Current Laboratory Techniques in Rabies Diagnosis, Research and Prevention, Volume 2

USA

Part One

Rabies Diagnosis

Outline

Section A Demonstration of Virus

Section B Demonstration of Viral Subunits and Antigens

Section C Demonstration of Viral Nucleic Acids

Section D Demonstration of Viral Antibodies: Binding Assays

Section E Demonstration of Viral Antibodies: Functional Assays

Section F Typing/Differentiation of Lyssaviruses

Section A

Demonstration of Virus

Outline

Chapter One Demonstration of Lyssaviruses by Electron Microscopy

Chapter Two Virus Isolation in Animals: The Mouse Inoculation Test

Chapter Three Virus Isolation in Cell Culture: The Rabies Tissue Culture Infection Test

Chapter Four A Rat Basophilic Leukemia Cell Sensor for the Detection of Rabies Viruses

Chapter One

Demonstration of Lyssaviruses by Electron Microscopy

Cynthia S. Goldsmith and Sherif R. Zaki,    Infectious Diseases Pathology Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA

Because of time constraints, electron microscopy has not typically been used on the front lines for diagnosis of lyssavirus infections such as rabies virus (RABV). However, the ability to use rapid microwave processing for thin section samples, and the possibility of conducting negative stain electron microscopy on fixed, homogenized brain tissue, has reduced the turnaround time for such testing.

Keywords

Lyssavirus; Microwave Processing; Negative Stain Electron Microscopy; Rabies Virus; Thin Section Electron Microscopy

Chapter Contents

1.1 Introduction 5

1.2 Materials 6

1.2.1 Reagents 6

1.2.2 Equipment 6

1.2.3 Biological Materials 7

1.3 Methods 7

1.3.1 Fixation 7

1.3.1.1 Central Nervous System Tissue 7

1.3.1.2 Infected Cell Culture 7

1.3.2 Processing 8

1.3.2.1 Thin Section Technique 8

1.3.2.2 Negative Staining Technique 9

1.3.3 Interpretation of Results 9

1.4 Discussion 9

1.4.1 Experimental Tips 9

1.4.2 Precautions 9

1.4.3 Alternative Methods 10

1.4.4 Time Considerations 10

1.4.5 Limitations 11

References 11

1.1 Introduction

The conventional electron microscopic (EM) technique for detecting lyssaviruses such as rabies virus (RABV) in the central nervous system (CNS) has been thin-section EM. Tissues where Negri bodies and RABV particles have been found include Ammon’s horn of the hippocampus, the cerebellum, the brain stem, and cerebral tissue. This chapter describes the use of fixed tissues for rapid microwave processing for same-day results of thin-section EM and a protocol for negative stain EM of fixed, homogenized CNS tissue for results within about an hour.

EM has long been used in the characterization of lyssaviruses. In 1962, using thin-section EM, Matsumoto confirmed the viral nature of the Negri bodies, describing a matrix containing elongated and bullet-shaped particles.¹ The morphology of the viral nucleocapsid was found by negative stain EM to be a single-stranded helix.² Immunofluorescence and EM were instrumental in understanding the pathogenesis of lyssaviruses in experimental animals.³ EM has assisted in the description of cases in which RABV was transmitted by transplantation of a cornea and of solid organs from infected donors to recipients.⁴,⁵ More recently, EM was used to confirm confocal microscopy studies on the presence of Toll-like receptor-3 (TLR3) in Negri bodies in a RABV-infected neuroblastoma cell line.⁶ Confocal microscopy showed that antibodies against TLR3 identified large perinuclear aggregates, and surrounding the aggregates immunostaining for RABV nucleocapsid was detected. EM preparations found amorphous Negri bodies, sometimes associated with viral particles, in a similar perinuclear location in RABV-infected cells, and immunoelectron microscopy demonstrated the presence of RABV nucleocapsid and TLR3 proteins within the Negri bodies. Hence, EM has been used historically and recently in both diagnostic and research applications.

1.2 Materials

1.2.1 Reagents

 0.1 M Phosphate buffer, pH 7.3.

 2.5% Phosphate-buffered glutaraldehyde.

 5% Phosphate-buffered paraformaldehyde.

 1% Osmium tetroxide.

 4% Uranyl acetate.

 Ethanols: 70%, 80%, 95%, and 100%.

 Acetone.

 Epon-Araldite epoxy resin (see Additional Information).

 Toluidine blue-azur II (see Additional Information).

 Lead citrate stain.

 5% Ammonium molybdate, pH 6.9, and 0.1% trehalose.

1.2.2 Equipment

 Microfuge tube and pestle (see Additional Information).

 Small polypropylene vials.

 Chemical safety cabinet.

 EM-grade laboratory microwave oven with variable wattage that can eliminate hot spots and has a vacuum chamber.

 BEEM® capsules.

 Oven.

 Ultramicrotome.

 Diamond Knife.

 EM grids, 75/300 mesh (for thin section EM).

 EM grids, formvar/carbon coated, 300 mesh (for negative stain EM).

 Microfuge (refrigerated preferred).

 Transmission electron microscope (TEM).

1.2.3 Biological Materials

 CNS tissue.

 RABV-infected and uninfected cell culture.

1.3 Methods

1.3.1 Fixation

1.3.1.1 Central Nervous System Tissue

At a minimum biosafety level 2 (BSL-2) precaution (because of the risk of other potential pathogens in diagnostic samples), small (1 mm×1 mm) pieces of tissue are placed in 2.5% phosphate-buffered glutaraldehyde for 2 hrs at 4°C (or up to overnight if the pieces are larger). The glutaraldehyde is removed and disposed of as a hazardous chemical waste, replaced with 0.1 M phosphate buffer, and the tissue is stored at 4°C. Alternatively, tissue that has been fixed in 10% formalin can be rinsed with 0.1 M phosphate buffer, fixed in 2.5% buffered glutaraldehyde, and stored in phosphate buffer, as above.

For a negative stain preparation, using BSL-2 precautions, unfixed CNS tissue can be placed in a microfuge tube, ground with a pestle, and the suspension mixed 1:1 with 5% buffered paraformaldehyde and allowed to fix for at least 2 hours.

1.3.1.2 Infected Cell Culture

For thin section preparation, and within a biohazard cabinet using BSL-2 precautions, infected cells are scraped from a flask, transferred to a conical centrifuge tube, and pelleted for 10 min at 1,200×RPM in a tabletop centrifuge. The media is pipetted off and disposed of as infectious waste, the cells are re-suspended in 0.1 M phosphate buffer, and pelleted again for 10 min at 1,200×RPM. The phosphate buffer is pipetted off and disposed of as an infectious waste, and 2.5% phosphate-buffered glutaraldehyde is added carefully to the centrifuge tube, without disturbing the pellet. The cells are fixed for 2 hrs at 4°C. The glutaraldehyde is removed and disposed of as a hazardous chemical waste, carefully replaced with 0.1 M phosphate buffer, without disturbing the pellet, and the cells are stored at 4°C.

For a negative stain preparation of infected cell culture, and using BSL-2 precautions in a biohazard cabinet, the supernatant is mixed 1:1 with 5% buffered paraformaldehyde, and allowed to fix at least for 2 hours at 4°C.

1.3.2 Processing

1.3.2.1 Thin Section Technique

Microwave (at 250 watts): 1% osmium tetroxide for 40 sec on/20 sec off/40 sec on. Place the vials on ice on a rotator for 15 min (while mixing the first 4 chemicals of the epoxy resin, see Additional Information). Then, perform two water rinses in the microwave, 40 sec each. Add DMP-30 to the epoxy resin, mix for 5 min, and place under vacuum.

At 550 watts: 4% uranyl acetate for 40 sec on/20 sec off/40 sec on. Perform two water rinses for 40 sec each.

Perform alcohol exchanges in ascending concentrations (70%, 80%, 95%, 100%, 100%) for 40 sec each. Perform two rinses in acetone, 40 sec each.

At this point, place 900 mL of H2O at the front of the microwave, putting in fresh water with each chemical exchange. Mix acetone-to-resin at 2:1, and microwave for 5 min. Mix acetone-to-resin at 1:2, and microwave for 5 min. Make two small holes in the top of each vial, and place the vials under a vacuum for each proceeding step. Use pure resin for four exchanges, 7 min each.

Place labels into BEEM® capsules with a small amount of resin. Place one piece of the specimen into each capsule and fill almost to the top with resin. Polymerize at 58°C overnight or for up to 48 hrs. If speed is necessary, specimens can be polymerized at 90°C for 75 min, or until the resin has cured.

Tissue blocks are first sectioned at the LM-level, dried on a glass slide, and stained with toluidine blue-azur II. Sections are examined to select the blocks with the appropriate neuronal cells (these steps are not necessary for cell culture specimens). Cut sections at the EM-level (60–80 nm), place on mesh grids, and stain with 4% uranyl acetate and lead citrate stain. Examine grids under a TEM.

1.3.2.2 Negative Staining Technique

The fixed homogenized brain tissue should be centrifuged in a microfuge at 5,000–14,000 RPM for 10 min. A 300-mesh formvar/carbon-coated EM grid is placed on 2 microliters of supernatant from either a cell culture or from the homogenized brain tissue and allowed to adsorb for 10 min. The grid is blotted, rinsed, stained briefly with a solution of 5% ammonium molybdate, pH 6.9, and 0.1% trehalose, and blotted. Examine grids under a TEM.

1.3.3 Interpretation of Results

In thin section samples, lyssavirus inclusions, known as Negri bodies, can be found in neurons and neuronal processes, and consist of a granular matrix that contains virus particles either within the matrix or at the periphery (Figure 1.1(1)). Typical virions are sometimes bullet-shaped when cut tangentially or appear as a bulls-eye when cut in cross-section, and are approximately 60 nm in diameter, with short surface projections. Mature particles can also be found budding upon intracytoplasmic membranes or the plasma membrane (Figure 1.1(2)). Some of the matrices contain larger tubular particles (approx. 130 nm in diameter), with dense material under the membrane, and with some particles containing granules (Figure 1.1(3)). Using the EM technique of negative staining of fixed, ground brain tissue, the virions appear bullet-shaped with short projections, and the coiled nucleocapsid is visible (Figure 1.1(4)).

Figure 1.1 Electron microscopic images of CNS from fatal human rabies cases. (1) Typical Negri body composed of granular material and virions cut tangentially (arrowhead) and in cross-section (arrow). (2) RABV particles, cut in cross-section, within the cisternae of intracytoplasmic membranes. (3) Another Negri body, with large RABV particles enmeshed in the matrix. (4) Negative stain of a bullet-shaped RABV virion from homogenized brain tissue, with short surface projections and an internal nucleocapsid. Bars, 100 nm. (Image (4) is courtesy of Maureen Metcalfe and Atis Muehlenbachs, Centers for Disease Control and Prevention.)

1.4 Discussion

1.4.1 Experimental Tips

 All chemical exchange steps are performed within a chemical safety cabinet.

 Osmium can be disposed of as a hazardous chemical in a plastic bottle filled 1/3 with corn oil.

 After mixing the epoxy resin, place it under vacuum to remove the air bubbles.

1.4.2 Precautions

 As an additional safety precaution, samples can be gamma irradiated with 2×10⁶ rads on wet ice.

 All work with unfixed lyssaviruses needs to be conducted following BSL-2 precautions.

1.4.3 Alternative Methods

Different EM laboratories may use slightly different protocols for processing specimens.

1.4.4 Time Considerations

Once a specimen is fixed, negative stain EM can give results in less than an hour. Thin section EM, using rapid microwave processing and epoxy polymerization in a hot oven, can give same-day results.

1.4.5 Limitations

By thin section EM, some Negri bodies may not contain virions. If these are found, the operator should continue searching the specimen to find viral particles. By negative stain EM, virions in other virus families also have surface projections, but lyssavirus particles are consistently bullet-shaped. Different lyssavirus species are not differentiable by EM.

Additional Information

Composition of Epoxy Resin (may scale back the amounts to make less resin)

 25 mL Epon-substitute.

 13 mL Araldite.

 65 mL Dodecenylsuccunic anhydride (DDSA).

 1.2 mL Dibutyl phthalate (DBP).

 1.5% 2,4,6-Tris(dimethylaminomethyl) phenol (DMP-30).

Mix the first 4 chemicals for 5 min, add DMP-30, mix for 5 min, and place under vacuum.

Composition of Toluidine Blue-Azur II Stain

 0.1% Toluidine blue.

 1% Azur II.

 1% Sodium borate.

Source of Pestle

 Fisher Scientific, Catalog K749521 1590.

References

1. Matsumoto S. Electron microscopy of nerve cells infected with street rabies virus. Virology. 1962;17:198–202.

2. Hummeler K, Tomassini N, Sokol F, Kuwert E, Koprowski H. Morphology of the nucleoprotein component of rabies virus. J Virol. 1968;2:1191–1199.

3. Murphy FA, Harrison AK, Winn WC, Bauer SP. Comparative pathogenesis of rabies and rabies-like viruses: infection of the central nervous system and centrifugal spread of virus to peripheral tissues. Lab Invest. 1973;29:1–16.

4. Baer GM, Shaddock JH, Houff SA, Harrison AK, Gardner JJ. Human rabies transmitted by corneal transplant. Arch Neurol. 1982;39:103–107.

5. Srinivasan A, Burton EC, Kuehnert MJ, et al. Transmission of rabies virus from an organ donor to four transplant recipients. N Engl J Med. 2005;352:1103–1111.

6. Menager P, Roux P, Megret F, et al. Toll-like receptor 3 (TLR3) plays a major role in the formation of rabies virus Negri Bodies. PLoS Pathog. 2009;5:e1000315.

Chapter Two

Virus Isolation in Animals

The Mouse Inoculation Test

Ivan V. Kuzmin,    Global Alliance for Rabies Control, Manhattan, KS, USA

Mouse inoculation is one of the classical methods for isolation of lyssaviruses. Although cell culture can replace animal use in many applications, animals are still most suitable when high titers of virus should be amplified without excessive passaging, in various pathogenicity studies in animal models, or for evaluation of potency of rabies biologics. For diagnostic purposes, the inoculation is usually performed in suckling or weanling mice (10–15 or 30 μL of inoculum, respectively) intracranially. The inoculum is prepared as 5–20% homogenized tissue suspension or cell culture supernatant, and is injected by an insulin syringe with a 26–27 gauge needle. Proper inoculation technique will help to minimize trauma and reflux of the inoculum out of the skull. Observation should continue for 14 days, and clinically ill animals should be euthanized based on pain scores. Rabies diagnosis must be confirmed by detection of lyssavirus antigens or RNA in the mouse brains.

Keywords

Animal Model; Diagnosis; Inoculation; Isolation; Lyssavirus; Mouse; Rabies; Test

Chapter Contents

2.1 Ethical Considerations 13

2.2 Choice of Mice and their Husbandry 14

2.3 Preparation of Inoculum 15

2.4 Inoculation Procedure 17

2.5 Observation 19

2.6 Postmortem Examination 20

2.7 Troubleshooting 20

2.8 Virus Titration 21

References 23

2.1 Ethical Considerations

Isolation of pathogens in animal models, including the mouse inoculation test (MIT) for rabies diagnosis, has been used in global laboratory practice for over a century.¹ The advent of cell cultures facilitated significant reduction of animal use. However, there is still no alternative in many areas of infectious disease research, including: pathogenicity studies,² evaluation of safety and efficacy of biologics and other pharmaceutical preparations,³ etc. Although weanling and adult mice demonstrate less susceptibility to lyssaviruses than some other mammals (such as Syrian hamsters, particularly via peripheral inoculation routes), they offer operational convenience because of the small body size, which allows housing of several animals in a cage, relatively inexpensive husbandry, and ease-of-handling because of their long tails. Inoculated intracranially, mice (particularly 1–3 days old) demonstrate very high susceptibility to lyssaviruses, which is similar to the susceptibility of neuronal cell lines.

Isolation of lyssaviruses in mice has several advantages and disadvantages compared to the isolation in cell culture. For example, mice are usually more resistant to bacterial contaminants and toxic substances, which may be present in the field diagnostic material obtained from dead animals. Mice allow accumulation of high virus titers at first inoculation, as the infection will cause disease only after significant replication of virus in the mouse brain. In contrast, the majority of conventional cell cultures used for the rapid tissue culture inoculation test (RTCIT)⁴ must be split every 3–5 days, thus not providing sufficient time for virus accumulation until several passages (sometimes 10 or more) have been made. In fact, the opposite and disadvantageous side of this difference is that MIT requires much greater time for test completion compared to RTCIT to ensure a negative result. In pathogen discovery studies, animal inoculation will allow isolation of different infectious agents which cause diseases.⁵ These agents could be missed in cell culture if they do not produce cytopathic effects and are not demonstrated by specific techniques. The MIT does not require a sterile environment or a CO2 incubator. It can be performed in relatively basic field laboratories with a standard vivarium. Therefore, an operational choice between MIT and RTCIT depends on the aim of the study, quality of samples, and laboratory