Practical considerations for high-throughput influenza A virus surveillance studies of wild birds by use of molecular diagnostic tests

Abstract

Influenza A virus surveillance studies of wild bird populations are essential to improving our understanding of the role of wild birds in the ecology of low-pathogenic avian influenza viruses and their potential contribution to the spread of H5N1 highly pathogenic avian influenza viruses. Whereas the primary results of such surveillance programs have been communicated extensively, practical considerations and technical implementation options generally receive little attention. In the present study, the data obtained from 39,490 samples were used to compare the impacts of variables such as the sampling procedure, storage and transport conditions, and the choice of molecular and classical diagnostic tests on the outcome of the results. Molecular diagnostic tests allowed estimation of the virus load in samples, which has implications for the ability to isolate virus. Virus isolation in embryonated eggs was more sensitive than virus isolation in cell cultures. Storage and transport conditions had less of an impact on diagnostics by the use of molecular tests than by the use of classical approaches. These findings indicate that molecular diagnostic tests are more sensitive and more reliable than classical tests. In addition, molecular diagnostic tests facilitated analyses in real time and allowed the discrimination of H5 influenza viruses with low and high pathogenicities without the need for virus isolation. Critical assessment of the methods used in large surveillance studies like this will facilitate comparison of the results between studies. Moreover, the lessons learned from current large-scale influenza A virus surveillance activities could be valuable for other pathogen surveillance programs in the future.

FIG. 1.

C_T values and virus isolation. (A) Distribution of C_T values for 1,483 M RRT-PCR-positive samples obtained from 39,490 wild migratory birds during wild bird surveillance studies. (B) Correlation between C_T value and virus isolation results. Group I, all samples from which an influenza A virus was isolated during the first virus isolation attempt; group II, samples from which an influenza A virus was isolated only after blind passaging; negative, no influenza A virus was isolated after two isolation attempts. Isolation attempts were performed with embryonated hens' eggs. Each dot represents the C_T value for an individual bird sample. The 95% CI is represented by the red error bars for each of the three groups.

FIG. 2.

Likelihood of positive results during the first virus isolation attempt for 482 influenza A virus isolates obtained during wild bird surveillance studies. Blue bars, HA subtypes 1 to 16 (of note, HA subtypes 14 and 15 were not detected in this study); dotted line above the blue bars, the median (0.870) of the likelihood of positive results during the first virus isolation attempt for HA; red bars, NA subtypes 1 to 9; dotted line above the red bars, median (0.840) of the likelihood of positive results during the first virus isolation attempt for NA. The numbers above the bars indicate the numbers of viruses with a particular HA or NA subtype that were isolated during the first attempt.

FIG. 3.

Correlation between M RRT-PCR C_T values and H5 RRT-PCR C_T values. Black symbols, samples from the wild bird surveillance studies; red symbols, samples from a serially diluted virus stock of A/Mallard/Netherlands/3/99 (H5N2). Regression lines are shown for each series.

FIG. 4.

Entropy plots for oligonucleotide-annealing sites of the primers and probes of the M and H5 RRT-PCR assays. Nucleotide (nt) sequences from influenza A viruses isolated during the wild bird surveillance studies were aligned, and entropy was calculated for each nucleotide position of each oligonucleotide. Oligonucleotide positions are given in the 5′ to 3′ direction, with position 1 being the extreme 5′ nucleotide. (A) Analysis of 87 M gene sequences with primer RF1073, probe RF1293, and primer RF1074, shown from left to right, respectively. (B) Analysis of 64 H5 LPAI virus gene sequences with primer RF1151, probe RF1153, and primer RF1152, shown from left to right, respectively. The degree of heterogeneity (entropy) was defined as described elsewhere (33, 34).

FIG. 5.

Virus stability under different storage conditions. (A) Stability of influenza virus A/Mallard/Netherlands/3/99 (H5N2) stored at different temperatures. Three 10-fold serial dilutions of virus were tested. Blue dotted lines (⋄), samples stored at 4°C; red lines (□), samples stored at −20°C; and black lines (×), samples stored at −80°C. (B) Impact of multiple freeze-thaw cycles on virus viability and integrity of the RNA of influenza virus A/Mallard/Netherlands/3/99 (H5N2). Blue and red lines, samples stored at −80°C and −20°C, respectively; solid and dotted lines, C_T values (right axis) and virus titers (left axis), respectively. TCID₅₀, 50% tissue culture infective dose.