Low-pathogenicity influenza viruses replicate differently in laughing gulls and mallards.

Wild aquatic birds are natural reservoirs of low-pathogenicity avian influenza viruses (LPAIVs). Laughing gulls inoculated with four gull-origin LPAIVs (H7N3, H6N4, H3N8, and H2N3) had a predominate respiratory infection. By contrast, mallards inoculated with two mallard-origin LPAIVs (H5N6 and H4N8) became infected and had similar virus titers in oropharyngeal (OP) and cloacal (CL) swabs. The trend toward predominate OP shedding in gulls suggest a greater role of direct bird transmission in maintenance, whereas mallards shedding suggests importance of fecal-oral transmission through water contamination. Additional infectivity and pathogenesis studies are needed to confirm this replication difference for LPAI viruses in gulls.

Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses.

Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting AIV in swab samples from wild birds, but AIV detection rates are higher with RRT-PCR than isolation in ECE. In this study we tested duck embryos, turkey embryos, and multiple cell lines for AIV growth as compared to ECE for improved isolation and propagation of AIV for isolates representing all 16 hemagglutinin subtypes. There were no differences in low pathogenicity AIV (LPAIV) propagation titers in duck or turkey embryos compared to ECE. The replication efficiency of LPAIV was lower in each of the cell lines tested compared to ECE. LPAIV titers were 1-3 log mean tissue-culture infective doses (TCID50) lower in Madin-Darby canine kidney (MDCK), primary chicken embryo kidney (CEK), and primary chicken embryo fibroblast (CEF) cell cultures, and 3-5 log TCID50 lower in chicken bone marrow macrophage (HD11), chicken fibroblast (DF-1), and mink lung epithelial (Mv1Lu) cells than the corresponding mean embryo infective doses (EID50) in ECE. The quail fibroblast (QT-35) and baby hamster kidney (BHK-21) cell lines produced titers 5-7 log TCID50 less than EID50 in ECE. Overall, ECEs were the most efficient system for growth of LPAIV. However, the savings in time and resources incurred with the use of the MDCK, CEK, and CEF cultures would allow a higher volume of samples to be processed with the same fiscal and financial resources, thus being potentially advantageous despite the lower replication efficiency and lower isolation rates.

Single and combination diagnostic test efficiency and cost analysis for detection and isolation of avian influenza virus from wild bird cloacal swabs.

Effective laboratory methods for identifying avian influenza virus (AIV) in wild bird populations are crucial to understanding the ecology of this pathogen. The standard method has been AIV isolation in chorioallantoic sac (CAS) of specific-pathogen-free embryonating chicken eggs (ECE), but in one study, combined use of yolk-sac (YS) and chorioallantoic membrane inoculation routes increased the number of virus isolations. In addition, cell culture for AIV isolation has been used. Most recently, real-time reverse transcriptase (RRT)-PCR has been used to detect AIV genome in surveillance samples. The purpose of this study was to develop a diagnostic decision tree that would increase AIV isolations from wild bird surveillance samples when using combinations of detection and isolation methods under our laboratory conditions. Attempts to identify AIV for 50 wild bird surveillance samples were accomplished via isolation in ECE using CAS and YS routes of inoculation, and in Madin-Darby canine kidney (MDCK) cells, and by AIV matrix gene detection using RRT-PCR. AIV was isolated from 36% of samples by CAS inoculation and 46% samples by YS inoculation using ECE, isolated from 20% of samples in MDCK cells, and detected in 54% of the samples by RRT-PCR. The AIV was isolated in ECE in 13 samples by both inoculation routes, five additional samples by allantoic, and 10 additional samples by yolk-sac inoculation, increasing the positive isolation of AIV in ECE to 56%. Allantoic inoculation and RRT-PCR detected AIV in 14 samples, with four additional samples by allantoic route alone and 13 additional samples by RRT-PCR. Our data indicate that addition of YS inoculation of ECE will increase isolation of AIV from wild bird surveillance samples. If we exclude the confirmation RT-PCR test, cost analysis for our laboratory indicates that RRT-PCR is an economical choice for screening samples before doing virus isolation in ECE if the AIV frequency is low in the samples. In contrast, isolation in ECE via CAS and YS inoculation routes without prescreening by RRT-PCR was most efficient and cost-effective if the samples had an expected high frequency of AIV.

Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction-positive wild bird surveillance samples.

Virus isolation rates for influenza A virus (FLUAV) and Avian paramyxovirus serotype 1 (APMV-1) from wild bird surveillance samples are lower than molecular detection rates for the specific viral genomes. The current study was conducted to examine the possibility of increased virus isolation rates from real-time reverse transcription polymerase chain reaction (real-time RT-PCR) using alternative virus isolation substrates such as embryonating duck eggs (EDEs), embryonating turkey eggs (ETEs), Madin-Darby canine kidney (MDCK) cell cultures, and African green monkey kidney (Vero) cell cultures. Rectal swabs of birds in the orders Anseriformes and Charadriiformes were tested by real-time RT-PCR for the presence of FLUAV and APMV-1 genomes, and virus isolation (VI) was attempted on all real-time RT-PCR-positive samples. Samples with threshold cycle (Ct) ≤ 37 had VI rates for FLUAV of 62.5%, 50%, 43.8%, 31.5%, and 31.5% in embryonating chicken eggs (ECEs), ETEs, EDEs, MDCK cells, and Vero cells, respectively. A higher isolation rate was seen with ECEs compared to either cell culture method, but similar isolation rates were identified between the different embryonating avian eggs. Virus isolation rates for APMV-1 on samples with real-time RT-PCR Ct ≤ 37 were 75%, 100%, 100%, 0%, and 37.5% in ECEs, ETEs, EDEs, MDCK cells, and Vero cells, respectively. Significantly higher VI rates were seen with ECEs as compared to either cell culture method for all real-time RT-PCR-positive samples. Because of the limited availability and high cost of ETEs and EDEs, the data support the continuing usage of ECEs for primary isolation of both FLUAV and APMV-1 from real-time RT-PCR-positive wild bird surveillance samples.