Experts are fearful about the emergence of a hybrid which combines the killing power of the H5N1 with the efficient transmissibility of H1N1. Guan, an expert on both the H5N1 and SARS, has analyzed the genetic sequences of the new virus, which is a triple reassortant containing genetic material from swine, human and bird.
He said there was a huge information gap due to a lack of regular surveillance on animal disease. Stability of influenza RNA at room temperature in different matrices at different spiking concentrations.
Comparison of RNA yield from different virus concentrations stored in buffy coats. Stability of influenza RNA at room temperature and 4 C when spiked into buffy coats at different concentrations. Stability of influenza RNA at 4 C in different matrices at different spiking concentrations.
Comparison of RNA recovery from different concentrations of virus stored in buffy coats. At higher concentrations 3. Although infectivity loss at lower concentrations 3.
Therefore, loss of virus infectivity was highest in PBS followed by buffy coats and plasma. Virus in plasma showed no significant loss in infectivity. Stability of influenza infectivity at 4 C in different matrices at different spiking concentrations. The possibility of an influenza pandemic has focused attention on the epidemiology and pathophysiology of influenza, with specific emphasis on strains of pandemic potential.
Viremia has been reported in some patients infected with influenza A virus [ 5 — 9 , 11 ]. Therefore, stability and infectivity of influenza virus in blood samples during collection, processing and transport is of potential concern. The novel pandemic influenza A H1N1 virus was used to study the effect of various storage conditions on the ability to detect influenza A viral RNA and virus infectivity in buffy coats relative to PBS and plasma, because the H1N1 virus has been found almost everywhere in the world [ 4 ].
Currently, influenza virus is detected and characterized by the use of real-time RT-PCR assays [ 13 ]. Virus culture is often used to confirm the presence of infectious virus in clinical samples.
Rapid influenza antigen tests have also been used to detect virus, although their specificity has hampered widespread for influenza diagnosis [ 14 ]. Our study focused on the use of RT-PCR and infectivity as a measure of influenza virus stability in the blood matrices.
The use of an RT-PCR assay directed at conserved regions of the influenza virus matrix gene [ 15 ] has proved invaluable as a frontline screening assay that delivers rapid, specific results. Following identification of an influenza virus positive result for the matrix gene sequences, the identification of potential viral HA gene subtypes and determination of their subtype is carried out using a combination of additional RT-PCR assays and sequence analysis [ 13 ].
Our results showed that the H1N1 RNA copy numbers in PBS, buffy coats, or plasma maintained for up to 3 days storage at room temperature or up to 35 days storage at 4 C was relatively stable, and showed no significant change.
Figure 1 , 3 , suggesting that storage time under these conditions may not be a major issue for viral RNA degradation. However, we found that RNA copy number declined more significantly in buffy coats at room temperature rather than at 4 C Figure 2. These findings could be taken into consideration when evaluating virus infectivity or stability in blood matrices such as buffy coats or plasma of infected persons.
Viral RNA copy numbers were significantly lower in buffy coats relative to plasma, or PBS under our experimental conditions and one possible reason may be due to virus adsorption to blood components such as platelets and red cells [ 16 ].
Higher temperature may cause more virions to associate with these blood derived components [ 7 ]. Further study will be needed to examine the effect of different anticoagulants in blood collection, such as EDTA, heparin and sodium citrate, on viral RNA recovery. Although the RT-PCR assay is a useful tool to identify influenza viral RNA stability, full characterization of virus infectivity using an EID 50 assay is necessary to establish the effects of storage on influenza viruses.
Our experiments showed that virus infectivity decays at the time-dependent manner, although viral RNA copy number was relatively stable during the experimental period. Proteins binding to viral RNA, which are required for infection of host cells, might be more prone to disassociation from each other or from viral RNA in PBS relative to plasma, and they may be easily bound to the fibrin network as well which decreases their recovery Figure 3. For these reasons, at lower concentrations of virus, there may be a higher likelihood for significant loss of infectivity over time comparison of Figure 4A with 4B.
We have assessed the stability of novel pandemic influenza A H1N1 virus in PBS, plasma and buffy coats subjected to conditions often encountered in specimen handling, transport, and storage. In conclusion, the findings of this study indicate that conditions of storage of blood plasma and buffy coats, including temperature and length of time could have an impact on influenza virus infectivity in these matrices and should be taken into consideration in handling and transport of samples for diagnostic testing and epidemiology studies.
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