David E. Stallknecht

Persistence of avian influenza viruses in water.

Persistence of five avian influenza viruses (AIVs) derived from four waterfowl species in Louisiana and representing five hemagglutinin and neuraminidase subtypes was determined in distilled water at 17 C and 28 C. Infectivity was determined over 60 days by microtiter endpoint titration. One AIV was tested over 91 days at 4 C. Linear regression models for these viruses predicted that an initial concentration of 1 x 10(6) TCID50/ml water could remain infective for up to 207 days at 17 C and up to 102 days at 28 C. Significant differences in slopes for AIV persistence models were detected between treatment temperatures and among viruses. Results suggest that these viruses are adapted to transmission on waterfowl wintering habitats. Results also suggest a potential risk associated with waterfowl and domestic poultry sharing a common water source.

Persistence of H5 and H7 Avian Influenza Viruses in Water

Abstract Although fecal–oral transmission of avian influenza viruses (AIV) via contaminated water represents a recognized mechanism for transmission within wild waterfowl populations, little is known about viral persistence in this medium. In order to provide initial data on persistence of H5 and H7 AIVs in water, we evaluated eight wild-type low-pathogenicity H5 and H7 AIVs isolated from species representing the two major influenza reservoirs (Anseriformes and Charadriiformes). In addition, the persistence of two highly pathogenic avian influenza (HPAI) H5N1 viruses from Asia was examined to provide some insight into the potential for these viruses to be transmitted and maintained in the environments of wild bird populations. Viruses were tested at two temperatures (17 C and 28 C) and three salinity levels (0, 15, and 30 parts per thousand sea salt). The wild-type H5 and H7 AIV persistence data to date indicate the following: 1) that H5 and H7 AIVs can persist for extended periods of time in water, with a duration of infectivity comparable to AIVs of other subtypes; 2) that the persistence of H5 and H7 AIVs is inversely proportional to temperature and salinity of water; and 3) that a significant interaction exists between the effects of temperature and salinity on the persistence of AIV, with the effect of salinity more prominent at lower temperatures. Results from the two HPAI H5N1 viruses from Asia indicate that these viruses did not persist as long as the wild-type AIVs.

Avian influenza virus in water: infectivity is dependent on pH, salinity and temperature.

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoir for avian influenza (AI) viruses. Transmission within these aquatic bird populations occurs through an indirect fecal-oral route involving contaminated water on shared aquatic habitats. In order to better understand the influence that aquatic environments exert on AI transmission and maintenance in the wild-bird reservoir system, we determined the duration of persistence for 12 wild-bird origin AI viruses under natural ranges of pH, salinity, and temperature. Viral persistence was measured using a laboratory-based distilled water model system. The AI viruses varied in their response to each of the examined variables, but, generally, the viruses were most stable at a slightly basic pH (7.4-8.2), low temperatures (<17 degrees C), and fresh to brackish salinities (0-20,000 parts per million (ppm)). Alternatively, the AI viruses had a much shorter duration of persistence in acidic conditions (pH<6.6), warmer temperatures (>32 degrees C), and high salinity (>25,000 ppm). The results of this research suggest that the pH, temperature, and salinity in natural aquatic habitats can influence the ability of AI viruses to remain infective within these environments. Furthermore, these results provide insight into chemical and physical properties of water that could enhance or restrict AI virus transmission on an aquatic bird habitat.

Effects of pH, Temperature, and Salinity on Persistence of Avian Influenza Viruses in Water

The combined effects of water temperature, salinity, and pH on persistence of avian influenza virus (AIV) were evaluated in a model distilled-water system using three isolates from ducks sampled in Cameron Parish, Louisiana. Variables were tested within the ranges normally associated with surface water. Differences were detected between temperature (17 C and 28 C), pH (6.2, 7.2, 8.2), and salinity (0 ppt and 20 ppt), with a strong interactive effect observed between pH and salinity. Estimated persistence of infectivity for 1 x 10(6) mean tissue-culture infective dose of A/mottled duck/LA/38M/87 (H6N2) was longest at 17 C/0 ppt/pH 8.2 (100 days) and shortest at 28 C/20 ppt/pH 8.2 (9 days). Differences in the response to these variables were apparent between viruses. The ability of AIV to persist in surface water was also evaluated using samples collected from varied waterfowl habitats in coastal Louisiana. Observations were consistent with the model system, with duration of infectivity decreasing with increased salinity and pH. This suggests that experimental results may have application to field conditions.

Phylogenetic Diversity among Low-Virulence Newcastle Disease Viruses from Waterfowl and Shorebirds and Comparison of Genotype Distributions to Those of Poultry-Origin Isolates

ABSTRACT Low-virulence Newcastle disease viruses (loNDV) are frequently recovered from wild bird species, but little is known about their distribution, genetic diversity, or potential to cause disease in poultry. NDV isolates recovered from cloacal samples of apparently healthy waterfowl and shorebirds (WS) in the United States during 1986 to 2005 were examined for genomic diversity and their potential for virulence (n = 249). In addition 19 loNDV isolates from U.S. live bird markets (LBMs) were analyzed and found to be genetically distinct from NDV used in live vaccines but related to WS-origin NDV. Phylogenetic analysis of the fusion protein identified nine novel genotypes among the class I NDV, and new genomic subgroups were identified among genotypes I and II of the class II viruses. The WS-origin viruses exhibited broad genetic and antigenic diversity, and some WS genotypes displayed a closer phylogenetic relationship to LBM-origin NDV. All NDV were predicted to be lentogenic based upon sequencing of the fusion cleavage site, intracerebral pathogenicity index, or mean death time in embryo assays. The USDA real-time reverse transcription-PCR assay, which targets the matrix gene, identified nearly all of the class II NDV tested but failed to detect class I viruses from both LBM and WS. The close phylogenetic proximity of some WS and LBM loNDV suggests that viral transmission may occur among wild birds and poultry; however, these events may occur unnoticed due to the broad genetic diversity of loNDV, the lentogenic presentation in birds, and the limitations of current rapid diagnostic tools.

Environmental transmission of low pathogenicity avian influenza viruses and its implications for pathogen invasion

Understanding the transmission dynamics and persistence of avian influenza viruses (AIVs) in the wild is an important scientific and public health challenge because this system represents both a reservoir for recombination and a source of novel, potentially human-pathogenic strains. The current paradigm locates all important transmission events on the nearly direct fecal/oral bird-to-bird pathway. In this article, on the basis of overlooked evidence, we propose that an environmental virus reservoir gives rise to indirect transmission. This transmission mode could play an important epidemiological role. Using a stochastic model, we demonstrate how neglecting environmentally generated transmission chains could underestimate the explosiveness and duration of AIV epidemics. We show the important pathogen invasion implications of this phenomenon: the nonnegligible probability of outbreak even when direct transmission is absent, the long-term infectivity of locations of prior outbreaks, and the role of environmental heterogeneity in risk.

Experimental Infection of Swans and Geese with Highly Pathogenic Avian Influenza Virus (H5N1) of Asian Lineage

Susceptibility to infection, duration of illness, and concentration of asymptomatic viral shedding vary between species of swans and geese.

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

Background Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. Methods and Principal Findings Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. Conclusion/Significance Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.

The Role of Environmental Transmission in Recurrent Avian Influenza Epidemics

Avian influenza virus (AIV) persists in North American wild waterfowl, exhibiting major outbreaks every 2–4 years. Attempts to explain the patterns of periodicity and persistence using simple direct transmission models are unsuccessful. Motivated by empirical evidence, we examine the contribution of an overlooked AIV transmission mode: environmental transmission. It is known that infectious birds shed large concentrations of virions in the environment, where virions may persist for a long time. We thus propose that, in addition to direct fecal/oral transmission, birds may become infected by ingesting virions that have long persisted in the environment. We design a new host–pathogen model that combines within-season transmission dynamics, between-season migration and reproduction, and environmental variation. Analysis of the model yields three major results. First, environmental transmission provides a persistence mechanism within small communities where epidemics cannot be sustained by direct transmission only (i.e., communities smaller than the critical community size). Second, environmental transmission offers a parsimonious explanation of the 2–4 year periodicity of avian influenza epidemics. Third, very low levels of environmental transmission (i.e., few cases per year) are sufficient for avian influenza to persist in populations where it would otherwise vanish.

Avian influenza viruses from migratory and resident ducks of coastal Louisiana.

Cloacal and tracheal swabs were collected from 1389 hunter-killed ducks in Cameron Parish, Louisiana, during the 1986 and 1987 waterfowl seasons. Twenty-eight avian influenza viruses (AIVs) were isolated from 605 blue-winged teal (Anas discors), 75 mottled ducks (A. fulvigula), 375 gadwalls (A. stepera) and 334 green-winged teal (A. crecca). Prevalence estimates of AIV in ducks sampled during September, November, and December through January were 3.1%, 2.0%, and 0.4%, respectively. Differences in prevalence were detected by season (P = 0.044) and age class (P = 0.036). Two isolations from resident mottled ducks document transmission of AIV on these wintering areas. Much subtype diversity was present, with nine of 13 hemagglutinin (HA) and nine of nine neuraminidase (NA) subtypes recovered. Predominant HA and NA subtypes were typical of AIVs commonly associated with waterfowl. Results indicate that AIVs are transmitted in the wintering areas, and, although prevalence is low, these viruses continue to circulate within these duck populations during winter.