The aim of this review was to summarize the findings from experiments that report persistence of influenza virus in the environment and to convey information about the quality of reporting for the body of work considered. The motivation was to provide better science-based information to advise policies that will impact livestock producers and surrounding communities. For example, to establish that a production site is free of influenza virus prior to repopulation, it may be necessary to sample the premises. The available literature should be able to inform which environmental matrices are associated with longer persistence and therefore should be targeted for testing for influenza virus. Recent outbreaks of avian influenza as well as the interest in the novel 2009 pandemic H1N1 influenza virus suggest that the need for high-quality information about the persistence of influenza virus in livestock environments will only increase.
The data, although limited, suggest that the half-life of influenza virus is significantly shorter in air than in other matrices and that in air, as in other matrices, persistence of influenza virus is longer at lower temperatures. Theoretically, this information and the accompanying estimates of virus half-life could be combined with estimates of virus concentration to predict aerosol dispersion between facilities. Such approaches have been used to predict aerosol transmission of other livestock pathogens, such as foot-and-mouth disease virus and porcine reproductive and respiratory syndrome virus (5
). However, although general associations can be described from the data, the estimates obtained from the review of virus half-life have wide confidence intervals (Tables , , and ). This limitation highlights the need for more applicable primary research into the feasibility of facility-to-facility transmission of influenza virus.
The data summation also suggests that influenza virus has an increased half-life in water compared with that in feces and fomites (Table ) and that persistence may be longer in cool, clean water than in buffered or lake water (P
= 0.0015). The application of this information is that in a depopulation situation, to understand whether influenza virus remains in a barn, water testing would appear to be the more sensitive evaluation, and sampling water from clean water sources, such as troughs or nipples, would be better than testing manure, waste, or contaminated water in the barn. Weber and Stilianakis (97
) also concluded that water might be considered a reservoir for influenza virus, given the similar data evaluated.
These conclusions are consistent with others (73
) regarding prolonged persistence at low temperatures and shortened persistence at extreme pHs and salinities. However, other studies have not previously tried to quantitatively summarize the magnitude of differences across multiple studies. More recent studies continue to demonstrate similar temperature and pH associations with influenza virus (11
). Weber and Stilianakis (97
) discussed the apparent short duration of persistence of influenza virus in the airborne state as well, particularly in low to moderate temperatures and low RH, although this statement was based on human transmission models, which may not be appropriate to apply to airborne persistence in the field between barns of pigs or poultry.
One potential source of bias in our summarized analysis was the number of studies ultimately evaluated, which may have resulted in correlations between results of the same study. The use of a nested random effect was incorporated to adjust for this issue; however, statistical adjustment post hoc is likely a poor substitute for more studies with greater variation. This particularly applies to the water data set, where, after adjusting for the between-study variation in the random effect (i.e., study × temperature), temperature was no longer a significant variable, likely due to the large discrepancy between observation contributions from each study (e.g., one of the seven water studies alone contributed 63 to the total 127 observations) (see Supplement S8 in the supplemental material). For the water model, if study was included as a main effect along with water source, temperature, salinity and pH, all main effects but water source became significant at P values of <0.0001.
Another source of potential bias was the diversity in measurements of viral concentration (i.e., TCID50
, PFU, and MP50
). We used conversion of all assays to viral half-life as a method to obtain a measure of persistence independent of specific assay; however, there was little overlap between measurement units even within the same matrix, unless an author provided continuity between papers (10
). Unless the research community agrees upon a standard method for quantification of virus, this issue will continue to arise for those needing to summarize results across studies.
Potentially, the most significant findings of the review were ancillary findings about data quantity and quality. The review documents the paucity of experiments reporting quantitative assays to assess the persistence of influenza virus in environmental matrices found in livestock facilities, a finding determined by Stallknecht and Brown (80
) as well. The application of systematic review principles to reviewing literature is not as widespread in the bench sciences as clinical sciences; however, others have applied similar approaches to the evaluation of the information about influenza virus and reached similar conclusions about the paucity and disparity of data (97
). To our knowledge, this systematic review is the first to also evaluate the quality of studies regarding influenza virus. Shahid et al. (73
) investigated inactivation rather than virus persistence in a narrative discussion, but they likewise noted that the aim of their review was to add evidence to the scant information available for biosecurity recommendations for poultry facilities. In this investigation, we had anticipated that persistence of influenza virus on surfaces and in feces and feces-like matrices would have generated more primary research; however, statistical synthesis of virus half-life on fomites and in feces was not possible, as so few observations were available (see Supplements S8, S10, and S11 in the supplemental material). Similarly, since no soil or compost study reported key features of a persistence study, it was not possible to report on the persistence of influenza virus in common methods of livestock mortality removal. More recent work has evaluated the persistence of avian influenza virus in land disposal (25
The lack of data may partly be a function of the systematic review methodology which uses predetermined parameters and criteria for the evaluation of citations for relevance, and these criteria are followed sequentially and strictly. As a consequence of this approach, relevant experiments would not be considered if the title or abstract did not discuss the pertinent topic of the persistence of influenza virus or were not evidently primary research. However, the potential for this bias seems unlikely, as few relevant studies were identified outside the electronic search, the search was comprehensive, and others have reported the paucity of data.
Further, in the experiments conducted, the variation in parameters assessed was narrow. Illustrative of the lack of range assessed is that only 30 observations in water, three observations for feces or diluted feces, and three observations in air were available at or below 12°C. This lack of data is particularly relevant, as low temperatures may occur in livestock facilities or manure storage units. Data on the persistence of influenza virus at extreme values of pH or salinity are of less importance, since it is likely the range of pH and salinity observed in livestock facilities is narrow.
The study designs and methods of reporting were also extremely heterogeneous and often limiting. Several studies were performed at room temperature, and descriptions of the sensitivity of the equipment were uniformly absent; therefore, there was significant interpretation necessary regarding the parameter values reported. Because of this, it was unfortunate but necessary to categorize naturally continuous variables like temperature, salinity, pH, and relative humidity. The continuous nature of these parameters may impact viral half-life in a progressive manner, and this could have been lost by our wide groupings. Likewise, even within the categories, there was insufficient representation to examine interactions between temperature and humidity, or temperature and pH, for example, and these are common questions about influenza virus persistence.
The evaluation of reporting quality is not as widespread in the bench sciences as clinical sciences, but it is useful to identify strengths and weaknesses in study reporting. In clinical research, there has been an increased focus in recent years on the quality of reporting and how closely reports adhere to the concept of reproducibility. Many studies have provided empirical evidence that clinical trials and observational studies frequently fail to report sufficient information for reproduction, assessment of bias, and research synthesis (14
). Articles or editorials have described poor reporting of statistical methods (57
); otherwise, there appears to be little empirical evaluation of the quality of reporting in the laboratory sciences.
This review suggests that, as has been documented in other fields, the reporting of these studies may be less than ideal to meet the requirements for a reproducible description of an executed study. Authors consistently failed to report sufficient information to fully understand the experiment design, execution, and results. Beyond looking at the reporting methods, this review identified what appeared to be common flaws in design execution as well. For example, sampling and replication, fundamental concepts and requirements for proper statistical assessment and reporting of standard deviation, were clearly insufficient in the studies under review to confidently extrapolate results to field application. Multiple replicates and samples per sampling time enable the expression of normal variation for estimates of continuous outcomes, and they improve confidence in parameter estimates for statistically meaningful results. Further detailed discussion of reporting gaps and replicate and sample numbers can be found in Supplements S13 and S14 in the supplemental material. Because of the absence of replicates, the uncertainty within studies clearly impacts the uncertainty when synthesizing information between studies for this review, as evidenced by the large confidence intervals around the persistence estimates (Tables , , and ).
Additional key areas that require considerable improvement in reporting are the descriptions of environmental conditions and the statistical methods, including data transformation. For baseline environmental conditions that did not vary during the experiment, such as temperature, pH, salinity, or RH, improved and more detailed descriptions are imperative to enable comparisons between studies. In this review, terms such as “room temperature” or “fresh water” were interpreted and estimates were assumed, because of lack of descriptions, to incorporate results into the cumulative data set. Similarly, experiments which portrayed data only graphically were interpreted and estimated to enable their inclusion in the review, and this estimation is not as accurate as data extracted from experiments presenting numerical results or statistical outcomes with well-described methodologies.
Finally, it was unexpected to find so few studies reporting results as decay rates or half-lives of the virus. Virus titer, percent virus remaining, and duration of persistence are not easily applicable to the field, as they can be useful only when exact starting concentrations are repeated. Alternatively, results reported as decay rates or half-lives have significantly more utility, as they can be applied to any starting concentration and therefore are able to be used in existing environmental settings and can be applied to any known starting concentration of virus.
The results of this study draw attention to potential needs for improved reporting of design and methods in the current scientific literature concerning influenza. Similar studies showing empirical evidence of poor reporting have provided the motivation for reporting guidelines in other areas of scientific research. There are numerous examples of disciplines in which guidelines have been published, where evidence of systemic problems with design execution and reporting in multiple fields led to guideline development (7
). The methodological assessment of the 19 studies included in the review confirms the need for additional but significantly improved studies regarding influenza virus persistence in the environment, the need for more transparency, with more focus on detailed reporting within sampling, and the need for attention to replication, to provide more robust outcome information to support decision-making and policy formation.
Ultimately, this review revealed that, although there is a significant amount of published literature regarding influenza virus, there are very few studies that can be used to support decision-making and policy formation. Although this study was comprehensive, the resultant data extracted for this synthesis leave a great deal of uncertainty for field application or management decisions and are outdated for certain matrices. Future work should use improved reporting of study designs and outcomes to enable a more thorough and robust meta-analysis of environmental persistence of influenza virus.