Laboratory Series#19: Viral Transport medium

In the current scenario of coping with large diagnostic load, a lot of people have asked me on use of VTM in Viral diagnostics. Here are the absolute basics.

Viral transport medium is a liquid media that can sustain the virus numbers, infectivity and reduce or inhibit any possible contaminants (such as bacteria or fungus). As already noted, VTM is not a universal requirement. In some cases, the sample may be submitted for analysis as it is. Samples including CSF, blood, BAL and urine samples can be simply sent in a cold chain. For other samples such as tissue biopsies, nasal swabs, skin swabs etc. VTM is preferred. There a large number of commercial and inhouse formulations of VTM available. Each of these supports good recovery of a few range of viruses. The basic components of all these viral transport media is a buffer solution with protective components such as proteins or charcoal and a PH indicator. A widely accepted principle is that VTMs are isotonic in nature. However, this is not true since there are several transport media which have high tonicity due to high sucrose content and have been successfully used. Note that VTM in itself dilutes the concentration of the virus from the specimen since the VTM is subsequently used for the testing procedure. Further, additional steps such as vigorous vortexing and centrifugation may be required to suspend the virus into medium especially for swabs. This allows for the removal of viruses that are otherwise strongly adsorbed onto the swab surface.

Figure 1: Universal Viral Transport Medium. Source

Cell culture medium:

Most cell culture medium (CCM) contain buffer solutions, a minimal set of nutrients, and a PH indicator. They are also used in the laboratory for attaining cell growth and supporting virus culture in-vitro. In this view, several CCMs have been studied and standardized for alternative use as VTM. The most common example include Eagle’s minimum essential medium with 10% fetal bovine albumin for recovery of HSV from genital swabs. However, they were found to be less efficient especially if longer time is involved. Currently, modified CCMs are used for transport of specimens only if it involves a very short time (< 30 min).

Balanced Salt Solutions:

Balanced Salt solutions (BSS) are extremely useful in controlling the PH which is a major determinant of stability. The most commonly used formulation is phosphate-buffered saline or Hanks BSS. The solutions were combined with other proteins (ex: Albumin, gelatin), buffers (Ex: HEPES buffer) or others (Ex: Agar, Mucopolysaccharides, gelatin, Charcoal) to enhance the virus viability.

Buffered Sugar solutions:

Combining of buffers with sugars such as Sorbitol or Sucrose in concentrations served as a strong cryoprotectant and have been previously used as transport mediums. However, they cause a significant reduction in the number of enveloped viruses and hence their use has been limited.

Bentonite based Solution:

Bentonite is an absorbent clay that acts as an excellent cation- exchange resin. They have been used as a plain or with a combination of proteins that enhance stability. The combination has been found to provide extreme stability for several viruses for up to 21 days.

Virus Transport medium	Main Contents	Examples of Best Supported Viruses
Stuart’s Medium	Agar Thioglycolic acid Sodium glycerophosphate Calcium chloride Methylene blue	Herpes Simplex Virus- 1 & 2
Virocult combination	Modified Stuart medium Phosphate buffer D-glucose Lactalbumin hydrolysate Chloramphenicol & Cycloheximide	Herpes Simplex Virus Varicella-Zoster Virus Influenza Type A& B.
Hanks Buffered BSA	Hanks BSS 1% Bovine Serum Albumin (BSA) Sodium bicarbonate Phenol Red *With or without antibiotics (Amphotericin B, Penicillin G and streptomycin).	Parainfluenza virus Enterovirus Adenovirus
Bentonite Transport medium	Tris buffer EDTA Bentonite coated with rabbit serum	HSV Influenza viruses Rubella viruses
Carr-Scarborough	Phosphate-buffered sucrose L-15 medium Glutamic acid Bovine albumin Gentamicin	Most commonly used for Chlamydia recovery. Varicella-Zoster Virus
Leibovitz-SPG	Leibovitz Buffer Sucrose Phosphate buffer Glutamate BSA	HSV Rhinovirus
Charcoal VTM (CVTM)	Phosphate-buffered saline Potassium chloride Charcoal Agar	Adenovirus
Modified Leibovitz-Emory	Same as CVTM. Agarose use instead of Agar	Adenovirus
Richards media	Phosphate buffer Sucrose Amino acids Bovine serum phenol red	Most respiratory viruses

Table 1: Various types of VTMs available for use and their contents.

Since many different VTMs do not support the recovery of a plethora of viruses more recently formulations have been made that support almost every virus. These are commercially available and known as UVTM (Universal VTMs) and are available from most of the standard suppliers. Alcohol-based VTM (Ex: CyMol VTM) are also commercially available. They rapidly inactivate the virus but maintain the integrity and structure of the virus. They are compatible with downstream applications such as PCR and immunofluorescence, but not culture.