgments We thank Dr. F. Kobune, National Institutes of Health, Japan, for his generous supply of B95a cells and measles virus strains and are grateful to Dr. S. Makino, The Kitasato Institute, for his enthusiastic discussion.

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