Routine virology samples

Three sample types were collected for routine testing at the NHLS Virology Division laboratory as part of SUDI case investigations at the Tygerberg Medico-Legal Mortuary. These include whole blood, as well as left lung and liver tissue.

Lung and liver tissue collection

Small pieces of the liver and left lung tissue were collected for routine centrifugation-enhanced SVC according to the institutional protocol. A sterile blade and scalpel handle was used to cut a 5 mm x 5 mm piece of left lung tissue, which was placed into red top glass tube. A new, sterile blade was also used to cut a 5 mm x 5 mm piece of liver tissue, which was also placed in an appropriately labelled red top glass tube. The samples were transported to the laboratories in 1.5 ml of Universal Transport Medium (UTM), aliquoted into each of the two glass tubes to preserve any viruses present.

Heart blood collection

The pericardial sac was cut open to expose the heart for macroscopic examination by a forensic pathologist prior to whole blood collection. The heart apex was then lifted using sterile forceps to allow blood to be drawn from the base of the heart using a 5 ml syringe and sterile 15-gauge needle.

The collected blood was transferred to a red top glass tube. The tissue and blood specimens were transported to the Virology Division for processing.

Routine SVC

SVCs were routinely used to test for nine viruses⁴ in all SUDI cases at the Tygerberg Medico-Legal Mortuary. Lung tissue was screened for all nine viruses, while the liver was only screened for HAdV and CMV. Three different cell lines stored at 37°C in an ESCO CelCulture CO2 incubator were used for culture, depending on the target virus. Human Fibroblast (HF) cells were used to culture CMV, while a combination of Madin-Darby Canine Kidney (MDCK) and Hep 2 cells was used to culture HAdV, HMPV, Influenza A and B and HPIV 1-3.

Sample Processing for culture

After reception of samples at the Virology laboratory, lung and liver specimens were placed into separate mortars and ground with a pestle. Antibiotic cocktail (0.2 ml) was added to 6 ml viral transport medium (VTM) and the mixture was then added to the ground tissue and allowed to stand for five minutes. Thereafter, it was transferred to appropriately labelled 15 ml centrifuge tubes and centrifuged at 2 000 rpm for 10 minutes at 4°C. The specimens were then passed through a 0.2 µm disposable syringe filter attached to a 10 ml syringe (Figure 3.2) to trap any bacteria that might be present.

Figure 3.2 Syringe and filter for filtering bacteria possibly present in the ground lung and liver tissue in preparation for culture in the shell vials

Inoculation and incubation of prepared sample

Two vials with coverslips inside were labelled with the appropriate sample number. Each vial contained cell lines, media and antibiotics according to the target viruses as shown below:

• HF cells: Rosswell Park Memorial Institute (RPMI) 1640 medium, 10% foetal calf serum, penicillin and streptomycin.

• MDCK and Hep 2 cell combination: Eagle’s Minimum Essential Medium (EMEM), 10% foetal calf serum, penicillin and streptomycin.

The prepared tissue samples (200 µl) were inoculated into the appropriate shell vials according to the target viruses. The shell vials were then centrifuged at 3 000 rpm for 45 minutes in a Heraeus Megafuge 40 centrifuge to enhance viral entry into the cells. After centrifugation, 1 ml of EMEM was added to the vial with MDCK and Hep 2 cell combination and incubated at 33°C for 48 hours.

In the other vial with HF cells, 1 ml of RPMI 1640 medium was added and incubated at 37°C for 48 hours.

After incubation, the coverslips were taken out of the shell vials and fixed in 2 ml cold acetone for 10 minutes to prevent the cells from being washed off.

Determination of culture results

A Respiratory Panel Viral Screening and Identification Immunofluorescence Assay kit (Light Diagnostics, United States of America) was used to determine the culture results. Initially, the cover slips were mounted on a glass slide with Entellan. Afterwards, one drop of a cocktail of monoclonal antibodies against all target viruses was added to the appropriate coverslips and incubated at 37°C for 30 minutes. The coverslips were washed with PBS and anti-mouse IgG:Fluorescein isothiocyanate (FITC) antibodies added and the coverslips incubated at 37°C for 30 minutes. Afterwards, the coverslips were washed with phosphate-buffered saline (PBS), air dried, mounted and read under a fluorescence microscope in a dark room.

Confirmation of positive culture results

Positive screening results after culturing of tissue using a cocktail of monoclonal antibodies were confirmed and the virus identified as follows:

• The supernatant fluid from the SVC was transferred to labelled 2 ml tubes and the shell vials rinsed with PBS.

• 2 ml of PBS was poured into the shell vials and a plastic bulb used to scrape the cells off the coverslip.

• The solution was pipetted into the labelled 2 ml tubes and centrifuged briefly for 30 seconds.

• After centrifugation, supernatant fluid was discarded and 150 µl of PBS added to the cell pellet.

• The cell pellet and PBS were then thoroughly mixed, after which 15 µl was pipetted into each of the nine wells on a multi-well Teflon slide and allowed to air dry for 30 minutes.

• The slide was then fixed in cold acetone for 10 minutes.

• After fixation, an immunofluorescence test was conducted by adding antisera specific to each target viruses to the wells on the Teflon slide to determine which specific virus was responsible for the initial positive immunofluorescent assay (IFA) screening result.

IFA Interpretation

Positive result – apple green fluorescence in nucleus and/or cytoplasm (Figures 3.3 and 3.4).

Negative result – dull red staining of cells (Figure 3.5).

Figure 3.3 CMV positive result on HF cells characterised by apple green fluorescence under a fluorescence microscope

Figure 3.4 HAdV positive result on HF cells characterised by apple green fluorescence under a

Figure 3.5 CMV negative result on HF cells. Note the absence of apple green fluorescence