Since the geographical spread of highly pathogenic avian influenza infection is very fast in case of an outbreak, early diagnosis and accurate confirmation of the infection are of paramount importance. This, in turn, is possible only if a fast, reliable, and full-proof sample transportation mechanism is available to transport the samples from the affected area to the laboratory. In the case of HPAI, a preliminary diagnosis of the outbreak is carried out with molecular tests such as RT-PCR and Real-Time RT-PCR, followed by actual confirmation of the disease by virus isolation [9]. It is therefore, very important to ensure that the transportation of the virus from the field to the laboratory causes minimum damage to the integrity of the RNA and the virus itself present in the sample. For proper diagnosis of avian influenza, proper collection, preservation, storage, and transportation of samples is very important from the time of collection until the sample is processed in the laboratory [6]. It has been recognized for a long time that the infectivity of the virus decreases over time and generally the decay rate is a function of temperature and the stability of all the viruses is enhanced by cooling [10]. The other basic requirement for the successful isolation of viruses from the field samples is the availability of a suitable transport medium that provides the virus with a stable environment to remain infective [5]. It is therefore very important to maintain the cold chain throughout the transportation of the clinical material and provide suitable media which protect the virus from environmental damage. For diagnosis of avian influenza, cloacal swabs, oropharyngeal/ tracheal swabs, lungs, brain, and digestive organs are mainly collected. WHO has recommended two different transport media, viz., transport medium based on tissue culture medium 199 and glycerol-based medium for transportation of samples of AIVs [6]. Systematic studies and specific recommendations for the transportation of HPAI, however, are singularly lacking. In the present study we evaluated the survivability of the H5N1 avian influenza virus in the samples preserved under different transport media and studied the effect of the cold chain method that has been primarily adopted by the majority of cases to transport the HPAI suspected samples to NIHSAD, Bhopal. The study revealed that the inside temperature of the thermocol boxes reached above 30ºC within three days, after which there was no difference in the internal temperature and the outside temperature. Since only a single size of thermocol box (18cm x18cm x15cm) was used in the study, it was difficult to ascertain if the size of the boxes and the amount of ice packs added in these thermocol boxes would alter temperature situation inside the boxes. The linear regression analysis indicated very little difference between the observed and expected value for survivability in case of PBS, NaCl and CVTM as the R2 value was high (> 0.95) for all the three transport media (Fig. 2,4 and5 ).
In this study, it was found that PBS and NaCl were relatively more effective in maintaining the stability of the virus. Wolfel et al. (2006) who studied two commercially available transport media in comparison to NaCl moisturized swabs reported that commercially available viral transport media performed significantly worse than the NaCl-moisturised swabs with regard to virus recovery rates in cell culture as well as viral genome copy numbers in the qPCR [11]. The cause for the effectiveness of normal saline is difficult to be ascertained, however, a reduction in the pH to below 6.0 in the normal saline could have helped in maintaining the infectivity of the virus [3]. However, NS solution should not be used as with BSA and antibiotics as it may lead to changes that will destroy viruses [6]. Stabilization of acid-base conditions at or near physiological pH is considered desirable for the preservation of virus activity in clinical specimens [5]. Phosphate-buffered saline has been used as a buffer in a wide variety of media as it is considered to approximate the osmotic pressure required by viruses for optimal stability. The stability of the H5N1 virus in PBS in the present study could be because of this buffering action. The relative effectiveness of normal saline and PBS in maintaining the infectivity of the virus as compared to the commercially available transport media though surprising, was an important finding, particularly in the Indian context, since these solutions can be easily prepared at the field level and the ingredients are readily available at economical cost. The stabilizing effect of charcoal has been one of the important reasons for its inclusion in the transport media. CVTM was devised in an effort to formulate a single all-purpose medium and has been useful in preserving a large number of viruses. Many workers have questioned the usefulness of this medium in preserving the influenza virus [12]. However, this study has demonstrated that the H5N1 virus remained infective for up to 6 days indicating the usefulness of this medium as a transport medium. Viral transport medium is one of the most popular mediums used for the maintenance of viruses in field samples. In the present study, the H5N1 virus remained viable only up to 60 hr in the VTM when stored in the simulated transportation conditions as against the longer survivability of PBS, Normal saline, and CVTM. The relatively lower survivability of the virus in VTM could be a problem if the transportation time of the sample despatch is more than two days. VTM is the most commonly referred transport media for the storage of specimens for viral detection diagnosis. This medium majorly is based on Hanks Balanced Salt Solution (HBSS) with Magnesium and Calcium and have heat-inactivated Fetal Bovine Serum, with some antibiotic including Gentamycin and Amphotericin B. Most of the VTM claim to preserve the virus for up to 72 hrs at room temperature and 7–8 days at refrigerator temperature in accordance with our findings. As is in most field conditions availability and affordability of VTM is not sure. Jensen and Johnson (1994) reported that subsequent culture isolation was possible using viral transport media if transport times are kept to a minimum (< 1 day) [13]. Studies on virus stability showed that most viruses tested are sufficiently stable in transport media to withstand a transport time of 1 to 3 days [12]. Glycerol has been used for a long time as a viral preservation medium in tissue samples at 50% concentration, however, the dehydrating action of glycerol and its possible influence in the enzymatic processes of nucleic acid breakdown, raises the suspicion that glycerol, in a higher concentration, could be virucidal particularly in the enveloped viruses [14]. Inactivating effect of glycerol in storage media on the enveloped viruses has also been reported. Monoglycerides such as glycerol monolaurate can reduce the concentrations of enveloped viruses (vesicular stomatitis virus, herpes simplex virus, visna virus, and HIV) by over 10,000 fold indicating an antiviral effect. This antiviral effect of these monoglycerides may be due to their disruptive effect on viral lipid membranes causing leakage and destruction [15].
The linear regression analysis of the effect of 50% glycerol-PBS on the survivability of the H5N1 avian influenza virus indicated that the observed survivability of the virus was only 24 hr as against the expected survivability of 117 hr with a determinant coefficient (R2) value of only 0.254. This indicated a very low fit of the regression model compared to other transport media. Brown et al. (2007) have also reported this aberration in their studies on the persistence of H5 and H7 in water and attributed it to the effect of outliers on the coefficient of determinants in experiments with small sample sizes. Since there were only two observations in the study as the data was collected at 12 hourly intervals, the number of observations was too small to give a good fit to the linear regression model [16]. Data observation with smaller intervals, for e.g., 3 hourly could explain the model more effectively. Temperature is one of the most important determents which have a negative correlation with the survival of AIV [3, 17, 4]. The temperature of the inside of the boxes had reached above 30ºC within 72 hr. At 4ºC infectivity of the virus in the control group was maintained for up to 7 days for all the transport media except in the case of 50% glycerol saline. On comparing the effect of temperature on the survivability of the virus in feces in the experiment it was observed that transport media increased the survivability of the virus even when the temperature was above 30ºC for up to 6 days. In contrast, the control group fecal samples without transport media kept at 37 ºC showed that the survivability of the virus was only for one day while at 4ºC virus could be recovered up to the last day of an experiment which is the 7th day. However, swabbing material too affect the detection rate as flocked and foam swabs were better with BHI and more effectively protected the virus compared to PBS in the same conditions [18]. The flocked swabs are specially designed by an exclusive spray-on nylon flocked fiber technology which allows the improved collection and sufficient release of specimens [19, 20]. This study was carried out on one subtype of avian influenza but as all avian influenza viruses are structurally similar so the finding may be applied to similar viruses. However, the experiment was carried out in fecal samples, but the result may differ for other types of samples like nasal swabs and blood samples.
In India, the temperature varies very much according to season with the lowest up to 2–4 ºC to 40–45 ºC in summer in many parts of the country. This study revealed that the temperature is an important factor in determining the stability of the virus and the type of transport media may affect survivability of the virus and substantially have some protective effect on the virus with reference to temperature but the ability of transport media to support the survivability of the virus was not independent of temperature.