Comparison of spike and aerosol challenge tests for the recovery of viable influenza virus from non-woven fabrics.

BACKGROUND: To experimentally determine the survival kinetics of influenza virus on personal protective equipment (PPE) and to evaluate the risk of virus transfer from PPE, it is important to compare the effects on virus recovery of the method used to contaminate the PPE with virus and the type of eluent used to recover it. METHODS: Avian influenza virus (AIV) was applied as a liquid suspension (spike test) and as an aerosol to three types of non-woven fabrics [polypropylene (PP), polyester (PET), and polyamide (Nylon)] that are commonly used in the manufacture of PPE. This was followed by virus recovery using eight different eluents (phosphate-buffered saline, minimum essential medium, and 1.5% or 3.0% beef extract at pH 7, 8, or 9). RESULTS: For spike tests, no statistically significant difference was found in virus recovery using any of the eluents tested. Hydrophobic surfaces (PP and PET) yielded higher spiked virus recovery than hydrophilic Nylon. From all materials, the virus recovery was much lower in aerosol challenge tests than in spike tests. CONCLUSIONS: Significant differences were found in the recovery of viable AIV from non-woven fabrics between spike and aerosol challenge tests. The findings of this study demonstrate the need for realistic aerosol challenge tests rather than liquid spike tests in studies of virus survival on surfaces where airborne transmission of influenza virus may get involved.

Comparison of cloacal and oropharyngeal samples for the detection of avian influenza virus in wild birds.

This study was conducted to compare oropharyngeal (OP) and cloacal samples of wild birds (n = 137) for the detection and isolation of avian influenza virus (AIV). A total of 39 (28.5%) cloacal and 85 (62.0%) OP samples were positive for AIV by real-time reverse transcription-PCR (RRT-PCR). The AIV nucleic acid was detected in both cloacal and OP samples from 27 (19.7%) birds, in cloacal samples only from 12 (8.8%) birds, and in OP samples only from 58 (42.3%) birds. Thus, a total of 97 (70.8%) birds were AIV positive by RRT-PCR. The cycle threshold values for the cloacal samples ranged from 16.6 to 36.9 (mean 31.5), and those for OP samples ranged from 18 to 38.9 (mean 34.9). Of the cloacal samples, 12 were positive for H5 subtype influenza virus by RRT-PCR, with one being low pathogenic H5N1. In contrast, five of the OP samples were H5 positive, but none was H5N1. None of the cloacal or OP samples was H7 positive. Eight cloacal samples yielded AIV on inoculation in embryonated chicken eggs, while only one isolate was obtained from OP samples. Thus, from testing of 137 birds, only nine (6.6%) AIV isolates were obtained. The isolates from cloacal samples were subtyped as H6N1 (n = 5), H3N8 (n = 2), and H4N8 (n = 1), and the isolate from OP sample was subtyped as H6N1. No virus was isolated from the corresponding cloacal sample of the bird whose OP sample yielded AIV on virus isolation. These results suggest that surveillance programs for detection of AIV by RRT-PCR may include both sample types (cloacal and OP) to obtain a better picture of AIV prevalence, and OP samples may yield additional isolates of AIV when tested in conjunction with cloacal samples.

Phylogenetic analysis of Newcastle disease viruses isolated from waterfowl in the upper midwest region of the United States.

BACKGROUND: This study was conducted to characterize Newcastle disease virus (NDV) isolates obtained from waterfowl from the Upper Midwest region of the United States. A total of 43 NDVs were isolated by inoculation of cloacal samples in embryonated chicken eggs. These isolates were obtained from 24 mallards, seven American green-winged teals, six northern pintails, four blue-winged teals, and two wood ducks. Partial sequences of fusion gene were analyzed to determine the pathotypes and genotypes involved. RESULTS: Deduced amino acid sequence of the cleavage site of fusion (F) protein revealed that all isolates had avirulent motifs. Of the 43 isolates, 23 exhibited sequence motif of (111)GGKQGRL(117) at the cleavage site, 19 exhibited (111)GEKQGRL(117) while one isolate showed (111)GERQGRL(117). Phylogenetic analysis based on comparison with different classes of NDVs revealed that all 43 isolates clustered with class II NDVs and none with class I NDVs. Within class II, five isolates were phylogenetically close to genotype I NDVs while the remaining 38 were close to genotype II. CONCLUSION: We conclude that more than one genotype of NDV circulates in waterfowl in the Upper Midwest region of the US. Continuous surveillance may help better understand the epidemiology of NDVs maintained in wild bird populations and their relationship to NDVs in domestic poultry, if any.

Comparative infection efficiency of Porcine reproductive and respiratory syndrome virus field isolates on MA104 cells and porcine alveolar macrophages.

Isolation of Porcine reproductive and respiratory syndrome virus (PRRSV) on MA104 or MARC-145 cells is frequently used in PRRS diagnosis. However, the ability of recent field isolates to grow on these established simian cell lines has not been determined. The aim of this study was to characterize the growth of PRRSV field isolates on primary porcine alveolar macrophages (PAMs) and MA104 cells in comparison with the growth of the laboratory-adapted strain VR-2332. A cytopathic effect was observed in 70% of serum samples after 1 passage on PAMs and was verified by immunofluorescent staining or reverse transcriptase-polymerase chain reaction. Field isolate growth was observed on MA104 cells for only 1 of 50 serum samples after 14 d. Strain VR-2332 grew readily in MA104 cells [maximum titer, 10(7) TCID(50) (median tissue culture infective dose) per milliliter at 30 h] but not in PAMs (10(2) TCID(50)/mL at 72 h). These results show that PAMs are superior to simian cells for diagnostic isolation of current field PRRSV strains.

Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method.

We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1-15) and 9 NA (1-9) subtypes of influenza A viruses of avian (n=16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.