Selective Isolation of Avian Influenza Virus (AIV) from Cloacal Samples Containing AIV and Newcastle Disease Virus

Abstract

Avian influenza viruses (AIVs) are important zoonotic pathogens whose natural reservoir is waterfowl. In addition to AIV, waterfowl are often coinfected with other viruses, such as the paramyxoviruses, of which Newcastle disease virus (NDV) is of particular importance because of the highly virulent nature of certain strains of this virus for domestic poultry. In routine surveillance of waterfowl for AIV, a number of cloacal samples were encountered that were positive for AIV by real-time reverse transcription polymerase chain reaction (RT-PCR), but did not yield AIV by inoculation in embryonated chicken eggs. On further testing, these samples were also positive for NDV by conventional RT-PCR. It was hypothesized that if both NDV and AIV are present in a sample, the former may overgrow AIV yielding false-negative AIV results. Such samples were treated with chicken anti-NDV polyclonal antiserum and then inoculated in embryonated chicken eggs. Several samples were found to be positive for different subtypes of AIV, indicating that, in the presence of mixed infection with NDV and AIV, it is imperative to remove the influence of NDV, so a true picture of AIV prevalence emerges. An additional benefit is that information on the circulation of NDV in these birds sheds light on their epidemiologic and ecologic significance.

Highly pathogenic Avian influenza virus (HPAIV) and velogenic strains of Newcastle disease virus (NDV) are included on the reportable disease list of the World Organization for Animal Health (OIE). 21,22 Several studies have reported the isolation of AIV 2,5 and NDV 12,17 from waterfowl. Influenza is one of the most important zoonotic diseases of global importance and can cause epidemics and pandemics in poultry and human beings. Based on their virulence, AIV is classified into 2 pathotypes, high pathogenicity (HPAIV) and low pathogenicity (LPAIV). 3,20

Recent outbreaks of avian H5N1 13 and novel H1N1 swine influenza 6 indicate the importance of influenza virus surveillance in wild birds and animals. All known subtypes of AIV can be found in waterfowl or aquatic birds of the orders Anseriformes (ducks, geese, and swan species) and Charadriiformes (shorebird species including gulls), which may help spread the virus to widespread locations and to other species. 14 Therefore, the surveillance of circulating strains of AIV among these populations is necessary to determine their epidemiology and ecology, which could be useful in the effective control of this virus.

In waterfowl, Influenza A virus is occasionally found to coexist with other viruses especially the avian paramyxo viruses (APMVs) 7,10 including NDV, APMV-2, APMV-4, and APMV-6. 5,11 Newcastle disease virus (order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae, genus Avulavirus) is a single-strand, negativesense, nonsegmented RNA virus. 22 On the basis of pathogenicity, strains of NDV are divided into lentogenic (low virulence), mesogenic (medium virulence), and velogenic (high virulence). The coexistence of NDV and AIV in field samples might present a diagnostic problem when isolating and characterizing only AIV is desired for surveillance and epidemiologic purposes. One concern is that the overwhelming growth of NDV may inhibit AIV virus isolation in embryonated chicken eggs (ECEs), causing a skewing of the surveillance data. In AIV surveillance studies, a sample found positive for NDV is usually not processed further for AIV detection. 18,19

The authors know of no study in which attempts were made to systematically improve the isolation and detection of AIV from samples with mixed infection. Under a National Institutes of Health surveillance program, 7,260 cloacal swabs of waterfowl collected from the states of Minnesota and North Dakota in the summer of 2008 were screened. Some of the samples were found to contain both AIV and NDV by molecular tests. On inoculation of these samples in ECEs, NDV could be recovered but not AIV. The current study reports success of a schema in which pretreatment of samples with polyclonal anti-NDV antibodies resulted in AIV isolation from samples with mixed infection.

In the above comprehensive AIV study in Minnesota, cloacal swabs obtained from migrating waterfowl were screened by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) 19 in pools of 5 samples per pool. Pools found to be positive for AIV by real-time RT-PCR were individually tested for AIV by real-time RT-PCR. Individual samples found to be AIV positive were screened for H5-AIV and H7-AIV by real-time RT-PCR. In most laboratories that perform AIV surveillance, samples found to be negative for AIV by real-time RT-PCR and those found to be positive for NDV are not tested further. Hence, the present study was conducted by inoculation of 1,369 AIV real-time RT-PCR–negative and 298 AIV real-time RT-PCR–positive samples in 9-day-old ECEs for virus isolation. Allantoic fluids obtained from 222 samples (147 from real-time RT-PCR–negative samples and 75 from real-time RT-PCR–positive samples) were found positive for hemagglutinin (HA) with 0.5% chicken erythrocytes. 8 The HA-positive allantoic fluids tested positive for AIV by real-time RT-PCR, but the amount of virus was not adequate for sub typing of the isolates. On further testing by conventional gel-based RT-PCR, most of them (n = 201; 90.5%) were found to be NDV positive. A total of 72 samples with mixed infection were randomly selected, and suppression of NDV was attempted by treating the samples with anti-NDV serum so enough AIV could be obtained for subtype determination. Initially, these samples were AIV-positive by real-time RT-PCR. However, when tested for AIV by virus isolation in ECEs without treatment with NDV antiserum, none of them yielded AIV, although NDV was detectable in many of them on virus isolation.

For initial screening, RNA was extracted from cloacal samples using a commercial isolation kit. a From allantoic fluids, RNA was extracted using TRIzol. b For the detection of AIV in cloacal samples and allantoic fluids, a previously described 19 real-time RT-PCR method was used. Samples were considered positive for AIV if the threshold cycle (Ct) values obtained were ≤36. 1

The cloacal samples were inoculated into 9-day-old ECEs (2 eggs per sample) by the allantoic route using 200 μl of sample per egg. The eggs were incubated at 37°C for 4 days and candled daily to determine embryo viability. At the end of 4 days of incubation or upon embryo death, eggs were chilled at 4°C for 24 hr, and the allantoic fluids were harvested aseptically and then reinoculated in fresh 9-day-old ECEs for a second passage. The allantoic fluids harvested after the second egg passage were tested for HA using chicken red blood cells (0.5% in 0.01 M phosphate buffered saline [PBS], pH 7.2). All HA-positive allantoic fluids were again tested by real-time RT-PCR to confirm the presence of AIV.

The HA-positive allantoic fluids were tested for NDV by conventional RT-PCR. 15 The PCR products of approximately 330 base pairs (bp) were electrophoresed on 1.2% agarose gel. Chicken polyclonal antiserum c against NDV was diluted in sterile PBS (pH 7.2) at 1:100, 1:500, and 1:1,000. In a preliminary study, 1:100 dilution of serum was found to suppress the growth of NDV and was, therefore, used for all the remaining samples. To 300-μl aliquots of allantoic fluids, an equal amount of 1:100 anti-NDV serum was added, followed by incubation at 37°C for 1 hr. After centrifugation at 1,200 × g for 10 min, the supernatant was inoculated in 9-day-old ECEs (200 μl of supernatant in each of 2 eggs).

If an allantoic fluid was found positive for the matrix (M) gene of AIV using real-time RT-PCR, further subtyping was performed by conventional, gel-based RT-PCR using hemagglutinin and neuraminidase primers. To amplify the hemagglutinin and neuraminidase, a one-step RT-PCR kit d was performed in a total volume of 50 μl with Influenza A virus H and N primers described previously. 4,9 Bands of H and N genes were excised from the gel and purified using a commercial gel extraction kit. d The purified DNA fragments were sequenced, and the sequence data thus obtained were aligned with the existing influenza database using the BLAST search tool (www.ncbi.nlm.nih.gov).

Polyclonal antiserum was used to neutralize NDV in samples containing both NDV and AIV to improve the isolation and subtyping of AIV. Of the 72 samples treated, 10 yielded fully subtypeable AIVs. The subtypes obtained were H1N1, H2N2, H3N8, H4N1, H4N2, H4N6, H5N2, H7N1, and H7N2. Of the remaining 62 samples, 12 could be N typed with N-specific primers 4 : 11 were N2 and 1 was a mixture of N1 and N2. The H5- and H7-AIV isolates were pathotyped and were found to be LPAIV. Thus, 22 AIV subtypes (31%) were isolated from 72 samples with mixed infection.

Detection of AIV in cloacal samples of waterfowl is a problematic process especially if other hemagglutinating viruses are also present. The most commonly isolated “extraneous” virus in AIV surveillance studies is NDV, whose presence in cloacal samples may hinder the isolation of AIV. 7,11,12 This suppression of AIV by NDV could jeopardize valuable information needed for AIV ecology and epidemiology. In the present proof-of-concept study, it was demonstrated that pretreatment of such samples with anti-NDV antiserum overcomes this problem in many cases. In many previous studies, mixed infections of waterfowl with NDV and AIV have been reported. 11,19 However, it is customary that samples positive for NDV are labeled as NDV positive and are not tested further. 16,21 Based on the results of the current study, a modification in the AIV surveillance procedures is suggested in that if both AIV and NDV are detected by molecular methods, then NDV in these samples should be neutralized by treatment with anti-NDV antiserum, followed by AIV isolation in eggs. A true picture of AIV prevalence can only emerge if such a procedure is adopted.

In the current study, AIV could not be isolated and characterized from all HA-positive allantoic fluids. This lack of AIV recovery could be due to the presence of inactivated virus. It is also possible that such samples need additional blind passages in ECEs or permissive cell cultures (e.g., Madin–Darby canine kidney cells) to enhance the rate of AIV recovery. Similarly, N could be subtyped from 12 samples from which H could not be subtyped using Hoffman primers. 9 Again, further passages may help alleviate this problem. Because the present study was a proof-of-concept study, such samples were not pursued further. It should also be noted that certain strains of AIV may not readily adapt to growth to detectable titers in ECEs. Further studies are being conducted to determine whether samples that were virus isolation–negative in eggs could yield virus on inoculation in cell cultures. In conclusion, pretreatment of samples with mixed infections may yield additional AIV isolates that could be of epidemiologic importance.

Acknowledgements. This work has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Department of Health and Human Services, under contract HHSN266200700007C. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The authors thank the Egyptian Cultural and Educational Bureau in Washington, DC, Zagazig University in Egypt, and the Egyptian Ministry of Higher Education and State for Scientific Research for financial support of the PhD research scholar Mr. M. Ezzat El Zowalaty.