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Molecular and immunological methods to detect rotavirus in formalin-fixed tissue
Journal of Virological Methods 105 (2002) 305 /319 www.elsevier.com/locate/jviromet
Molecular and immunological methods to detect rotavirus in formalin-fixed tissue Kathleen M. Tatti a,*, Jon Gentsch b, Wun-Ju Shieh a, Tara Ferebee-Harris a, Maureen Lynch b, Joseph Bresee b, Baoming Jiang b, Sherif R. Zaki a, Roger Glass b a
Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Infectious Disease Pathology Activity, 1600 Clifton Road, NE, Mailstop G30, Atlanta, GA 30333, USA b Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Respiratory and Enteric Virus Branch, Gastroenteritis Section, Atlanta, GA 30333, USA Received 17 January 2002; received in revised form 23 May 2002; accepted 28 May 2002
Abstract In 1999, a tetravalent rhesus-based rotavirus vaccine was withdrawn from the market after reports of intussusception cases among vaccinated infants. Methods to detect rotavirus in formalin-fixed pathology specimens from such patients will be important in examining the possible associations between the vaccine and intussusception, in investigating fatalities caused by natural rotavirus infection, and in furthering our understanding of the pathogenesis of rotavirus disease. Three different methods, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and in situ hybridization (ISH), were developed to detect rotavirus in infected cell lines that were fixed in formalin and embedded in paraffin. Using specific primer pairs to identify the VP4 gene with a one-step RT-PCR method, we detected simian rotavirus strains RRV and YK-1 in the liver of an RRV-infected SCID mouse and in the small intestine of an YK-1 infected macaque, respectively. Using a two-step indirect immunoalkaline phosphatase technique, we found RRV antigens in the liver of an infected SCID mouse with a rabbit polyclonal anti-group A rotavirus antibody and a murine monoclonal anti-rotavirus VP2 antibody. Using riboprobes designed to detect RRV genes, VP4 and NSP4, we obtained a positive hybridization signal in the same area of the infected SCID mouse liver as the area in which rotavirus antigens were localized. These techniques should prove valuable to detect rotavirus antigens and nucleic acids in tissues from patients infected naturally with rotavirus or with intussususception associated with rotavirus vaccine. # 2002 Published by Elsevier Science B.V. Keywords: Rotavirus; Immunohistochemistry; RT-PCR; In situ hybridization
1. Introduction * Corresponding author. Tel.: /1-404-639-3797; fax: /1404-639-1377 E-mail address: [email protected] (K.M. Tatti).
Human rotavirus, the major etiologic agent of acute gastroenteritis in infants and young children, causes the death of 600 000 children per year,
0166-0934/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 1 6 6 - 0 9 3 4 ( 0 2 ) 0 0 1 2 4 - 6
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mostly in developing countries (Glass et al., 1997). Rotaviruses were discovered in 1963 in mice and associated immediately with gastroenteritis (Adams and Kraft, 1963). The subsequent recovery of rotaviruses from many animal species demonstrated its wide host range. Despite the extensive burden of rotavirus diarrhea among children worldwide, few studies have examined in depth the pathology of rotavirus infection in humans (Davidson et al., 1975; Davidson and Barnes, 1979; Morrison et al., 2001). Piglets infected with rotavirus developed desquamation of the epithelial cells of the villi of the small intestine, resulting in severe stunting, extensive damage to the microvilli, and accumulation of lipid in the cytoplasm (Woode et al., 1974, 1976; Hall et al., 1976) with the jejunum being the most severely affected region. Human rotavirus was first visualized by thin-section electron microscopy in the epithelial cells of duodenal mucosa from children with non-bacterial gastroenteritis in 1973 (Bishop et al., 1973). The pathogenesis of rotavirus infection is unclear, but several different mechanisms have been proposed to explain how rotavirus induces diarrhea. One mechanism suggests that the nonstructural protein NSP4 produced by rotavirus acts as a viral enterotoxin that induces a signal transduction pathway, culminating in diarrhea (Ball et al., 1996; Zhang et al., 2000). A second concept proposes that rotavirus activates the enteric nervous system stimulating cells of the intestinal wall to increase water secretion, resulting in diarrhea (Lundgren et al., 2000). The third mechanism has rotavirus infecting the mature enterocytes in the villous epithelium of the small intestine by direct cell membrane penetration, resulting in villous atrophy and cell death (Kaljot et al., 1988; Osborne et al., 1988). Malabsorption secondary to the enterocyte death induces diarrhea. Notably, the first two concepts presented to explain pathogenesis of rotavirus diarrhea do not involve observable tissue damage. In 1998, the first live-attenuated oral rhesushuman reassortant rotavirus tetravalent vaccine against the globally common serotypes G1 to G4 was licensed. Subsequent post-licensure investigations demonstrated that the incidence of intussus-
ception increased significantly among vaccinees during the 2 weeks after the first immunization dose (Centers for Disease Control and Prevention, 1999a). The mechanism of vaccine-associated intussusception is unknown, but the vaccine appears to be a contributory agent (Murphy et al., 2001). National recommendations for administering the vaccine were withdrawn in November 1999 (Centers for Disease Control and Prevention, 1999b). To examine the pathogenesis of intussusception after rotavirus vaccination, a group of molecular and/or immunological assays was needed to detect and to localize rotavirus in formalin-fixed, paraffin-embedded tissue. This study describes three techniques that were developed: reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern hybridization, immunohistochemistry (IHC), and in situ hybridization (ISH). These assays should not only help investigate the relationship of vaccine with intussusception, but also aid in understanding the pathogenesis of the severe and fatal cases of rotavirus diarrhea.
2. Materials and methods 2.1. Cell lines and virus strains The human rotavirus strains tested in this study were Wa (P1A[8], G1), DS-1 (P1B[4], G2), and US1205 (P2A[6], G9), and the simian rotavirus strains were RRV (P5B[3], G3) and YK-1 (P[?], G?), a recently isolated strain (B. Jiang, unpublished data). By nucleotide sequencing, strain YK1 is related to strain RRV (P5B[3], G3) in both its VP4 hypervariable region and VP7 protein (/89 / 90% amino acid identity), suggesting that they are antigenically closely related (B. Jiang et al., unpublished data). However, the P and G serotypes of YK-1 have not yet been studied by crossneutralization experiments. The VP4 genes of reference strains Wa (Accession number M96825), and RRV (Accession number M18736) were used for VP4 primer design. Probes were designed from the RRV NSP1 (Accession number Z32535) and NSP4 (Accession number L41247) genes.
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Fetal rhesus monkey kidney cells (MA104) were grown to confluence in roller bottles, washed three times with serum-free medium, and infected at about 0.1 PFU per cell with human rotavirus strains Wa, or DS-1, or US1205, or the rhesus rotavirus vaccine strain MMU 18006 (RRV) as described previously (Gentsch et al., 1993; Kirkwood et al., 1999). The final concentration of Type IX porcine trypsin (Sigma, St Louis, MO) was 1 mg/ml, except for RRV it was 0.25 mg/ml. The infected cells were incubated on a roller apparatus for 16 /17 h at 37 8C before harvesting. The cells were scraped from the monolayer with sterile plastic spatulas, combined with the infection media, and centrifuged at 1000 /g at room temperature (RT). The cell pellets were washed three times with phosphate-buffered saline (PBS), resuspended in 10% buffered formalin, and incubated for 24 h at RT. Finally, the cells were centrifuged and each cell pellet mixed with minced normal human tissue and embedded in paraffin. 2.2. Animal tissue SCID mice, strain CB-17, were infected with 1.2 /105 PFU of strain RRV and were euthanized with Metofane 17 days after inoculation. The liver tissue was excised, fixed in formalin, and embedded in paraffin. Liver tissue from a SCID mouse not infected with RRV served as a negative control. A macaque monkey, PFM-1, which was infected naturally with rotavirus, developed severe diarrhea and was euthanized. Simian rotavirus strain YK-1 was isolated from feces collected from the macaque during the illness (B. Jiang, unpublished data). The stomach, pylorus, duodenum, jejunum, ileum, colon, pancreas, and liver were removed immediately after necropsy, fixed in formalin for 3 weeks, and embedded in paraffin. After necropsy, normal intestinal tissue from a macaque was fixed in formalin for 3 days, embedded in paraffin, and served as a negative control in these experiments. 2.3. RT-PCR One 10-mm section of formalin-fixed, paraffinembedded tissue was placed in a microcentrifuge
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tube for each extraction. Paraffin was extracted with xylene and washed with ethanol to remove residual xylene. RNA was extracted from the formalin-fixed tissue with a commercial RNaid Plus kit purchased from Bio101 (Vista, CA). The tissue was lysed in a salt solution containing guanidine thiocyanate, protein extracted with phenol, and the RNA purified on a silica-based matrix. The manufacturer’s protocol was followed with two modifications, (1) the tissue was homogenized briefly with a Teflon pestle in the cell lysis buffer to allow adequate resuspension and enhance lysis. (2) The RNA was resuspended in only 30/40 ml of either 10 mM Tris buffer pH 7.5 or H2O. Two primer pairs were used to obtain partial products of gene 4 of strains RRV and YK-1 and two probes to confirm the identity of the PCR products (Table 1). A one-step RT-PCR kit (Qiagen, Valencia, CA) was used according to the manufacturer’s instructions with 30 pmol of each primer in the RT-PCR reaction. After denaturation of the dsRNA as described previously (Gentsch et al., 1992), reverse transcription was carried out for 30 min at 50 8C, followed by 15 min at 95 8C to inactivate the RT and activate the HotstarTaqTM DNA polymerase. Once cDNA was synthesized, 30 /40 cycles of PCR were performed with the subsequent step-cycles: 30 s, 94 8C; 30 s, 42 8C; 45 s, 72 8C, followed by a 7min extension at 72 8C and a 4 8C cooling step. The procedures employed for Southern hybridization and chemiluminescent detection of bound digoxigenin-labeled probe using commercial reagents from Boehringer Mannheim Biochemicals (Elkhart, IN) have been described previously (Ando et al., 1995; Leite et al., 1996). To determine the sensitivity of detection of RRV VP4 RNA by RT-PCR, rhesus rotavirus RNA was isolated from purified virions (Gentsch et al., 1992) and RNA was extracted from a rotavirus-negative, formalin-fixed simian tissue using a commercial RNaid Plus kit purchased from Bio101. RRV RNA was diluted serially from 1.16 /104 to 1.16 /10 3 viral particles in RNA from a rotavirus-negative tissue and analyzed with the RRV VP4 primer pair (Table 1) using the onestep RT-PCR assay aforementioned. To confirm
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Table 1 Oligonucleotide primer nomenclature, sequence and nucleotide position Primer designation
Gene target
RRV for RRV rev YK-1for YK-1rev
VP4 VP4 VP4 VP4
Nucleotide position
Primer sequence (5? /3?)
Melting temperature (8C)c,d
369 /387 549 /568 274 /292 536 /552
gct att tgg aac gca aga g tag tgg tca cat tcg gag tt gct ggt gta gtg gtt gaa g cgt ttc tcc gtt gta tg
60.7 59.6 57.5 53.1
Digoxigenin probes YK-1 VP4 RRV VP4
443 /460 466 /486
aaa cca cgc aga atg gaa tca caa tac gga cca tta caa
61.1 61.1
Primers for RNA probes RRVa VP4 RRVa VP4
186 /227 689 /705
85.1 86.4e
Waa
VP4
676 /723
Waa
VP4
1177 /1193
DS-1a DS-1a
VP4 VP4
1480 /1524 1984 /2001
RRVa RRVa
NSP1 NSP1
527-560 757 /773
RRVa RRVa
NSP4 NSP4
313 /344 687 /704
tac tgt aga acc ggt act tga tgg tcc tta tca acc aac ttc cgc aat taa tac gac tca cta tag gga gag ttt cgt gta ttc tga ab ctg cca cca att caa aat act aga aat gta gtt cca ttg cca tta tca cgc aat taa tac gac tca cta tag gga gaa ctg gcc atg cac cta cb cag gat tta gaa cgc cag ctt aat gat ttg cga gaa gag ttt a cgc aat taa tac gac tca cta tag gga gat tca gac gct tca gta acb tca tca cca tgc ata gat ttg cca tat aga ctt g cgc aat taa tac gac tca cta tag gga gat aag aac tat gcg act tb cta aag atg aaa ttg aaa ggc aaa tgg aca ga cgc aat taa tac gac tca cta tag gga gaa ttg gtt aaa cgg gat tab
88.8 90.5 87.7 87.7 79.7 85.4 77.9 87.7
a
The VP4 genes of reference strains Wa (P1A[8], G1; accession number M96825), DS-1 (P1B[4], G2) and RRV (P5B[3], G3; accession number M18736) were used for VP4 primer design; 1 probe each was designed from the RRV NSP1 (accession number Z32535) and NSP4 (accession number L41247) genes. b The underlined sequence represents the T7 RNA polymerase promoter sequence while the rotavirus-specific sequence is not underlined. c RT-PCR primer melting temperatures given for a salt concentration of 1 M and an oligonucleotide concentration of 100 pM. d The predicted melting temperatures (3/SSC, 50% formamide, homologous template) of the riboprobes were: 72.4 (RRV VP4), 72.7 (Wa VP4), 72.8 (DS-1 VP4), 71.2 (RRV NSP1), and 71.6 (RRV NSP4). e The melting temperature calculation included both the rotavirus-specific nucleotides and those of the T7 RNA polymerase promoter.
the identity of the PCR product from the RRV strain, Southern hybridization and chemiluminescent detection with the RRV-specific VP4 gene probe (Table 1) was performed (Ando et al., 1995; Leite et al., 1996). 2.4. ISH assays For ISH, the RNA probes were generated from PCR products amplified from the rotaviral genes VP4, NSP4, and NSP1 and tailed with the T7 promoter (Cone and Schlaepfer, 1997). This pro-
cedure was employed because the signal intensity was superior and the background signal reduced when compared with signals of plasmid-derived probes (Cone and Schlaepfer, 1997). The nucleotide position and sequence of the primers are given in Table 1. The T7 promoter is underlined in the primer sequence (Table 1) and occurs at the 5? end of the anti-sense strand, generating anti-sense RNA probe. Five anti-sense probes, two human rotavirus RNA probes for VP4 from strains Wa (518 bases) and DS-1 (522 bases) and three RRV RNA probes for VP4 (520 bases), NSP4 (392
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bases), and NSP1 (247 bases), were designed by this method (Table 1). Digoxigenin-labeled RNA probes were generated by in vitro transcription of the PCR products with T7 RNA polymerase, using a digoxigenin RNA-labeling kit from Boehringer Mannheim Biochemicals. To determine that adequate labeling occurred, the concentration of the digoxigenin-labeled RNA probes was ascertained by a DIG quantification test from Boehringer Mannheim Biochemicals. Thin (3 mm) sections of tissue were placed on Fisher Plus slides (Fisher Scientific, Pittsburgh, PA) and ISH was performed overnight at 37 8C as previously described (Burt et al., 1997) with two modifications: (1) Digestion of the tissues was conducted for 15 min with 0.1 mg/ml proteinase K in 0.6 M Tris (pH 7.5)/0.1% CaCl2 for cells minced with normal tissue and for 30 min at RT with animal tissue. A series of pilot experiments was performed to determine the optimal proteinase K concentration and the optimal digestion time for adequate penetration of the probe into either cells or tissue. (2) The optimal concentration of the RNA probe to be used on the tissue was determined by a series of titration experiments with cell controls. The RNA probes were diluted at 1:100 in hybridization buffer from Boehringer Mannheim Biochemicals containing 50% deionized formamide and 3/SSC. Any cross-hybridization was ascertained by hybridizing the digoxigenin-labeled human rotavirus Wa VP4 probe with RRVinfected MA104 cells and the digoxigenin-labeled RRV probe with Wa-infected MA104 cells. In addition, the specificity of the RRV VP4, NSP4 and NSP1 probes was determined by hybridizing the RRV probes to uninfected tissue and uninfected cell controls. 2.5. IHC assays For IHC assays, antibodies were tested on formalin-fixed, paraffin-embedded cell controls to determine the specificity and cross-reactivity of the antibodies (Table 2). The suitability of the following antibodies was ascertained: (1) rabbit polyclonal IgG antibodies enriched by IgG capture to Protein A Sepharose (Amersham, Piscataway, NJ) directed against the Wa strain, (2) a guinea pig
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hyperimmune antiserum, and (3) a panel of eight monoclonal antibodies (MAb) directed against rotaviral antigens (Table 2). Preliminary experiments were performed using serial dilutions of the antibody on control cells in order to determine the appropriate dilution of antibody to detect rotaviral antigens without a background reaction. The rabbit polyclonal antibody directed against the Wa strain and two MAbs, 3A-8 and B4-2, directed against viral proteins VP2 and NSP4, respectively, were chosen for the IHC assays on animal tissue. The appropriate negative control was performed in parallel for each experiment. The negative controls included preimmune rabbit serum, enriched for IgG by IgG capture to Protein A Sepharose (Amersham), normal mouse ascites fluid (NMAF) and normal guinea pig serum. IHC assays were carried out as described previously with a streptavidin-biotin alkaline phosphatase technique (Burt et al., 1997) with two modifications: (1) The optimal concentration of antibodies was ascertained by performing a preliminary series of titration experiments. (2) The specificity of staining was confirmed with NMAF or preimmune rabbit serum as the primary antibody. These primary antibodies were applied to non-rotavirus infected tissue sections and uninfected MA104 cells.
3. Results 3.1. RT-PCR Three methods to purify RNA from formalinfixed, paraffin-embedded tissue were evaluated. The RT-PCR results (data not shown) demonstrated that the most effective RNA recovery method generating PCR-amplifiable RNA required three steps. Cell lysis occurred in a guanidine salt-based system, protein was extracted with phenol, and contaminants were removed by binding the RNA to a silica-based matrix. Pilot studies demonstrated that binding to the silica resin enhanced the quality of the RNA preparation. The RNaid Plus kit from Bio101 incorporated all of these steps and was used for all subsequent RNA extractions. This method reproducibly
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Table 2 Antibodies tested in this study Antibody designation
Rotavirus immunogen Specificity
Preimmune rabbit serum, enriched for IgG / Rabbit polyclonal antibodies enriched by IgG capture Wa Normal guinea pig serum / Rotavirus guinea pig hyperimmune serum RRV MAb 631/9 Wa MAb 3A-8 Wa MAb B4-2 Wa MAb 631/24 Wa MAb 255/60 RRV MAb1A9 RRV MAb 2G4 OSU Normal mouse ascites fluid Negative control
yielded a PCR-amplifiable RNA preparation from formalin-fixed, paraffin-embedded tissue. The RT-PCR and probe hybridization results with the RRV and YK-1 primers yielded PCR amplicons in all infected tissues (Fig. 1). The correct-size PCR amplicon (200 bp) was obtained from RRV-infected SCID mouse liver tissue (Fig. 1A, lane 3) with RRV VP4 primers and confirmed to be RRV-derived by Southern hybridization with the RRV internal VP4 probe (data not shown). A faint PCR amplicon of 279 bp was obtained with the YK-1 VP4 primers in the RRVinfected SCID mouse (Fig. 1A, lane 10), however, this amplicon was not detected by Southern hybridization with a strain-specific digoxigeninlabeled YK-1 probe (Fig. 1B, lane 10), demonstrating that the product resulted from crosspriming of the YK-1 primers on the RRV template. The strain-specific binding of the YK-1 probes to the YK-1-positive control amplicon (Fig. 1B, lane 18), but not to the RRV-positive control amplicon (Fig. 1B, lane 17), was clearly demonstrated. Uninfected SCID mouse liver tissue did not generate a PCR amplicon with either RRV or YK-1 primers (Fig. 1A, lane 2 and 9, respectively) or hybridization signal with either RRV or YK-1 probes. A matrix is illustrated in Fig. 1C to indicate the primer pairs and the tissue or control RNA present in each lane of Fig. 1A and B. RNA extracts from two different blocks of YK1-infected monkey intestinal tissue were amplified with the YK-1 primers generating the correct-size
Working dilution Source
/ 1:400 Group A rotavirus 1:400 / 1:1000 Group A rotavirus 1:400 VP6 (SGII) 1:800 VP2 1:2000 NSP4 1:2000 VP6 (SGI and II) 1:100 VP6 (SGI) 1:800 VP4 (neutralizing) 1:250 VP4 (neutralizing) 1:250 1:1000
CDC CDC NIH NIH H Greenberg H Greenberg H Greenberg H Greenberg H Greenberg H Greenberg H Greenberg CDC
PCR amplicon of 279 bp (Fig. 1A, lanes 12 and 13) and confirmed by Southern hybridization (Fig. 1B, lanes 12 and 13). Amplification of RNA extracted from one block of YK-1-infected monkey intestinal tissue with the RRV primers generated a PCR amplicon of 200 bp (Fig. 1A, lane 5) that was confirmed by Southern hybridization with the strain-specific YK-1 probe (Fig. 1B, lane 5). The other RNA extract from the block of YK-1infected monkey intestinal tissue that generated less PCR amplicon with YK-1 primers (Fig. 1A, lane 13) did not generate a PCR amplicon with the RRV primers (Fig. 1A, lane 6). Uninfected monkey intestinal tissue did not generate an amplicon with either RRV or YK-1 primers (Fig. 1A, lanes 4, 7, 11 and 14) or a hybridization signal with either RRV or YK-1 probes. RRV and YK-1 RNA amplified with RRV and YK-1 primer pairs generated the correct size amplicon (Fig. 1A, lanes 17 and 18, respectively), and the water controls did not generate an amplicon with either primer pair (Fig. 1A, lane 8 and 15). The RT-PCR and probe hybridization results with the RRV VP4 primers yielded PCR amplicons only in samples containing RRV (Fig. 2). The correct-size PCR amplicon of 200 bp from the RRV VP4 gene was amplified by a one-step RTPCR method from 1.16 /104 viral particles (Fig. 2A, lane 2) to 1.16 /102 viral particles (Fig. 2A, lane 4) as determined by ethidium bromide staining of the agarose gel. After analysis of RT-PCR products on agarose gels, the RRV VP4 gene
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Fig. 1. Detection of simian rotaviruses RRV and YK-1 in infected tissues by RT-PCR. (A) Ethidium bromide stained agarose gel: lanes 2 /8 and 17 (RRV VP4 primer pair) and 9 /15 and 18 (YK-1 VP4 primer pair), products amplified from dsRNA extracts of uninfected (lanes 2 and 9) or RRV-infected (lanes 3, 10) SCID mouse tissue or uninfected (lanes 4, 7, 11, 14) or YK-1-infected (lanes 5, 6, 12, 13) simian tissue. Lanes 17 and 18, RRV and YK-1 RNA amplified with RRV and YK-1 primer pairs, respectively. Lanes 8 and 15 contained additional negative controls (H2O). Lane 16 was not loaded with a sample. (B) Results of Southern hybridization and chemiluminescent detection of the PCR products are shown in panel A using an YK-1-specific VP4 probe. Lanes 1 and 19, digoxigeninlabeled and unlabeled molecular weight markers, respectively, with the molecular size (base pairs) indicated for selected fragments. (C) A matrix is illustrated which indicates the primer pairs and control sample or tissue present in each lane. In this panel, MWM designates both digoxigenin-labeled and unlabeled molecular weight markers, UIM designates uninfected SCID mouse tissue, IM designates infected SCID mouse tissue, UIS designates uninfected simian tissue, and IS designates infected simian tissue.
product was confirmed by probe hybridization with a digoxigenin-labeled RRV-specific VP4 gene probe (Fig. 2B, lanes 2/5). The RRV VP4 gene can be detected by RT-PCR followed by probe hybridization in 12 viral particles (Fig. 2B, lane 5). A rotavirus-negative extract did not generate an amplicon with the RRV VP4 primer pair (Fig. 2A and B, lane 9).
3.2. ISH assays The hybridization signal from each probe was assessed on Wa-infected, RRV-infected, and uninfected cells and the intensity of the signal strength (0 to /4) was graded for each probe (Table 3). The VP4 probe designed from the Wa strain hybridized intensely with Wa-infected cells
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312
Fig. 2. Sensitivity of detection of rhesus rotavirus VP4 gene RNA by RT-PCR. Rhesus rotavirus RNA from purified virions was diluted serially in an RNA extract from a rotavirusnegative, formalin-fixed simian tissue specimen and then analyzed with RRV VP4 primer pair. (A) Ethidium bromide stained agarose gel: lane DM, digoxigenin-labeled molecular size markers; lanes 1 /8 products amplified from dsRNA extracted from the number of virus particles indicated: 1.16/ 104 (lane 1), 1.16/103 (lane 2), 1.16/102 (lane 3), 1.16/101 (lane 4), 1.16 /100 (lane 5). 1.16 /10 1 (lane 6), 1.16/10 2 (lane 7), 1.16/10 3 (lane 8); lane 9, product amplified from a rotavirus negative stool RNA extract. (B) Results of Southern Hybridization and chemiluminescent detection of the PCR products shown in panel A using an RRV-specific VP4 gene probe (Table 1). The molecular size (base pairs) of the digoxigenin-labeled molecular weight markers is indicated on the left side of Panel B.
(Fig. 3A), but not with RRV-infected cells (Fig. 3B) or uninfected cells. The VP4 probe from the DS-1 strain hybridized with DS-1 and Wa-infected
cells (data not shown), perhaps due to the 89.7% (Gorziglia et al., 1988) sequence identity of the VP4 genes from the two strains and to the fact that we constructed the probe in a region of DS-1 VP4 (nt 1480/2001) in a relatively conserved region of the VP4 gene. Only the VP4 probe from the Wa strain was used further in the animal studies. The RRV NSP4 probe hybridized to both RRVand Wa-infected cells, but the intensity of the signal from the RRV-infected cells was stronger, /4, than that from the Wa-infected cells, /2 (Table 3). Based on the 81% identity of the RRV and Wa NSP4 genes, these results are reasonable. The RRV NSP1 and RRV VP4 (Fig. 3C) probes hybridized only to the RRV-infected cells, but the intensity of the signal from the NSP1 probe was less than that of the VP4 probe. The RRV VP4 gene is 59% identical to the Wa VP4 gene (Fig. 3D) and the RRV NSP1 gene is about 52% identical to the Wa NSP1 gene, explaining the lack of hybridization to the Wa-infected cells. None of the three RRV probes hybridized with uninfected cells (Table 3). Based on intensity of the hybridization signal, the RRV VP4 probe was the most specific while the RRV NSP4 probe was the most sensitive for the detection of RRV nucleic acids. Therefore, the RRV VP4 and NSP4 probes were selected for further analysis. The RRV NSP4 probe hybridized efficiently to the superficial epithelial cells of the ileum and jejunum in the YK-1-infected monkey (Fig. 3E), while no hybridization was evident with the RRV VP4 probe. These results are consistent with the
Table 3 Rotavirus ISH results Cell line or tissuea/infecting strainb Probes VP4 from Wa strain (human) VP4 from RRV strain (simian) NSP4 from RRV strain (simian) MA104/Wa MA104/RRV Macaque/YK-1 SCID mouse/RRV a b c d
////c / / /
/d //// / //
// //// /// ////
The uninfected cell control, uninfected macaque intestinal tissue, and uninfected SCID mouse liver were negative. Wa (P1A[8], G1) is a human rotavirus strain, while RRV (P5B[3], G3) and YK-1 (P[?], G?) are simian strains. / Is equal to the presence of granular staining, ranging from / to //// as the most intense. / Is equal to the absence of granular staining.
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313
Fig. 3
specificities of the two probes, because NSP4 genes from different strains exhibit a greater degree of sequence conservation than do the corresponding VP4 genes. No signal was evident in the uninfected
monkey tissue with either the RRV VP4 or the NSP4 probe (Fig. 3F). The Wa VP4 probe did not hybridize to either the YK-1- infected or the uninfected monkey tissue.
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Table 4 Rotavirus IHC results Cell line or tissuea/infecting strainb
MA104/US1205 MA104/Wa MA104/DS-1 MA104/RRV Macaque/YK-1 SCID mouse/RRV
Antibody Rabbit polyclonal antibody
Preimmune rabbit serum
Rota VP2, MAb 3A-8
Rota NSP4, MAb B4-2
/c / / / / /
/d / / / / /
/ / / / / /
/ / / / / /
a
The uninfected cell control, uninfected macaque intestinal tissue, and uninfected SCID mouse liver were negative. US1205 (P2A[6], G9), Wa (P1A[8], G1) and DS-1 (P1B[4], G2) are human rotavirus strains, while RRV (P5B[3], G3) and YK-1 (P[?], G?) are simian strains. c / Is equal to the presence of granular staining. d / Is equal to the absence of granular staining. b
The RRV NSP4 probe hybridized intensely in the regions of the liver where inflammatory infiltrates and necrosis were visible in the RRVinfected SCID mouse (Fig. 3G). Similarly, the RRV VP4 probe hybridized in the same region of the RRV-infected SCID mouse, but less intensely than the NSP4 probe. No signal was evident in the uninfected SCID mouse from either the RRV VP4 (Fig. 3H) or the NSP4 probe. The Wa VP4 probe did not hybridize to either the RRV-infected SCID mouse or the uninfected SCID mouse.
3.3. IHC assays Eleven antibodies were evaluated in the IHC tests for their ability to detect rotavirus antigens in formalin-fixed tissue. Based on their reactivities with positive cell controls (Table 4), the rabbit polyclonal antiserum and two MAbs, VP2 (MAb 3A-8) and NSP4 (MAb B4-2), were selected for use in the IHC assays for the detection of RRV in animal tissues. The polyclonal hyperimmune gui-
nea pig antiserum reacted with the uninfected cell controls and was not used. The rabbit polyclonal antibody and the NSP4 MAb reacted with antigens from human rotavirus strains Wa (Fig. 4A) and DS-1 and from simian rotavirus strains RRV and YK-1 in infected cell controls (Table 4). The characteristic granular staining in the cytoplasm of the infected cells is absent in the adjacent human control tissue. No staining was present utilizing the preimmune rabbit antiserum in the Wa-infected or RRVinfected cell controls (Fig. 4B and E, respectively). The VP2 MAb reacted with RRV antigens, but not YK-1 antigens, generating granular staining in the cytoplasm of the RRV-infected cells, and with human rotavirus, DS-1 antigens, but not Wa antigens (Fig. 3D, Table 4). No staining occurred for each antibody source with uninfected cell controls (Fig. 4C and F). The polyclonal rabbit antibody and NSP4 MAb showed diffuse granular staining in the superficial epithelial cells of the ileum and jejunum from the macaque, infected with the simian strain YK-1
Fig. 3. Localization of rotaviral nucleic acid in infected and uninfected cells and tissues by ISH. (A /B) MA104 cells infected with the Wa strain (A) or RRV strain (B) that are hybridized with VP4 digoxigenin-labeled pro1be from Wa. (C /D) MA104 cells infected with the RRV strain (C) or Wa strain (D) that are hybridized with VP4 digoxigenin-labeled probe from RRV. (E /F) Small intestine tissue of macaque monkey infected with YK-1 strain (E) and small intestine tissue of uninfected monkey (F) hybridized with NSP4 digoxigenin-labeled probe from RRV. (G /H) Liver tissue of SCID mouse infected with RRV strain (G) and liver tissue of uninfected SCID mouse (H) hybridized with VP4 digoxgenin-labeled probe from RRV. Immunoalkaline phosphatase staining was performed using a naphthol fast red substrate with light hematoxylin counterstain. (A /H) Original magnification, /50.
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Fig. 4
(Fig. 4G and H). The immunostaining by the VP2 MAb was focal in nature in the epithelial cells of the mucosa in the macaque and absent in the mucosa of the uninfected tissue (Fig. 4I). The immunostaining was not observed in the large
intestine, pancreas, liver, lymphoid follicles, or uninfected cells. Rotavirus antigens were demonstrated in the liver in the SCID mouse by the polyclonal rabbit antibody (Fig. 4J) and VP2 MAb (Fig. 4K), but
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not with the NSP4 MAb. Immunostaining also occurred in the periportal areas with inflammation and necrosis, similar to the areas showing hybridization by ISH. The VP2 MAb generated more intense staining in the intestinal epithelial cells of the macaque than in the necrotic region of the SCID mouse liver. No immunostaining occurred in the uninfected SCID mouse liver (Fig. 4L and data not shown).
4. Discussion In this study, RT-PCR, ISH, and IHC assays were developed to detect and localize rotavirus nucleic acids and antigens in formalin-fixed, paraffin-embedded tissue. We detected simian rotavirus RNA from formalin-fixed, paraffinembedded infected cells and tissues by RT-PCR with primers designed to a region of the VP4 gene of simian rotavirus strains. RT-PCR followed by Southern hybridization with probes that hybridize internally to the PCR-amplified region of the VP4 gene significantly enhanced this detection. The ISH results with the infected cell controls demonstrate that the VP4 riboprobes in ISH differentiated between human and rhesus rotavirus VP4 regions for the strains tested. One human rotavirus RNA probe, VP4, could be used to localize viral nucleic acid in natural human rotavirus infections, while two RRV RNA probes, NSP4 and VP4, could be used in cases associated with rhesus rotavirus infections. As the rabbit polyclonal antibody preparation reacted with all the human and simian rotavirus strains tested, it could be used for diagnostic purposes to screen for rotavirus infections. Based on cell controls, the rabbit polyclonal antibody and two MAbs, VP2 and NSP4, could be used to localize RRV antigens in
cases associated with rhesus rotavirus infections while preserving tissue morphology. In the macaque infected with the simian strain YK-1, rotavirus antigens and RNA were localized to the epithelial cells of the ileum and jejunum using three rotavirus antibody reagents and the NSP4 riboprobe, respectively. Cross-hybridization between the RRV NSP4 probe and YK-1 RNA was expected due to high identity (91%) between RRV and YK-1 NSP4 genes in the probe region. In contrast, the RRV VP4 riboprobe did not hybridize to the YK-1 RNA in the epithelial cells of the intestinal mucosa probably because of the lower identity (83%) of that probe to the YK-1 VP4 in the probe region. Histopathological analysis of the RRV-infected SCID mouse liver identified regions containing necrotic tissue and infiltrate that were the same areas in which RRV RNA was localized by the NSP4 and VP4 riboprobes, and RRV antigens were localized with VP2 MAb and the rabbit polyclonal antibody. As the RT-PCR and ISH analysis was comparable to the IHC results in both animal tissues, these results demonstrate the value of these three techniques in documenting RRV infections. Previously, an indirect immunoperoxidase technique to detect rotavirus antigen in paraffinembedded calf intestinal tissue (Parsons et al., 1984) and human biopsy intestinal tissue (Graham and Estes, 1979) demonstrated that rotavirus infected the enterocytes of the villus epithelium in the small intestine. More recently, employing an indirect immunoperoxidase test with a commercially available polyclonal rabbit antibody (DAKO, Carpenteria, CA), rotavirus was detected in formalin-fixed, paraffin-embedded cells infected with human rotavirus (Cartun et al., 1993) and in the intestinal tissue of two human fatalities (Morrison et al., 2001). In our indirect immunoalkaline
Fig. 4. Immunohistochemical detection of rotaviral antigens in infected and uninfected cells and tissues with polyclonal and monoclonal antibodies. (A /C) MA104 cells infected with human rotavirus strain Wa incubated with rabbit polyclonal antibody (A), with preimmune rabbit serum (B) and uninfected MA104 cells with rabbit polyclonal antibody (C). (D /F) MA104 cells infected with simian rotavirus RRV and incubated with MAb VP2 (D) and with preimmune rabbit serum (E). Uninfected MA104 cells incubated with MAb VP2 (F). (G /I) Macaque small intestine infected with YK-1 strain incubated with rabbit polyclonal antibody (G) and MAb NSP4 (H) and uninfected monkey incubated with rabbit polyclonal antibody (I). (J /L) SCID mouse liver infected with RRV strain incubated with rabbit polyclonal antibody (J), MAb VP2 (K) and uninfected SCID mouse incubated with polyclonal rabbit antibody (L). Immunoalkaline phosphatase staining was performed using a naphthol fast red substrate with light hematoxylin counterstain. (A / G and I /L) Original magnification, /50; (H) original magnification, /100.
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phosphatase IHC method, two MAbs, VP2 and NSP4, as well as a polyclonal rabbit antibody reagent were effective in localizing rotavirus infections. We performed numerous cell controls, tissue controls, and cross-reactivity studies to address specificity issues in order to achieve a well-defined and well-characterized IHC protocol with several antibodies. The ISH method reported here for simian rotavirus-specific riboprobes allowed the localization of rotavirus infections to the epithelial cells of the small intestine and to the liver in formalin-fixed, paraffin-embedded tissues. Recently, a report describing an RT in situ PCR hybridization analysis with an human rotavirusspecific riboprobe has been published which localized human rotavirus to the mucosal cells of the small intestine and other tissues (Morrison et al., 2001). In an earlier study, Uhnoo et al. (1990) detected RRV strain MMU18006 in the liver of SCID mice by cell culture, immunofluorescence, and electron microscopy, illustrating that the tissue tropism of rotavirus was not restricted to the villus epithelium of the small intestine in an immunocompromised host. Similarly, Gilger et al. (1992) demonstrated by immunohistochemical evidence that rotavirus infects the liver and kidney of immunocompromised children. Our analyses demonstrated that RRV could also be detected in formalin-fixed liver tissues of RRV-infected SCID mice, supporting rotavirus hepatotropism. RTPCR is commonly performed to detect RNA in fecal samples (Wilde et al., 1990, 1991; Pang et al., 1999) and a previous study performed by Koopmans et al. (1993) demonstrated that rotavirus could be detected by RT-PCR with RNA extracted from formalin-fixed, paraffin-embedded tissue. In our present RT-PCR analysis, we reduced the quantity of formalin-fixed paraffinembedded tissue employed to extract RNA (one 10-mm section) and all rotavirus-infected tissues were positive by RT-PCR. It has been documented previously that PCR is 100 000 times more sensitive than standard electropherotype identification of rotavirus and 5000 times more sensitive than dot-blot hybridization (Xu et al., 1990; Kapikian and Chanock, 1996). In our study, amplification of RNA by a one-step RT-PCR method followed by Southern hybridiza-
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tion with chemiluminescent detection of bound probe allows the detection of the VP4 gene in approximately 12 viral particles, which illustrates the enhanced sensitivity of our probe-hybridization method. The increased sensitivity of the RTPCR assay is exemplified here, as one of the YK-1infected macaque tissue sections from the small intestine was negative by both ISH and IHC, but positive by RT-PCR. The most sensitive method developed in our study is RT-PCR, the most specific is the ISH, and the one with the broadest range of reactivity is the IHC assay. Taken together, these techniques have significant sensitivity and specificity to detect and to distinguish human and rhesus rotaviruses in naturally occurring infections. One caveat exists. Only the IHC and the ISH methods can be used to localize rotavirus antigens and nucleic acids in specific cells while preserving tissue morphology. Therefore, it may be useful to develop additional antibodies and in situ riboprobes to increase the sensitivity of the IHC and ISH tests, allowing either intracellular or intraluminal localization of antigens and nucleic acids in suspected cases with reduced concentrations of rotavirus. This study demonstrates the feasibility of undertaking IHC, ISH, and RT-PCR tests to detect and to localize the presence of animal and human rotavirus antigens and RNA in formalin-fixed, paraffin-embedded tissues from patients with intussusception associated with RRV vaccine and those infected with natural rotavirus. The development of these methods to detect rotavirus in tissue provides powerful tools to understand the pathogenesis of the severe and fatal cases of rotavirus diarrhea. Analysis of pathological specimens in retrospective studies with these sensitive and specific methods would allow us to determine the extent of infection by identifying rotavirusinfected tissues and to ascertain if such findings reflect an actual tissue infection or a systemic viremia.
Acknowledgements We wish to thank the following individuals: Dr Harold McClure and Dr Marie Riepenhoff-Talty
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for kindly providing the tissue specimens; Dr Tamie Ando and Dr Taka Hoshino for generously giving us the polyclonal antibodies; Dr Harry Greenberg for making available the many monoclonal antibodies used in this study; and Dixie Griffin for her technical assistance in sequencing gene fragments of the YK-1 strain. We also want to thank John O’Connor for his editorial assistance.
References Adams, W.R., Kraft, L.M., 1963. Epizootic diarrhea of infant mice: identification of the etiologic agent. Science 141, 359 / 360. Ando, T., Jin, Q., Gentsch, J.R., Monroe, S.S., Noel, J.S., Dowell, S.F., Cicirello, H.G., Kohn, M.A., Glass, R.I., 1995. Epidemiologic applications of novel molecular methods to detect and differentiate small round structured viruses (Norwalk-like viruses). J. Med. Virol. 47, 145 /152. Ball, J.M., Tian, P., Zeng, C.Q.-Y., Morris, A.P., Estes, M.K., 1996. Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272, 101 /104. Bishop, R.F., Davidson, G.P., Holmes, I.H., Ruck, B.J., 1973. Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet ii, 1281 /1283. Burt, F.J., Swanepoel, R., Shieh, W.-J., Smith, J.F., Leman, P.A., Greer, P.W., Coffield, L.M., Rollin, P.E., Ksiazek, T.G., Peters, C.J., Zaki, S.R., 1997. Immunohistochemical and in situ localization of Crimean-Congo Hemorrhagic Fever (CCHF) virus in human tissues and implications for CCHF pathogenesis. Arch. Pathol. Lab. Med. 121, 839 / 846. Cartun, R.W., Van Kruiningen, H.J., Pedersen, C.A., Berman, M.M., 1993. An immunocytochemical search for infectious agents in Crohn’s disease. Mod. Pathol. 6, 212 /219. Centers for Disease Control and Prevention, 1999a. Intussusception among recipients of rotavirus vaccine-United States, 1998 /1999. MMWR 48, 577 /581. Centers for Disease Control and Prevention, 1999b. Withdrawal of rotavirus vaccine recommendation. MMWR 48, 1007. Cone, R.W., Schlaepfer, E., 1997. Improved in situ hybridization to HIV with RNA probes derived from PCR products. J. Hist. Cytochem. 45, 721 /727. Davidson, G.P., Barnes, G.L., 1979. Structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis. Acta Paediatr. Scand. 68, 181 /186. Davidson, G.P., Bishop, R.F., Townley, R.R., Holmes, I.H., 1975. Importance of a new virus in acute sporadic enteritis in children. Lancet i, 242 /246.
Gentsch, J.R., Glass, R.I., Woods, P., Gouvea, V., Gorziglia, M., Flores, J., Das, B.K., Bhan, M.K., 1992. Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30, 1365 /1373. Gentsch, J.R., Das, B.K., Jiang, B., Bhan, M.K., Glass, R.I., 1993. Similarity of the VP4 protein of rotavirus strain 116E to that of the bovine B223 strain. Virology 194, 424 /430. Gilger, M.A., Matson, D.O., Conner, M.E., Rosenblatt, H.M., Finegold, M.J., Estes, M.K., 1992. Extraintestinal rotavirus infections in children with immunodeficiency. J. Pediatr. 120, 912 /917. Glass, R.I., Bresee, J.S., Parashar, U., Miller, M., Gentsch, J.R., 1997. Rotavirus vaccines at the threshold. Nat. Med. 3, 1324 /1325. Gorziglia, M., Green, K., Nishikawa, K., Taniguchi, K., Jones, R., Kapikian, A.Z., Chanock, R.M., 1988. Sequence of the fourth gene of human rotaviruses recovered from asymptomatic or symptomatic infections. J. Virol. 62, 2978 /2984. Graham, D.Y., Estes, M.K., 1979. Comparison of methods for immunocytochemical detection of rotavirus infections. Infect. Immun. 26, 686 /689. Hall, G.A., Bridger, J.C., Chandler, R.L., Woode, G.N., 1976. Gnotobiotic piglets experimentally infected with neonatal calf diarrhoea reovirus-like agent (rotavirus). Vet. Pathol. 13, 197 /210. Kaljot, K.T., Shaw, R.D., Rubin, D.H., Greenberg, H.B., 1988. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis. J. Virol. 62, 1136 /1144. Kapikian, A.Z., Chanock, R.M., 1996. Rotaviruses. In: Fields, B.N., et al. (Ed.), Fields Virology, third ed.. LippincottRaven, Philadelphia, PA, p. 1683. Kirkwood, C.D., Gentsch, J.R., Hoshino, Y., Clark, H.F., Glass, R.I., 1999. Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the US. Virology 256, 45 /53. Koopmans, M., Monroe, S.S., Coffield, L.M., Zaki, S.R., 1993. Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives. J. Virol. Methods 43, 189 /204. Leite, J.P., Alfieri, A.A., Woods, P., Glass, R.I., Gentsch, J.R., 1996. Rotavirus G and P types circulating in Brazil: characterization by RT-PCR, probe hybridization, and sequence analysis. Arch. Virol. 141, 2365 /2374. Lundgren, O., Peregrin, A.T., Persson, K., Kordasti, S., Uhnoo, I., Svensson, L., 2000. Role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea. Science 287, 491 /495. Morrison, C., Gilson, T., Nuovo, G.J., 2001. Histologic distribution of fatal rotaviral infection: an immunohistochemical and reverse transcriptase in situ polymerase chain reaction analysis. Hum. Pathol. 32, 216 /221. Murphy, T.V., Gargiullo, P.M., Massoudi, M.S., Nelson, D.B., Jumaan, A.O., Okoro, C.A., Zanardi, L.R., Setia, S., Fair, E., LeBaron, C.W., Wharton, M., Livengood, J.R., 2001. Intussusception among infants given an oral rotavirus vaccine. New Engl. J. Med. 344, 564 /572.
K.M. Tatti et al. / Journal of Virological Methods 105 (2002) 305 /319 Osborne, M.P., Haddon, S.J., Spencer, A.J., Collins, J., Starkey, W.G., Wallis, T.S., Clarke, G.J., Worton, K.J., Candy, D.C., Stephen, J., 1988. An electron microscopic investigation of time-related changes in the intestine of neonatal mice infected with murine rotavirus. J. Pediatr. Gastroenterol. Nutr. 7, 236 /248. Pang, X.L., Joensuu, J., Hoshino, Y., Kapikian, A.Z., Vesikari, T., 1999. Rotaviruses detected by reverse transcription polymerase reaction in acute gastroenteritis during a trial of rhesus /human reassortant rotavirus tetravalent vaccine: implications for vaccine efficacy analysis. J. Clin. Virol. 13, 9 /16. Parsons, K.R., Wilson, A.M., Hall, G.A., Bridger, J.C., Chanter, N., Reynolds, D.J., 1984. Localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase. J. Clin. Pathol. 37, 645 /650. Uhnoo, I., Riepenhoff-Talty, M., Dharakul, T., Chegas, P., Fisher, J.E., Greenberg, H.B., Ogra, P.L., 1990. Extramucosal spread and development of hepatitis B in immunodeficient and normal mice infected with rhesus rotavirus. J. Virol. 64, 361 /368.
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Wilde, J., Eiden, J., Yolken, R., 1990. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J. Clin. Microbiol. 28, 1300 /1307. Wilde, J., Yolken, R., Willoughby, R., Eiden, J., 1991. Improved detection of rotavirus shedding by polymerase chain reaction. Lancet 337, 323 /326. Woode, G.N., Bridger, J.C., Hall, G., Dennis, M.J., 1974. The isolation of a reovirus-like agent associated with diarrhoea in colostrum-deprived calves in Great Britain. Res. Vet. Sci. 16, 102 /105. Woode, G.N., Bridger, J., Hall, G.A., Jones, J.M., Jackson, G., 1976. The isolation of reovirus-like agents (rota-viruses) from acute gastroenteritis of piglets. J. Med. Microbiol. 9, 203 /209. Xu, L., Harbour, D., McCrae, M.A., 1990. The application of polymerase chain reaction to the detection of rotaviruses in faeces. J. Virol. Methods 27, 29 /37. Zhang, M., Zeng, C.Q.-Y., Morris, A.P., Estes, M.K., 2000. A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells. J. Virol. 74, 11663 /11670.
Molecular and immunological methods to detect rotavirus in formalin-fixed tissue Kathleen M. Tatti a,*, Jon Gentsch b, Wun-Ju Shieh a, Tara Ferebee-Harris a, Maureen Lynch b, Joseph Bresee b, Baoming Jiang b, Sherif R. Zaki a, Roger Glass b a
Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Infectious Disease Pathology Activity, 1600 Clifton Road, NE, Mailstop G30, Atlanta, GA 30333, USA b Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Respiratory and Enteric Virus Branch, Gastroenteritis Section, Atlanta, GA 30333, USA Received 17 January 2002; received in revised form 23 May 2002; accepted 28 May 2002
Abstract In 1999, a tetravalent rhesus-based rotavirus vaccine was withdrawn from the market after reports of intussusception cases among vaccinated infants. Methods to detect rotavirus in formalin-fixed pathology specimens from such patients will be important in examining the possible associations between the vaccine and intussusception, in investigating fatalities caused by natural rotavirus infection, and in furthering our understanding of the pathogenesis of rotavirus disease. Three different methods, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and in situ hybridization (ISH), were developed to detect rotavirus in infected cell lines that were fixed in formalin and embedded in paraffin. Using specific primer pairs to identify the VP4 gene with a one-step RT-PCR method, we detected simian rotavirus strains RRV and YK-1 in the liver of an RRV-infected SCID mouse and in the small intestine of an YK-1 infected macaque, respectively. Using a two-step indirect immunoalkaline phosphatase technique, we found RRV antigens in the liver of an infected SCID mouse with a rabbit polyclonal anti-group A rotavirus antibody and a murine monoclonal anti-rotavirus VP2 antibody. Using riboprobes designed to detect RRV genes, VP4 and NSP4, we obtained a positive hybridization signal in the same area of the infected SCID mouse liver as the area in which rotavirus antigens were localized. These techniques should prove valuable to detect rotavirus antigens and nucleic acids in tissues from patients infected naturally with rotavirus or with intussususception associated with rotavirus vaccine. # 2002 Published by Elsevier Science B.V. Keywords: Rotavirus; Immunohistochemistry; RT-PCR; In situ hybridization
1. Introduction * Corresponding author. Tel.: /1-404-639-3797; fax: /1404-639-1377 E-mail address: [email protected] (K.M. Tatti).
Human rotavirus, the major etiologic agent of acute gastroenteritis in infants and young children, causes the death of 600 000 children per year,
0166-0934/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 1 6 6 - 0 9 3 4 ( 0 2 ) 0 0 1 2 4 - 6
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mostly in developing countries (Glass et al., 1997). Rotaviruses were discovered in 1963 in mice and associated immediately with gastroenteritis (Adams and Kraft, 1963). The subsequent recovery of rotaviruses from many animal species demonstrated its wide host range. Despite the extensive burden of rotavirus diarrhea among children worldwide, few studies have examined in depth the pathology of rotavirus infection in humans (Davidson et al., 1975; Davidson and Barnes, 1979; Morrison et al., 2001). Piglets infected with rotavirus developed desquamation of the epithelial cells of the villi of the small intestine, resulting in severe stunting, extensive damage to the microvilli, and accumulation of lipid in the cytoplasm (Woode et al., 1974, 1976; Hall et al., 1976) with the jejunum being the most severely affected region. Human rotavirus was first visualized by thin-section electron microscopy in the epithelial cells of duodenal mucosa from children with non-bacterial gastroenteritis in 1973 (Bishop et al., 1973). The pathogenesis of rotavirus infection is unclear, but several different mechanisms have been proposed to explain how rotavirus induces diarrhea. One mechanism suggests that the nonstructural protein NSP4 produced by rotavirus acts as a viral enterotoxin that induces a signal transduction pathway, culminating in diarrhea (Ball et al., 1996; Zhang et al., 2000). A second concept proposes that rotavirus activates the enteric nervous system stimulating cells of the intestinal wall to increase water secretion, resulting in diarrhea (Lundgren et al., 2000). The third mechanism has rotavirus infecting the mature enterocytes in the villous epithelium of the small intestine by direct cell membrane penetration, resulting in villous atrophy and cell death (Kaljot et al., 1988; Osborne et al., 1988). Malabsorption secondary to the enterocyte death induces diarrhea. Notably, the first two concepts presented to explain pathogenesis of rotavirus diarrhea do not involve observable tissue damage. In 1998, the first live-attenuated oral rhesushuman reassortant rotavirus tetravalent vaccine against the globally common serotypes G1 to G4 was licensed. Subsequent post-licensure investigations demonstrated that the incidence of intussus-
ception increased significantly among vaccinees during the 2 weeks after the first immunization dose (Centers for Disease Control and Prevention, 1999a). The mechanism of vaccine-associated intussusception is unknown, but the vaccine appears to be a contributory agent (Murphy et al., 2001). National recommendations for administering the vaccine were withdrawn in November 1999 (Centers for Disease Control and Prevention, 1999b). To examine the pathogenesis of intussusception after rotavirus vaccination, a group of molecular and/or immunological assays was needed to detect and to localize rotavirus in formalin-fixed, paraffin-embedded tissue. This study describes three techniques that were developed: reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern hybridization, immunohistochemistry (IHC), and in situ hybridization (ISH). These assays should not only help investigate the relationship of vaccine with intussusception, but also aid in understanding the pathogenesis of the severe and fatal cases of rotavirus diarrhea.
2. Materials and methods 2.1. Cell lines and virus strains The human rotavirus strains tested in this study were Wa (P1A[8], G1), DS-1 (P1B[4], G2), and US1205 (P2A[6], G9), and the simian rotavirus strains were RRV (P5B[3], G3) and YK-1 (P[?], G?), a recently isolated strain (B. Jiang, unpublished data). By nucleotide sequencing, strain YK1 is related to strain RRV (P5B[3], G3) in both its VP4 hypervariable region and VP7 protein (/89 / 90% amino acid identity), suggesting that they are antigenically closely related (B. Jiang et al., unpublished data). However, the P and G serotypes of YK-1 have not yet been studied by crossneutralization experiments. The VP4 genes of reference strains Wa (Accession number M96825), and RRV (Accession number M18736) were used for VP4 primer design. Probes were designed from the RRV NSP1 (Accession number Z32535) and NSP4 (Accession number L41247) genes.
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Fetal rhesus monkey kidney cells (MA104) were grown to confluence in roller bottles, washed three times with serum-free medium, and infected at about 0.1 PFU per cell with human rotavirus strains Wa, or DS-1, or US1205, or the rhesus rotavirus vaccine strain MMU 18006 (RRV) as described previously (Gentsch et al., 1993; Kirkwood et al., 1999). The final concentration of Type IX porcine trypsin (Sigma, St Louis, MO) was 1 mg/ml, except for RRV it was 0.25 mg/ml. The infected cells were incubated on a roller apparatus for 16 /17 h at 37 8C before harvesting. The cells were scraped from the monolayer with sterile plastic spatulas, combined with the infection media, and centrifuged at 1000 /g at room temperature (RT). The cell pellets were washed three times with phosphate-buffered saline (PBS), resuspended in 10% buffered formalin, and incubated for 24 h at RT. Finally, the cells were centrifuged and each cell pellet mixed with minced normal human tissue and embedded in paraffin. 2.2. Animal tissue SCID mice, strain CB-17, were infected with 1.2 /105 PFU of strain RRV and were euthanized with Metofane 17 days after inoculation. The liver tissue was excised, fixed in formalin, and embedded in paraffin. Liver tissue from a SCID mouse not infected with RRV served as a negative control. A macaque monkey, PFM-1, which was infected naturally with rotavirus, developed severe diarrhea and was euthanized. Simian rotavirus strain YK-1 was isolated from feces collected from the macaque during the illness (B. Jiang, unpublished data). The stomach, pylorus, duodenum, jejunum, ileum, colon, pancreas, and liver were removed immediately after necropsy, fixed in formalin for 3 weeks, and embedded in paraffin. After necropsy, normal intestinal tissue from a macaque was fixed in formalin for 3 days, embedded in paraffin, and served as a negative control in these experiments. 2.3. RT-PCR One 10-mm section of formalin-fixed, paraffinembedded tissue was placed in a microcentrifuge
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tube for each extraction. Paraffin was extracted with xylene and washed with ethanol to remove residual xylene. RNA was extracted from the formalin-fixed tissue with a commercial RNaid Plus kit purchased from Bio101 (Vista, CA). The tissue was lysed in a salt solution containing guanidine thiocyanate, protein extracted with phenol, and the RNA purified on a silica-based matrix. The manufacturer’s protocol was followed with two modifications, (1) the tissue was homogenized briefly with a Teflon pestle in the cell lysis buffer to allow adequate resuspension and enhance lysis. (2) The RNA was resuspended in only 30/40 ml of either 10 mM Tris buffer pH 7.5 or H2O. Two primer pairs were used to obtain partial products of gene 4 of strains RRV and YK-1 and two probes to confirm the identity of the PCR products (Table 1). A one-step RT-PCR kit (Qiagen, Valencia, CA) was used according to the manufacturer’s instructions with 30 pmol of each primer in the RT-PCR reaction. After denaturation of the dsRNA as described previously (Gentsch et al., 1992), reverse transcription was carried out for 30 min at 50 8C, followed by 15 min at 95 8C to inactivate the RT and activate the HotstarTaqTM DNA polymerase. Once cDNA was synthesized, 30 /40 cycles of PCR were performed with the subsequent step-cycles: 30 s, 94 8C; 30 s, 42 8C; 45 s, 72 8C, followed by a 7min extension at 72 8C and a 4 8C cooling step. The procedures employed for Southern hybridization and chemiluminescent detection of bound digoxigenin-labeled probe using commercial reagents from Boehringer Mannheim Biochemicals (Elkhart, IN) have been described previously (Ando et al., 1995; Leite et al., 1996). To determine the sensitivity of detection of RRV VP4 RNA by RT-PCR, rhesus rotavirus RNA was isolated from purified virions (Gentsch et al., 1992) and RNA was extracted from a rotavirus-negative, formalin-fixed simian tissue using a commercial RNaid Plus kit purchased from Bio101. RRV RNA was diluted serially from 1.16 /104 to 1.16 /10 3 viral particles in RNA from a rotavirus-negative tissue and analyzed with the RRV VP4 primer pair (Table 1) using the onestep RT-PCR assay aforementioned. To confirm
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Table 1 Oligonucleotide primer nomenclature, sequence and nucleotide position Primer designation
Gene target
RRV for RRV rev YK-1for YK-1rev
VP4 VP4 VP4 VP4
Nucleotide position
Primer sequence (5? /3?)
Melting temperature (8C)c,d
369 /387 549 /568 274 /292 536 /552
gct att tgg aac gca aga g tag tgg tca cat tcg gag tt gct ggt gta gtg gtt gaa g cgt ttc tcc gtt gta tg
60.7 59.6 57.5 53.1
Digoxigenin probes YK-1 VP4 RRV VP4
443 /460 466 /486
aaa cca cgc aga atg gaa tca caa tac gga cca tta caa
61.1 61.1
Primers for RNA probes RRVa VP4 RRVa VP4
186 /227 689 /705
85.1 86.4e
Waa
VP4
676 /723
Waa
VP4
1177 /1193
DS-1a DS-1a
VP4 VP4
1480 /1524 1984 /2001
RRVa RRVa
NSP1 NSP1
527-560 757 /773
RRVa RRVa
NSP4 NSP4
313 /344 687 /704
tac tgt aga acc ggt act tga tgg tcc tta tca acc aac ttc cgc aat taa tac gac tca cta tag gga gag ttt cgt gta ttc tga ab ctg cca cca att caa aat act aga aat gta gtt cca ttg cca tta tca cgc aat taa tac gac tca cta tag gga gaa ctg gcc atg cac cta cb cag gat tta gaa cgc cag ctt aat gat ttg cga gaa gag ttt a cgc aat taa tac gac tca cta tag gga gat tca gac gct tca gta acb tca tca cca tgc ata gat ttg cca tat aga ctt g cgc aat taa tac gac tca cta tag gga gat aag aac tat gcg act tb cta aag atg aaa ttg aaa ggc aaa tgg aca ga cgc aat taa tac gac tca cta tag gga gaa ttg gtt aaa cgg gat tab
88.8 90.5 87.7 87.7 79.7 85.4 77.9 87.7
a
The VP4 genes of reference strains Wa (P1A[8], G1; accession number M96825), DS-1 (P1B[4], G2) and RRV (P5B[3], G3; accession number M18736) were used for VP4 primer design; 1 probe each was designed from the RRV NSP1 (accession number Z32535) and NSP4 (accession number L41247) genes. b The underlined sequence represents the T7 RNA polymerase promoter sequence while the rotavirus-specific sequence is not underlined. c RT-PCR primer melting temperatures given for a salt concentration of 1 M and an oligonucleotide concentration of 100 pM. d The predicted melting temperatures (3/SSC, 50% formamide, homologous template) of the riboprobes were: 72.4 (RRV VP4), 72.7 (Wa VP4), 72.8 (DS-1 VP4), 71.2 (RRV NSP1), and 71.6 (RRV NSP4). e The melting temperature calculation included both the rotavirus-specific nucleotides and those of the T7 RNA polymerase promoter.
the identity of the PCR product from the RRV strain, Southern hybridization and chemiluminescent detection with the RRV-specific VP4 gene probe (Table 1) was performed (Ando et al., 1995; Leite et al., 1996). 2.4. ISH assays For ISH, the RNA probes were generated from PCR products amplified from the rotaviral genes VP4, NSP4, and NSP1 and tailed with the T7 promoter (Cone and Schlaepfer, 1997). This pro-
cedure was employed because the signal intensity was superior and the background signal reduced when compared with signals of plasmid-derived probes (Cone and Schlaepfer, 1997). The nucleotide position and sequence of the primers are given in Table 1. The T7 promoter is underlined in the primer sequence (Table 1) and occurs at the 5? end of the anti-sense strand, generating anti-sense RNA probe. Five anti-sense probes, two human rotavirus RNA probes for VP4 from strains Wa (518 bases) and DS-1 (522 bases) and three RRV RNA probes for VP4 (520 bases), NSP4 (392
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bases), and NSP1 (247 bases), were designed by this method (Table 1). Digoxigenin-labeled RNA probes were generated by in vitro transcription of the PCR products with T7 RNA polymerase, using a digoxigenin RNA-labeling kit from Boehringer Mannheim Biochemicals. To determine that adequate labeling occurred, the concentration of the digoxigenin-labeled RNA probes was ascertained by a DIG quantification test from Boehringer Mannheim Biochemicals. Thin (3 mm) sections of tissue were placed on Fisher Plus slides (Fisher Scientific, Pittsburgh, PA) and ISH was performed overnight at 37 8C as previously described (Burt et al., 1997) with two modifications: (1) Digestion of the tissues was conducted for 15 min with 0.1 mg/ml proteinase K in 0.6 M Tris (pH 7.5)/0.1% CaCl2 for cells minced with normal tissue and for 30 min at RT with animal tissue. A series of pilot experiments was performed to determine the optimal proteinase K concentration and the optimal digestion time for adequate penetration of the probe into either cells or tissue. (2) The optimal concentration of the RNA probe to be used on the tissue was determined by a series of titration experiments with cell controls. The RNA probes were diluted at 1:100 in hybridization buffer from Boehringer Mannheim Biochemicals containing 50% deionized formamide and 3/SSC. Any cross-hybridization was ascertained by hybridizing the digoxigenin-labeled human rotavirus Wa VP4 probe with RRVinfected MA104 cells and the digoxigenin-labeled RRV probe with Wa-infected MA104 cells. In addition, the specificity of the RRV VP4, NSP4 and NSP1 probes was determined by hybridizing the RRV probes to uninfected tissue and uninfected cell controls. 2.5. IHC assays For IHC assays, antibodies were tested on formalin-fixed, paraffin-embedded cell controls to determine the specificity and cross-reactivity of the antibodies (Table 2). The suitability of the following antibodies was ascertained: (1) rabbit polyclonal IgG antibodies enriched by IgG capture to Protein A Sepharose (Amersham, Piscataway, NJ) directed against the Wa strain, (2) a guinea pig
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hyperimmune antiserum, and (3) a panel of eight monoclonal antibodies (MAb) directed against rotaviral antigens (Table 2). Preliminary experiments were performed using serial dilutions of the antibody on control cells in order to determine the appropriate dilution of antibody to detect rotaviral antigens without a background reaction. The rabbit polyclonal antibody directed against the Wa strain and two MAbs, 3A-8 and B4-2, directed against viral proteins VP2 and NSP4, respectively, were chosen for the IHC assays on animal tissue. The appropriate negative control was performed in parallel for each experiment. The negative controls included preimmune rabbit serum, enriched for IgG by IgG capture to Protein A Sepharose (Amersham), normal mouse ascites fluid (NMAF) and normal guinea pig serum. IHC assays were carried out as described previously with a streptavidin-biotin alkaline phosphatase technique (Burt et al., 1997) with two modifications: (1) The optimal concentration of antibodies was ascertained by performing a preliminary series of titration experiments. (2) The specificity of staining was confirmed with NMAF or preimmune rabbit serum as the primary antibody. These primary antibodies were applied to non-rotavirus infected tissue sections and uninfected MA104 cells.
3. Results 3.1. RT-PCR Three methods to purify RNA from formalinfixed, paraffin-embedded tissue were evaluated. The RT-PCR results (data not shown) demonstrated that the most effective RNA recovery method generating PCR-amplifiable RNA required three steps. Cell lysis occurred in a guanidine salt-based system, protein was extracted with phenol, and contaminants were removed by binding the RNA to a silica-based matrix. Pilot studies demonstrated that binding to the silica resin enhanced the quality of the RNA preparation. The RNaid Plus kit from Bio101 incorporated all of these steps and was used for all subsequent RNA extractions. This method reproducibly
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Table 2 Antibodies tested in this study Antibody designation
Rotavirus immunogen Specificity
Preimmune rabbit serum, enriched for IgG / Rabbit polyclonal antibodies enriched by IgG capture Wa Normal guinea pig serum / Rotavirus guinea pig hyperimmune serum RRV MAb 631/9 Wa MAb 3A-8 Wa MAb B4-2 Wa MAb 631/24 Wa MAb 255/60 RRV MAb1A9 RRV MAb 2G4 OSU Normal mouse ascites fluid Negative control
yielded a PCR-amplifiable RNA preparation from formalin-fixed, paraffin-embedded tissue. The RT-PCR and probe hybridization results with the RRV and YK-1 primers yielded PCR amplicons in all infected tissues (Fig. 1). The correct-size PCR amplicon (200 bp) was obtained from RRV-infected SCID mouse liver tissue (Fig. 1A, lane 3) with RRV VP4 primers and confirmed to be RRV-derived by Southern hybridization with the RRV internal VP4 probe (data not shown). A faint PCR amplicon of 279 bp was obtained with the YK-1 VP4 primers in the RRVinfected SCID mouse (Fig. 1A, lane 10), however, this amplicon was not detected by Southern hybridization with a strain-specific digoxigeninlabeled YK-1 probe (Fig. 1B, lane 10), demonstrating that the product resulted from crosspriming of the YK-1 primers on the RRV template. The strain-specific binding of the YK-1 probes to the YK-1-positive control amplicon (Fig. 1B, lane 18), but not to the RRV-positive control amplicon (Fig. 1B, lane 17), was clearly demonstrated. Uninfected SCID mouse liver tissue did not generate a PCR amplicon with either RRV or YK-1 primers (Fig. 1A, lane 2 and 9, respectively) or hybridization signal with either RRV or YK-1 probes. A matrix is illustrated in Fig. 1C to indicate the primer pairs and the tissue or control RNA present in each lane of Fig. 1A and B. RNA extracts from two different blocks of YK1-infected monkey intestinal tissue were amplified with the YK-1 primers generating the correct-size
Working dilution Source
/ 1:400 Group A rotavirus 1:400 / 1:1000 Group A rotavirus 1:400 VP6 (SGII) 1:800 VP2 1:2000 NSP4 1:2000 VP6 (SGI and II) 1:100 VP6 (SGI) 1:800 VP4 (neutralizing) 1:250 VP4 (neutralizing) 1:250 1:1000
CDC CDC NIH NIH H Greenberg H Greenberg H Greenberg H Greenberg H Greenberg H Greenberg H Greenberg CDC
PCR amplicon of 279 bp (Fig. 1A, lanes 12 and 13) and confirmed by Southern hybridization (Fig. 1B, lanes 12 and 13). Amplification of RNA extracted from one block of YK-1-infected monkey intestinal tissue with the RRV primers generated a PCR amplicon of 200 bp (Fig. 1A, lane 5) that was confirmed by Southern hybridization with the strain-specific YK-1 probe (Fig. 1B, lane 5). The other RNA extract from the block of YK-1infected monkey intestinal tissue that generated less PCR amplicon with YK-1 primers (Fig. 1A, lane 13) did not generate a PCR amplicon with the RRV primers (Fig. 1A, lane 6). Uninfected monkey intestinal tissue did not generate an amplicon with either RRV or YK-1 primers (Fig. 1A, lanes 4, 7, 11 and 14) or a hybridization signal with either RRV or YK-1 probes. RRV and YK-1 RNA amplified with RRV and YK-1 primer pairs generated the correct size amplicon (Fig. 1A, lanes 17 and 18, respectively), and the water controls did not generate an amplicon with either primer pair (Fig. 1A, lane 8 and 15). The RT-PCR and probe hybridization results with the RRV VP4 primers yielded PCR amplicons only in samples containing RRV (Fig. 2). The correct-size PCR amplicon of 200 bp from the RRV VP4 gene was amplified by a one-step RTPCR method from 1.16 /104 viral particles (Fig. 2A, lane 2) to 1.16 /102 viral particles (Fig. 2A, lane 4) as determined by ethidium bromide staining of the agarose gel. After analysis of RT-PCR products on agarose gels, the RRV VP4 gene
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Fig. 1. Detection of simian rotaviruses RRV and YK-1 in infected tissues by RT-PCR. (A) Ethidium bromide stained agarose gel: lanes 2 /8 and 17 (RRV VP4 primer pair) and 9 /15 and 18 (YK-1 VP4 primer pair), products amplified from dsRNA extracts of uninfected (lanes 2 and 9) or RRV-infected (lanes 3, 10) SCID mouse tissue or uninfected (lanes 4, 7, 11, 14) or YK-1-infected (lanes 5, 6, 12, 13) simian tissue. Lanes 17 and 18, RRV and YK-1 RNA amplified with RRV and YK-1 primer pairs, respectively. Lanes 8 and 15 contained additional negative controls (H2O). Lane 16 was not loaded with a sample. (B) Results of Southern hybridization and chemiluminescent detection of the PCR products are shown in panel A using an YK-1-specific VP4 probe. Lanes 1 and 19, digoxigeninlabeled and unlabeled molecular weight markers, respectively, with the molecular size (base pairs) indicated for selected fragments. (C) A matrix is illustrated which indicates the primer pairs and control sample or tissue present in each lane. In this panel, MWM designates both digoxigenin-labeled and unlabeled molecular weight markers, UIM designates uninfected SCID mouse tissue, IM designates infected SCID mouse tissue, UIS designates uninfected simian tissue, and IS designates infected simian tissue.
product was confirmed by probe hybridization with a digoxigenin-labeled RRV-specific VP4 gene probe (Fig. 2B, lanes 2/5). The RRV VP4 gene can be detected by RT-PCR followed by probe hybridization in 12 viral particles (Fig. 2B, lane 5). A rotavirus-negative extract did not generate an amplicon with the RRV VP4 primer pair (Fig. 2A and B, lane 9).
3.2. ISH assays The hybridization signal from each probe was assessed on Wa-infected, RRV-infected, and uninfected cells and the intensity of the signal strength (0 to /4) was graded for each probe (Table 3). The VP4 probe designed from the Wa strain hybridized intensely with Wa-infected cells
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Fig. 2. Sensitivity of detection of rhesus rotavirus VP4 gene RNA by RT-PCR. Rhesus rotavirus RNA from purified virions was diluted serially in an RNA extract from a rotavirusnegative, formalin-fixed simian tissue specimen and then analyzed with RRV VP4 primer pair. (A) Ethidium bromide stained agarose gel: lane DM, digoxigenin-labeled molecular size markers; lanes 1 /8 products amplified from dsRNA extracted from the number of virus particles indicated: 1.16/ 104 (lane 1), 1.16/103 (lane 2), 1.16/102 (lane 3), 1.16/101 (lane 4), 1.16 /100 (lane 5). 1.16 /10 1 (lane 6), 1.16/10 2 (lane 7), 1.16/10 3 (lane 8); lane 9, product amplified from a rotavirus negative stool RNA extract. (B) Results of Southern Hybridization and chemiluminescent detection of the PCR products shown in panel A using an RRV-specific VP4 gene probe (Table 1). The molecular size (base pairs) of the digoxigenin-labeled molecular weight markers is indicated on the left side of Panel B.
(Fig. 3A), but not with RRV-infected cells (Fig. 3B) or uninfected cells. The VP4 probe from the DS-1 strain hybridized with DS-1 and Wa-infected
cells (data not shown), perhaps due to the 89.7% (Gorziglia et al., 1988) sequence identity of the VP4 genes from the two strains and to the fact that we constructed the probe in a region of DS-1 VP4 (nt 1480/2001) in a relatively conserved region of the VP4 gene. Only the VP4 probe from the Wa strain was used further in the animal studies. The RRV NSP4 probe hybridized to both RRVand Wa-infected cells, but the intensity of the signal from the RRV-infected cells was stronger, /4, than that from the Wa-infected cells, /2 (Table 3). Based on the 81% identity of the RRV and Wa NSP4 genes, these results are reasonable. The RRV NSP1 and RRV VP4 (Fig. 3C) probes hybridized only to the RRV-infected cells, but the intensity of the signal from the NSP1 probe was less than that of the VP4 probe. The RRV VP4 gene is 59% identical to the Wa VP4 gene (Fig. 3D) and the RRV NSP1 gene is about 52% identical to the Wa NSP1 gene, explaining the lack of hybridization to the Wa-infected cells. None of the three RRV probes hybridized with uninfected cells (Table 3). Based on intensity of the hybridization signal, the RRV VP4 probe was the most specific while the RRV NSP4 probe was the most sensitive for the detection of RRV nucleic acids. Therefore, the RRV VP4 and NSP4 probes were selected for further analysis. The RRV NSP4 probe hybridized efficiently to the superficial epithelial cells of the ileum and jejunum in the YK-1-infected monkey (Fig. 3E), while no hybridization was evident with the RRV VP4 probe. These results are consistent with the
Table 3 Rotavirus ISH results Cell line or tissuea/infecting strainb Probes VP4 from Wa strain (human) VP4 from RRV strain (simian) NSP4 from RRV strain (simian) MA104/Wa MA104/RRV Macaque/YK-1 SCID mouse/RRV a b c d
////c / / /
/d //// / //
// //// /// ////
The uninfected cell control, uninfected macaque intestinal tissue, and uninfected SCID mouse liver were negative. Wa (P1A[8], G1) is a human rotavirus strain, while RRV (P5B[3], G3) and YK-1 (P[?], G?) are simian strains. / Is equal to the presence of granular staining, ranging from / to //// as the most intense. / Is equal to the absence of granular staining.
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Fig. 3
specificities of the two probes, because NSP4 genes from different strains exhibit a greater degree of sequence conservation than do the corresponding VP4 genes. No signal was evident in the uninfected
monkey tissue with either the RRV VP4 or the NSP4 probe (Fig. 3F). The Wa VP4 probe did not hybridize to either the YK-1- infected or the uninfected monkey tissue.
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Table 4 Rotavirus IHC results Cell line or tissuea/infecting strainb
MA104/US1205 MA104/Wa MA104/DS-1 MA104/RRV Macaque/YK-1 SCID mouse/RRV
Antibody Rabbit polyclonal antibody
Preimmune rabbit serum
Rota VP2, MAb 3A-8
Rota NSP4, MAb B4-2
/c / / / / /
/d / / / / /
/ / / / / /
/ / / / / /
a
The uninfected cell control, uninfected macaque intestinal tissue, and uninfected SCID mouse liver were negative. US1205 (P2A[6], G9), Wa (P1A[8], G1) and DS-1 (P1B[4], G2) are human rotavirus strains, while RRV (P5B[3], G3) and YK-1 (P[?], G?) are simian strains. c / Is equal to the presence of granular staining. d / Is equal to the absence of granular staining. b
The RRV NSP4 probe hybridized intensely in the regions of the liver where inflammatory infiltrates and necrosis were visible in the RRVinfected SCID mouse (Fig. 3G). Similarly, the RRV VP4 probe hybridized in the same region of the RRV-infected SCID mouse, but less intensely than the NSP4 probe. No signal was evident in the uninfected SCID mouse from either the RRV VP4 (Fig. 3H) or the NSP4 probe. The Wa VP4 probe did not hybridize to either the RRV-infected SCID mouse or the uninfected SCID mouse.
3.3. IHC assays Eleven antibodies were evaluated in the IHC tests for their ability to detect rotavirus antigens in formalin-fixed tissue. Based on their reactivities with positive cell controls (Table 4), the rabbit polyclonal antiserum and two MAbs, VP2 (MAb 3A-8) and NSP4 (MAb B4-2), were selected for use in the IHC assays for the detection of RRV in animal tissues. The polyclonal hyperimmune gui-
nea pig antiserum reacted with the uninfected cell controls and was not used. The rabbit polyclonal antibody and the NSP4 MAb reacted with antigens from human rotavirus strains Wa (Fig. 4A) and DS-1 and from simian rotavirus strains RRV and YK-1 in infected cell controls (Table 4). The characteristic granular staining in the cytoplasm of the infected cells is absent in the adjacent human control tissue. No staining was present utilizing the preimmune rabbit antiserum in the Wa-infected or RRVinfected cell controls (Fig. 4B and E, respectively). The VP2 MAb reacted with RRV antigens, but not YK-1 antigens, generating granular staining in the cytoplasm of the RRV-infected cells, and with human rotavirus, DS-1 antigens, but not Wa antigens (Fig. 3D, Table 4). No staining occurred for each antibody source with uninfected cell controls (Fig. 4C and F). The polyclonal rabbit antibody and NSP4 MAb showed diffuse granular staining in the superficial epithelial cells of the ileum and jejunum from the macaque, infected with the simian strain YK-1
Fig. 3. Localization of rotaviral nucleic acid in infected and uninfected cells and tissues by ISH. (A /B) MA104 cells infected with the Wa strain (A) or RRV strain (B) that are hybridized with VP4 digoxigenin-labeled pro1be from Wa. (C /D) MA104 cells infected with the RRV strain (C) or Wa strain (D) that are hybridized with VP4 digoxigenin-labeled probe from RRV. (E /F) Small intestine tissue of macaque monkey infected with YK-1 strain (E) and small intestine tissue of uninfected monkey (F) hybridized with NSP4 digoxigenin-labeled probe from RRV. (G /H) Liver tissue of SCID mouse infected with RRV strain (G) and liver tissue of uninfected SCID mouse (H) hybridized with VP4 digoxgenin-labeled probe from RRV. Immunoalkaline phosphatase staining was performed using a naphthol fast red substrate with light hematoxylin counterstain. (A /H) Original magnification, /50.
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Fig. 4
(Fig. 4G and H). The immunostaining by the VP2 MAb was focal in nature in the epithelial cells of the mucosa in the macaque and absent in the mucosa of the uninfected tissue (Fig. 4I). The immunostaining was not observed in the large
intestine, pancreas, liver, lymphoid follicles, or uninfected cells. Rotavirus antigens were demonstrated in the liver in the SCID mouse by the polyclonal rabbit antibody (Fig. 4J) and VP2 MAb (Fig. 4K), but
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not with the NSP4 MAb. Immunostaining also occurred in the periportal areas with inflammation and necrosis, similar to the areas showing hybridization by ISH. The VP2 MAb generated more intense staining in the intestinal epithelial cells of the macaque than in the necrotic region of the SCID mouse liver. No immunostaining occurred in the uninfected SCID mouse liver (Fig. 4L and data not shown).
4. Discussion In this study, RT-PCR, ISH, and IHC assays were developed to detect and localize rotavirus nucleic acids and antigens in formalin-fixed, paraffin-embedded tissue. We detected simian rotavirus RNA from formalin-fixed, paraffinembedded infected cells and tissues by RT-PCR with primers designed to a region of the VP4 gene of simian rotavirus strains. RT-PCR followed by Southern hybridization with probes that hybridize internally to the PCR-amplified region of the VP4 gene significantly enhanced this detection. The ISH results with the infected cell controls demonstrate that the VP4 riboprobes in ISH differentiated between human and rhesus rotavirus VP4 regions for the strains tested. One human rotavirus RNA probe, VP4, could be used to localize viral nucleic acid in natural human rotavirus infections, while two RRV RNA probes, NSP4 and VP4, could be used in cases associated with rhesus rotavirus infections. As the rabbit polyclonal antibody preparation reacted with all the human and simian rotavirus strains tested, it could be used for diagnostic purposes to screen for rotavirus infections. Based on cell controls, the rabbit polyclonal antibody and two MAbs, VP2 and NSP4, could be used to localize RRV antigens in
cases associated with rhesus rotavirus infections while preserving tissue morphology. In the macaque infected with the simian strain YK-1, rotavirus antigens and RNA were localized to the epithelial cells of the ileum and jejunum using three rotavirus antibody reagents and the NSP4 riboprobe, respectively. Cross-hybridization between the RRV NSP4 probe and YK-1 RNA was expected due to high identity (91%) between RRV and YK-1 NSP4 genes in the probe region. In contrast, the RRV VP4 riboprobe did not hybridize to the YK-1 RNA in the epithelial cells of the intestinal mucosa probably because of the lower identity (83%) of that probe to the YK-1 VP4 in the probe region. Histopathological analysis of the RRV-infected SCID mouse liver identified regions containing necrotic tissue and infiltrate that were the same areas in which RRV RNA was localized by the NSP4 and VP4 riboprobes, and RRV antigens were localized with VP2 MAb and the rabbit polyclonal antibody. As the RT-PCR and ISH analysis was comparable to the IHC results in both animal tissues, these results demonstrate the value of these three techniques in documenting RRV infections. Previously, an indirect immunoperoxidase technique to detect rotavirus antigen in paraffinembedded calf intestinal tissue (Parsons et al., 1984) and human biopsy intestinal tissue (Graham and Estes, 1979) demonstrated that rotavirus infected the enterocytes of the villus epithelium in the small intestine. More recently, employing an indirect immunoperoxidase test with a commercially available polyclonal rabbit antibody (DAKO, Carpenteria, CA), rotavirus was detected in formalin-fixed, paraffin-embedded cells infected with human rotavirus (Cartun et al., 1993) and in the intestinal tissue of two human fatalities (Morrison et al., 2001). In our indirect immunoalkaline
Fig. 4. Immunohistochemical detection of rotaviral antigens in infected and uninfected cells and tissues with polyclonal and monoclonal antibodies. (A /C) MA104 cells infected with human rotavirus strain Wa incubated with rabbit polyclonal antibody (A), with preimmune rabbit serum (B) and uninfected MA104 cells with rabbit polyclonal antibody (C). (D /F) MA104 cells infected with simian rotavirus RRV and incubated with MAb VP2 (D) and with preimmune rabbit serum (E). Uninfected MA104 cells incubated with MAb VP2 (F). (G /I) Macaque small intestine infected with YK-1 strain incubated with rabbit polyclonal antibody (G) and MAb NSP4 (H) and uninfected monkey incubated with rabbit polyclonal antibody (I). (J /L) SCID mouse liver infected with RRV strain incubated with rabbit polyclonal antibody (J), MAb VP2 (K) and uninfected SCID mouse incubated with polyclonal rabbit antibody (L). Immunoalkaline phosphatase staining was performed using a naphthol fast red substrate with light hematoxylin counterstain. (A / G and I /L) Original magnification, /50; (H) original magnification, /100.
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phosphatase IHC method, two MAbs, VP2 and NSP4, as well as a polyclonal rabbit antibody reagent were effective in localizing rotavirus infections. We performed numerous cell controls, tissue controls, and cross-reactivity studies to address specificity issues in order to achieve a well-defined and well-characterized IHC protocol with several antibodies. The ISH method reported here for simian rotavirus-specific riboprobes allowed the localization of rotavirus infections to the epithelial cells of the small intestine and to the liver in formalin-fixed, paraffin-embedded tissues. Recently, a report describing an RT in situ PCR hybridization analysis with an human rotavirusspecific riboprobe has been published which localized human rotavirus to the mucosal cells of the small intestine and other tissues (Morrison et al., 2001). In an earlier study, Uhnoo et al. (1990) detected RRV strain MMU18006 in the liver of SCID mice by cell culture, immunofluorescence, and electron microscopy, illustrating that the tissue tropism of rotavirus was not restricted to the villus epithelium of the small intestine in an immunocompromised host. Similarly, Gilger et al. (1992) demonstrated by immunohistochemical evidence that rotavirus infects the liver and kidney of immunocompromised children. Our analyses demonstrated that RRV could also be detected in formalin-fixed liver tissues of RRV-infected SCID mice, supporting rotavirus hepatotropism. RTPCR is commonly performed to detect RNA in fecal samples (Wilde et al., 1990, 1991; Pang et al., 1999) and a previous study performed by Koopmans et al. (1993) demonstrated that rotavirus could be detected by RT-PCR with RNA extracted from formalin-fixed, paraffin-embedded tissue. In our present RT-PCR analysis, we reduced the quantity of formalin-fixed paraffinembedded tissue employed to extract RNA (one 10-mm section) and all rotavirus-infected tissues were positive by RT-PCR. It has been documented previously that PCR is 100 000 times more sensitive than standard electropherotype identification of rotavirus and 5000 times more sensitive than dot-blot hybridization (Xu et al., 1990; Kapikian and Chanock, 1996). In our study, amplification of RNA by a one-step RT-PCR method followed by Southern hybridiza-
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tion with chemiluminescent detection of bound probe allows the detection of the VP4 gene in approximately 12 viral particles, which illustrates the enhanced sensitivity of our probe-hybridization method. The increased sensitivity of the RTPCR assay is exemplified here, as one of the YK-1infected macaque tissue sections from the small intestine was negative by both ISH and IHC, but positive by RT-PCR. The most sensitive method developed in our study is RT-PCR, the most specific is the ISH, and the one with the broadest range of reactivity is the IHC assay. Taken together, these techniques have significant sensitivity and specificity to detect and to distinguish human and rhesus rotaviruses in naturally occurring infections. One caveat exists. Only the IHC and the ISH methods can be used to localize rotavirus antigens and nucleic acids in specific cells while preserving tissue morphology. Therefore, it may be useful to develop additional antibodies and in situ riboprobes to increase the sensitivity of the IHC and ISH tests, allowing either intracellular or intraluminal localization of antigens and nucleic acids in suspected cases with reduced concentrations of rotavirus. This study demonstrates the feasibility of undertaking IHC, ISH, and RT-PCR tests to detect and to localize the presence of animal and human rotavirus antigens and RNA in formalin-fixed, paraffin-embedded tissues from patients with intussusception associated with RRV vaccine and those infected with natural rotavirus. The development of these methods to detect rotavirus in tissue provides powerful tools to understand the pathogenesis of the severe and fatal cases of rotavirus diarrhea. Analysis of pathological specimens in retrospective studies with these sensitive and specific methods would allow us to determine the extent of infection by identifying rotavirusinfected tissues and to ascertain if such findings reflect an actual tissue infection or a systemic viremia.
Acknowledgements We wish to thank the following individuals: Dr Harold McClure and Dr Marie Riepenhoff-Talty
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for kindly providing the tissue specimens; Dr Tamie Ando and Dr Taka Hoshino for generously giving us the polyclonal antibodies; Dr Harry Greenberg for making available the many monoclonal antibodies used in this study; and Dixie Griffin for her technical assistance in sequencing gene fragments of the YK-1 strain. We also want to thank John O’Connor for his editorial assistance.
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