ABSTRACT
Human polyomaviruses are emerging pathogens that infect a large percentage of the human population and are excreted in urine. Consequently, urine that is collected for fertilizer production often has high concentrations of polyomavirus genes. We studied the fate of infectious double-stranded DNA (dsDNA) BK human polyomavirus (BKPyV) in hydrolyzed source-separated urine with infectivity assays and quantitative PCR (qPCR). Although BKPyV genomes persisted in the hydrolyzed urine for long periods of time (T90 [time required for 90% reduction in infectivity or gene copies] of >3 weeks), the viruses were rapidly inactivated (T90 of 1.1 to 11 h) in most of the tested urine samples. Interestingly, the infectivity of dsDNA bacteriophage surrogate T3 (T90 of 24 to 46 days) was much more persistent than that of BKPyV, highlighting a major shortcoming of using bacteriophages as human virus surrogates. Pasteurization and filtration experiments suggest that BKPyV virus inactivation was due to microorganism activity in the source-separated urine, and SDS-PAGE Western blots showed that BKPyV protein capsid disassembly is concurrent with inactivation. Our results imply that stored urine does not pose a substantial risk of BKPyV transmission, that qPCR and infectivity of the dsDNA surrogate do not accurately depict BKPyV fate, and that microbial inactivation is driven by structural elements of the BKPyV capsid.
IMPORTANCE We demonstrate that a common urinary tract virus has a high susceptibility to the conditions in hydrolyzed urine and consequently would not be a substantial exposure route to humans using urine-derived fertilizers. The results have significant implications for understanding virus fate. First, by demonstrating that the dsDNA (double-stranded DNA) genome of the polyomavirus lasts for weeks despite infectivity lasting for hours to days, our work highlights the shortcomings of using qPCR to estimate risks from unculturable viruses. Second, commonly used dsDNA surrogate viruses survived for weeks under the same conditions that BK polyomavirus survived for only hours, highlighting issues with using virus surrogates to predict how human viruses will behave in the environment. Finally, our mechanistic inactivation analysis provides strong evidence that microbial activity drives rapid virus inactivation, likely through capsid disassembly. Overall, our work underlines how subtle structural differences between viruses can greatly impact their environmental fate.
INTRODUCTION
Enteric viruses that arise from fecal contamination have long been known to be of serious concern for public health. Viruses that infect the urinary tract of humans can also be shed in large quantities, yet their concentrations and fate in the environment are unclear. Zika virus, for example, is excreted in urine (1) and can cause microcephaly in newborn children of infected mothers (2). Similarly, cytomegalovirus (CMV) is shed in the urine of infected individuals and is a risk to infants of infected mothers, as the virus can cause hearing and vision loss and other developmental disabilities (3).
Polyomaviruses are another class of emerging pathogens that commonly infect the urinary tract of humans (4–6). These nonenveloped, dsDNA viruses readily infect a vast majority of the public asymptomatically (7, 8) but can also cause severe diseases in immunocompromised individuals (9). Primary infection occurs in childhood, and the viruses persist for the entire life of the individual, mainly in epithelial cells in the kidneys and urinary tract and leukocytes in the blood (6, 7, 9–12). BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) are most commonly found excreted in urine (13, 14). The excretion of BKPyV by healthy individuals is asymptomatic, but in transplant patients, replication can cause severe disease (15–17). JCPyV and BKPyV concentrations have been reported as high as 1010 gene copies/ml in the urine of sick individuals, with healthy adults typically excreting lower concentrations (5, 18).
Despite the potential for abundant polyomavirus gene copies in excreted urine, its transmission pathways have not yet been fully determined. Respiratory and fecal-oral routes of transmission have been proposed for BKPyV (19–22), and urine may play a role (23). Ingestion of contaminated water and food has been implicated as an exposure route (21), indicating the potential significance of polyomavirus transmission via the environment.
The need to better understand polyomavirus transmission by urine is underscored by the growing trend of diverting urine from the waste stream and capturing nutrients in urine-derived fertilizers. Urine diversion can provide several environmental benefits, including a sustainable source of phosphorus (24–26), reduction in costs and pollution associated with wastewater treatment (27, 28), a potential reduction of water usage (29), and more efficient treatment of contaminants. Despite the benefits of diverting urine, biological contaminants need to be managed before urine can be reused. Biological contaminants in urine are mitigated with a number of treatment technologies, including long-term storage for several months, pasteurization, or nutrient precipitation (e.g., struvite) (30–32).
When urine is stored in sealed containers to inactivate biological contaminants, the urea in urine is hydrolyzed, resulting in high pH (∼9) and an increase in aqueous ammonia concentrations (2,000 to 8,000 mg N/liter) (33, 34). This transition to hydrolyzed urine can occur within a few hours or days depending on urease enzyme activity in the urine. The high pH and high aqueous ammonia levels have a biocidal impact on indicator organisms (35–37).
Research on biological contaminants in source-separated urine has primarily focused on the presence and fate of enteric pathogens (35, 38–43). Many enteric viruses are single-stranded RNA (ssRNA) viruses, so ssRNA viral surrogates are often used to predict enteric virus fate in urine. Inactivation of the ssRNA bacteriophage MS2, for example, correlated well with aqueous base (e.g., NH3 and OH−) activity, suggesting that inactivation is caused by transesterification of the ribose in RNA (44). Other ssRNA viruses are susceptible to ammonia activity, whereas the single-stranded DNA (ssDNA) bacteriophage ΦX174, the double-stranded RNA (dsRNA) reovirus, and the double-stranded DNA (dsDNA) human adenovirus and bacteriophage T4 were not susceptible to the same transesterification inactivation pathway (45). These results suggest that although common enteric ssRNA viruses are susceptible to the conditions in hydrolyzed urine, viruses commonly found in the urinary tract (polyomavirus, cytomegalovirus, etc.) are stable at the high aqueous ammonia concentrations found in hydrolyzed urine and therefore could pose risks in urine-derived fertilizers.
To identify the transmission risks that polyomavirus may pose in source-separated urine and urine-derived fertilizer production, we tracked the presence and fate of human polyomavirus in fresh and hydrolyzed urine using molecular and culture-based methods. We compared these results to the behavior of common bacteriophage surrogates in an effort to better understand how well surrogate infectivity predicts environmental virus fate and how capsid characteristics may influence inactivation in environmental matrices.
RESULTS AND DISCUSSION
Polyomavirus concentrations in urine and urine-derived fertilizers.Infectious polyomaviruses present in urine cannot be enumerated due to a lack of a compatible tissue culture for studying urine isolates. Consequently, BKPyV DNA concentrations in the collected urine before and after hydrolysis and pasteurization were enumerated by quantitative PCR (qPCR). The endogenous BKPyV DNA concentration in freshly collected source-separated urine sample A prior to hydrolysis was 7.0 × 105 gene copies ml−1 (Fig. 1), which is consistent with reported concentrations in urine of healthy individuals (5 × 100 to 1.24 × 108 gene copies ml−1) (5). These data are based on one fresh urine sample, as it is difficult to collect large fresh urine samples since hydrolysis can happen quickly. The average BKPyV gene copy concentration in hydrolyzed urine samples A, B, and C was 3.8 × 106 gene copies ml−1 and 1.2 × 107 gene copies ml−1 in pasteurized urine samples A, B, and C (Fig. 1) These data suggest that the polyomaviruses, or at least the polyomavirus DNA, survive the harsh conditions of hydrolyzed urine storage and pasteurization.
BKPyV gene copy (152 bp) concentrations detected by qPCR in urine A (fresh, hydrolyzed for 10 months, and pasteurized after being hydrolyzed for 10 months) and urine samples B and C (hydrolyzed for 1 month and 10 months, respectively, and pasteurized after the same hydrolysis times). Fresh urine was only available for urine A due to the rapid hydrolysis in urine samples B and C.
BKPyV and bacteriophage T3 inactivation in hydrolyzed urine.The gene copy concentrations measured by qPCR do not necessarily correspond to the concentrations of infectious viruses. Although infectivity assays for the polyomaviruses found in urine are not possible at this time, certain polyomavirus genome variants, such as BKPyV Dunlop, can be enumerated in vitro. BKPyV Dunlop is a rearranged variant of the archetype that is present in humans. This variant differs in the noncoding control region of the genome and is easily grown in cell culture models (46). The variant viral particles are structurally identical to the viruses found in urine. BKPyV Dunlop was spiked into various hydrolyzed urine samples, and inactivation kinetics were then measured. Source-separated urine characteristics vary depending on the donors' age, nutrition, amount of physical exercise, etc. (47). We therefore utilized a number of source-separated urine samples collected from different regions of the United States and stored for different amounts of time to capture these variations.
Control experiments confirmed that the unspiked urine samples contained no culturable BKPyV. In the spiked hydrolyzed urine samples, BKPyV inactivation rates ranged from 4.7 × 10−3 to 0.90 h−1, corresponding to T90 (time required for 90% reduction in infectivity) values from 1.1 to 210 h (Fig. 2 and see Table 2). Most of the hydrolyzed urine samples exhibited rapid inactivation rates (E, F, H, and I), with T90 of <11 h, but BKPyV was much more stable in hydrolyzed urine sample G. Hydrolyzed urine sample G did not differ from the other urine samples in its ammonia concentration (Table 1) but was collected from fewer donors than urine samples E, F, H, and I.
Infectivity of BK polyomavirus in hydrolyzed urine samples (E to I) measured over time. Initial infective virus concentrations in urine were 5 × 105 to 1 × 106 IU (infectious units) ml−1. Experiments were conducted until the detection limit was reached (3 × 10