AMPLIFICATION OF THE MUMPS VIRUS GENOME

Amplification of the mumps virus genome was performed in both intestinal specimens and peripheral blood lymphocytes (PBL) by RT-PCR. Intestinal specimens were obtained by endoscopic biopsy or surgical resection from 15 patients with ulcerative colitis, 15 patients with Crohn's disease, and 10 controls. The endoscopic biopsies contained mucosa and submucosa of the intestine, and the surgical biopsies contained the full thickness of the intestine. In each case the diagnosis was established using standard clinical, radiological, endoscopic, and histological criteria. Clinical details of the patients are shown in table 1. PBL were obtained from seven patients with ulcerative colitis, six patients with Crohn's disease, and three normal controls. All samples were taken with informed consent. Isolation of lymphocytes from peripheral blood cells was performed using a Lymphoprep kit (Nycomed Pharmaas, Oslo, Norway) according to the manufacturer's instructions.

RNA was extracted from intestinal specimens or PBL using the acid guanidinium-phenol-chloroform method with a RNaid Plus kit (BIO 101, California, USA). Reverse transcription of RNA was carried out by incubation at 42°C for 50 minutes using 3 μg of random hexamer (Takara, Otsu, Japan), 10 units of reverse transcriptase (RAV-2; Takara), and 10 nmol of dNTP in reverse transcription buffer containing 50 mM Tris HCl, pH 8.3, 75 mM KCl, 8 mM MgCl₂, and 10 mM DTT. Converted DNA was amplified with 1.25 units of Taq DNA polymerase (Takara Taq; Takara) in the presence of sets of primers (10 pmol), and 10 nmol of dNTP in the PCR buffer containing 10 mM Tris HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl₂. The mumps virus genome was amplified by nested PCR using primers specific to the portion of the viral genome encoding phosphoprotein (P)18 and haemagglutinin-neuraminidase (HN)19; both primers can also amplify vaccine strains. Nucleotide sequences of these primers are shown in table 2. Amplification of the P gene was performed as follows. After an initial four minutes of denaturation at 94°C, 35 PCR cycles (one minute at 94°C, 1.5 minutes at 44°C, and one minute at 72°C), followed by a seven minute extension step at 72°C using a thermal cycler (GeneAmp PCR system 9600; Perkin Elmer, Applied Biosystems Division, New Jersey, USA). For nested PCR, 1 μl of the first PCR product was mixed and the samples were subjected to PCR using the same thermal cycle programme.

Amplification of the HN gene was performed as follows. The first three cycles were two minutes at 92°C, three minutes at 55°C, and two minutes at 72°C. These were followed by 30 cycles (one minute at 93°C, one minute at 58°C, and two minutes at 72°C), with a final extension period of five minutes at 72°C. Nested PCR was performed using the same thermal cycle programme. The PCR products (5 μl) were electrophoresed on 2% agarose gel and visualised by staining with ethidium bromide and exposure to UV light. PCR products, which were amplified with P gene primer sets, were transferred subsequently to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's instructions. Then, Southern hybridisation was performed with an alkaline phosphatase labelled oligonucleotide probe of the mumps virus P gene, the same region of the nested PCR product, using an AlkPhos Direct kit (Amersham Pharmacia Biotech).

SEROLOGICAL TEST

Antimumps IgG titre was measured in 16 patients with ulcerative colitis, in 16 patients with Crohn's disease, and in 16 normal controls by enzyme linked immunosorbent assay (ELISA) using the mumps IgG (II)-EIA Seiken kit (Denka Seiken, Tokyo, Japan). Briefly, a plastic plate was coated with purified whole virion of mumps virus and covered with patient serum. The plate was then incubated with a peroxidase labelled antihuman IgG polyclonal antibody. Enzyme activity was measured spectrophotometrically after addition of 3, 3′, 5, 5′-tetramethylbenzidine as the specific chromogenic substrate. Clinical details of the patients are shown in table 3. None of the cases received mumps vaccination.

Statistical analysis was done using the Mann-Whitney U test, and p<0.05 was considered significant.

AMPLIFICATION OF THE MUMPS VIRUS GENOME

Sensitivity of RT-PCR

We detected the mumps virus P gene in 120 atto grams (ag) of RNA (fig 1A) and the HN gene in 12 femto grams (fg) of RNA (fig 1B) extracted from mumps virus infected Vero cells. We were also able to detect both P and HN genes of the mumps virus in a throat swab collected from a patient with mumps parotitis.

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Figure 1

Nested reverse transcription-polymerase chain reaction (RT-PCR) products from mumps virus infected cells. (A) Phosphoprotein (P) gene: molecular weight marker (lane 1), PCR product from 120 fg (lane 2), 12 fg (lane 3), 1.2 fg (lane 4), 120 ag (lane 5), and 12 ag (lane 6) of template RNA, and PCR product from a throat swab of mumps parotitis (lane 7). (B) Haemagglutinin- neuraminidase (HN) gene: molecular weight marker (lane 1), PCR product from 12 pg (lane 2), 1.2 pg (lane 3), 120 fg (lane 4), 12 fg (lane 5), and 1.2 fg (lane 6) of template RNA, and PCR product from a throat swab of mumps parotitis (lane 7).

Amplification of P gene

Single or multiple bands were observed in all samples encompassing intestinal specimens and PBL, but the sizes of these bands were not consistent with the expected band of mumps virus (199 bp). Eleven PCR products are shown in fig 2A. No hybrids were observed between these bands and labelled probe constructed of the mumps virus P gene (lanes 6–13; fig 2B) after Southern hybridisation. Strong hybrids were observed in positive controls of mumps virus (lanes 2, 3; fig 2B) and in the throat swab from the mumps parotitis patient (lane 15; fig 2B).

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Figure 2

(A) Nested polymerase chain reaction (PCR) products using primers to the phosphoprotein (P) gene. Molecular weight marker (lane 1), PCR products from intestinal specimens of ulcerative colitis (lanes 2–4), Crohn's disease (lanes 5–7), controls (lanes 8–10), PCR products from peripheral blood lymphocytes (PBL) of ulcerative colitis (lanes 11, 12), and PCR product without template DNA (lane 13), and from the throat swab of the patient with mumps parotitis (lane 14). (B) Southern hybridisation of PCR products using primers to P gene. Molecular weight marker (lane 1), PCR products from 1.2 fg (lane 2), 120 ag (lane 3), 12 ag (lane 4) of template RNA from mumps virus infected cells, blank (lane 5), PCR products from intestinal specimens of ulcerative colitis (lanes 6–8), Crohn's disease (lanes 9–11), PCR products from PBL of ulcerative colitis (lanes 12, 13), blank (lane 14), and PCR product from the throat swab of mumps parotitis (lane 15).

Amplification of HN gene

The expected band was not observed in any colonic mucosa or PBL sample. Four PCR products are shown in fig 3.

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Figure 3

Nested polymerase chain reaction (PCR) products using primers to the haemagglutinin-neuraminidase (HN) gene. Molecular weight marker (lane 1), PCR product from intestinal specimens of ulcerative colitis (lane 2), Crohn's disease (lane 3), control (lane 4), PCR product from peripheral blood lymphocytes (PBL) of ulcerative colitis (lane 5), PCR product without template DNA (lane 6), and PCR product from a throat swab of mumps parotitis (lane 7).

SEROLOGICAL TEST

Mean (SEM) age of the patients was 28.9 (2.0) years for those with ulcerative colitis, 25.3 (1.9) for those with Crohn's disease, and 28.3 (2.2) for the controls. Mean (SEM) birth year of the patients was 1965 (2.1) for ulcerative colitis, 1970 (2.1) for Crohn's disease, and 1968 (2.3) for the controls. There was no significant difference in age or birth year between the three groups. In the ELISA system, antimumps IgG titres showing over 4.0 EIA were judged positive, below 2.0 as negative, and between 2.0 and 4.0 as indeterminate. According to these criteria, 7/16 ulcerative colitis patients, 10/16 Crohn's disease patients, and 11/16 controls were judged to be positive for antimumps IgG. The mean (SEM) titre of antimumps IgG was 12.281 (7.831) EIA for ulcerative colitis, 7.675 (1.608) for Crohn's disease, and 8.637 (1.969) for the controls; there was no significant differenc between the three groups.