1Laboratory of Zoonoses, School of Veterinary Medicine, Kitasato University, 35-1 Higashi 23 Bancho, Towada, Aomori 034, JapanFind articles by
3Laboratory of Animal Health, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo 183-8509, JapanFind articles by
3Laboratory of Animal Health, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo 183-8509, JapanFind articles by
Background. This study investigates the viable persistence of avian influenza viruses (AIVs) in various types of artificially frozen environmental water and evaluates the feasibility of similar occurrence taking place in nature, and allowing for prolonged abiotic virus survival, with subsequent biotic viral recirculation. Methods. Fresh, brackish, and salty water, taken in Japan from aquatic biotopes regularly visited by migratory waterfowl, were seeded with AIVs. We monthly monitored the viability of the seeded viruses in the frozen state at ?20°C and ?30°C, for 12 months. We also monitored virus viability following repeatedly induced freezing and thawing. Results. The viruses exhibited considerable viable persistence all along that period of time, as well as during freezing-thawing cycles. Appreciable, yet noncrucial variances were observed in relation to some of the parameters examined. Conclusions. As typical waterborne pathogens of numerous northerly aquatic birds, AIVs are innately adapted to both the body temperature of their hosts (40°C to 42°C) and, presumably, to subzero temperatures of frozen lakes (down to ?54°C in parts of Siberia) occupied and virus-seeded by subclinically infected birds, prior to freezing. Marked cryostability of AIVs appears to be evident. Preservation in environmental ice has significant ecophylogenetic and epidemiological implications, potentially, and could account for various unexplained phenomena.
A wide diversity of bacteria, protozoa, and viruses are known to exist in various water bodies worldwide, including ponds, lakes, seas, and oceans [1]. In arctic and sub-arctic regions, those water bodies are frozen, entirely or partially, for 4 months (in the southern Taiga) up to 10 months (in the northern Tundra and Arctic Ocean), annually, in the form of seasonal ice. In the Arctic, perennial ice is found too across the Arctic Ocean and freshwater bodies located in Greenland and islands of the Arctic Ocean. All those water bodies are abundantly visited by migratory aquatic birds whenever partially or completely thawed. Consequently, microorganisms that are shed through feces by the birds into water become waterborne, until contracted again by a host or entrapped within refreezing water. In the case of viruses, as obligatory parasites, they are otherwise apt to perish, sooner or later; hence, whenever entrapped in ice, their cryotolerance might constitute a critical factor, in terms of persisting viability, meaning infectivity. The higher their cryotolerance, the longer is the period of time they are liable to viably be preserved in environmental ice, until thawing reoccurs.
Influenza A virus (IAV) is prevalent within some mammals, including man, and multiple avian host species. It is well established that wild birds infected with avian influenza viruses (AIVs)usually sub-clinicallyare infectious for approximately 610 days, during which they continuously shed vast concentrations of viral particles in their feces [2, 3]. Large portion of this viral mass is deposited into water by aquatic birds, which are highly permissive of AIVs and frequently infected. A part of the deposited virions are then ingested by susceptible birds occupying the water, thus completing the fecal/oral transmission route [4, 5]. This apparatus is well recognized throughout the Tundra and the Taiga belts. In arctic marine environments, various sea-birds, as well as seals and whales harboring IAVs, cyclically introduce them into local melting-freezing seawater. During spring and summer, sub-clinically AIV-infected migratory aquatic birds are prone to reach any frozen water body across the sub-arctic and arctic regions upon thawing, and seed those water bodies with viruses until freezing reoccurs. During fall, those avian populations are inclined to reach poultry farms, especially in Southeast Asia, thereby allowing for a dynamic viral interface with agricultural- and human-derived influenza strains.
Breban et al. [6] observed that abiotic environmental perpetuation of AIVs ought to regularly take place for periods of 2 years at least, due to offering a parsimonious explanation of the 24-year periodicity of avian influenza epidemics; provision of a virus persistence mechanism within small communities where epidemics cannot be sustained by direct transmission only (i.e., communities smaller than the critical community size); and the very low levels of environmental transmission (i.e., few cases per year) that are sufficient for avian influenza to endure within populations where it would otherwise vanish. Year-by-year perpetuation of AIVs in Alaskan lakes occupied during summertime by migratory waterfowl has indeed been pointed at by Ito et al. [7]
The feasibility of viable preservation of AIVs in lake ice has been proposed by Shoham [8, 9] and Webster et al. [10]. Experimentally, IAVs were indeed found to be fairly cryotolerant, in general [11, 12], as elaborated on below. In addition, occasional unconformities between expected and empirical rates of nucleotide substitutions evidenced within AIVs indicate possible pauses of years without virus replication [13]. At the same time, influenza viruses are not known to and most probably do not undergo dormant phases in their hosts, meaning that there has to be some abiotic mechanism accounting for such genetic conservation. Also, fundamentally, the following open question surfaces anyway: what practically happens to influenza virionswhich are known to commonly be found in lake water occupied by waterfowlwhen freezing takes place in arctic and subarctic regions, in terms of virus survival?
2.7. Repeated Freezing and Thawing
Two tubes containing the H7N1 virus Aomori/395/04 were seeded in fresh water and kept at ?20°C. Seven months after seeding (roughly the average lifespan of seasonal lake ice across the Tundra and Taiga) the tubes were removed and incubated at 10°C for 10 minutes to aid in melting. After melting, aliquot 250 ?L of sample from one tube and store it on ice. The remaining samples (from both tubes) were frozen at -20°C for a duration of 75 minutes. After freezing, the sample was removed once more and allowed to melt, aliquoting as previously mentioned, up to four times. Samples were prepared as previously mentioned, with the exception of adding 250 ?L of 100x antibiotic instead of 500 ?L, and titrating as previously mentioned.
2.3. Cells Used for Virus Assaying
AIV propagation has been demonstrated to be well supported by human colon adenocarcinoma (Caco-2) cells [15]. The cells were kept in Nissui Pharmaceutical Company’s Dulbeccos Modified Eagle Medium (DMEM). Ltd. Tokyo, Japan), with the addition of penicillin 100 units/mL, streptomycin 100 ?g/mL, and amphotericin B 0. The 5%20%CE%BCg/mL, 4%20mM%20L-glutamine, and heat-inactivated 2010%%20(V/V)%20fetal bovine serum (FBS) were incubated in a CO2%20(5%)%20incubator in a humidified environment at a temperature of 2037°C to 2029°F. When necessary, cells were maintained in serum-free medium.
Virkon solution (2%, w/v) containing 0. Potassium monopersulfate (MPS) was prepared as an active ingredient using 43% (w/v) of Virkon tablets (Antec International, Sudbury, England). Accel solution (5% or 6. 25%, v/v) containing 0. 35% or 0. 44% (w/w) of the HP was prepared as an active ingredient using Accell Prevention Concentrate (Virox Technologies, Oakville, Ontario). 400 parts per million of calcium carbonate was the standard hardness of the hard water used in the study (11) The antifreeze agents, PG (1,2-propanediol; Sigma-Aldrich, Oakville, Ontario), MeOH (Caledon, Georgetown, Ontario), and anhydrous CaCl2%20 (Sigma-Aldrich), were added during the preparation of the disinfectant solutions to the final concentrations of 2010% to %2040%%20(v/v)%20for PG and MeOH and 2015% to %2030%%20(w/v)%20for CaCl2. The solutions were used immediately after preparation. 2-mL Nalgene cryogenic vials (Thermo Fisher Scientific, Ottawa, Ontario) were filled with aliquots (1 mL) of the disinfectant solutions containing one of the antifreeze agents. The vials were then placed in a freezer with the temperature set at ?20°C. The solutions were observed to see if they remained liquid or had frozen after one hour of incubation. 30% PG, 2020% MeOH, and 2020% CaCl2 were the lowest concentrations at which the disinfectant solutions remained liquid and were therefore chosen for additional testing.
According to what was reported, the Virkon and Accel solutions were sufficiently for the addition of 2020%%CaCl2%, 2020%%PG, or 2020%MeOH to keep the solutions from freezing for at least one hour at 20E2%88%9220%C2%B0C. After neutralization, the antifreeze agents had no negative effects on the ECEs. The virus was resistant to treatment with either 2020%%MeOH or 2030% PG alone for up to 30 minutes (and so); consequently, 2020%MeOH or 2030% PG did not kill AIV. However, when the control disks were treated with 2020% CaCl2% in PBS for 5 minutes or 2010% min at 20°E2.88%9220%C2%B0C, there was a reduction of 0 and 4. 7 log10 EID50 of AIV was noted (and ) when compared to control disks with PBS alone. It’s possible that the interactions between calcium cations and viral proteins produced the killing effect. As seen in the production of cheese and tofu, these interactions may lead to protein aggregation, precipitation, and denaturation (16). Seventy years ago, it was reported that CaCl2 at 0 05 N and 0. 5 N (equivalent to 0. 55% and 5. 5%, w/v) resulted in a mild to moderate decline in the influenza virus within hours (17). The current study found that a significantly higher concentration of CaCl2%(20%, w/v) inactivated roughly 5% of log10%EID50 of the influenza virus in minutes (%) This caliber of a 2020%%20CaCl2%20solution to denature viral proteins is in line with the report stating that the same kind of solution caused coagulative necrosis of living animal tissues (18). It is notable that CaCl2 is widely accessible and used to melt ice on Canadian roads during the winter. But it corrodes metal very easily, so you have to wash it off to avoid damage.
Utilizing the second-tier quantitative carrier test (13) allowed for an assessment of the disinfectant solutions’ virucidal activity. Disks (1 cm in diameter; 0. 75 mm thick) of brushed stainless steel (AISI no. 430; Muzeen Single time point experiments with a contact time of five minutes and contact time course experiments with time points of ten, twenty, and thirty minutes were conducted. Triplicate sample disks and control disks were prepared for each time point. Each disk was surface-applied with 10 ?L of the virus inoculum, which contained roughly 5 to 6 log10 EID50 of AIV based on virus recovered from the control disks. The disk was then allowed to air dry in a biosafety cabinet for one hour. Next, the disk, with the inoculum side up, was placed in a 30-mL polypropylene straightside vial. The vials were then placed inside wells of specially made metal blocks that were preconditioned to maintain the test temperatures. To cover the dried inoculum on the sample disks, a disinfectant solution (50 ?L) preconditioned to the test temperatures was added. For the control disks, phosphate-buffered saline (PBS, pH 7. 0) with or without antifreeze agent (50 ?L) was added. For varying durations of up to half an hour, the vials holding the disks were incubated at -20°C or 21°C, mimicking both warm and cold conditions. Immediately after each contact time, a neutralizer solution (950 ?L) was added to every vial (including those containing control disks) to halt the disinfectant’s action. Nine volumes of Difco D/E neutralizing broth (Thermo Fisher Scientific) and one volume of antibiotic-antimycotic (Invitrogen Canada, Burlington, Ontario) were combined to create the neutralizer. Following a 10-fold serial dilution, the suspension from each vial was examined for AIV infectivity in ECEs (14) Prior to testing the disinfectant solutions, pilot studies were carried out to evaluate the impact of the neutralizer and antifreeze agents on the survival of the virus and ECEs, as well as the impact of the neutralizer on the disinfectant’s activity.
In other studies, disinfectants were able to kill the AIV and Newcastle disease virus at roughly 20°C after only 5 to 10 minutes of contact (4,5,9) However, chemical reactions slow down with decreasing temperature, meaning that more contact time is needed for effective disinfection (9) Increasing a disinfectant’s concentration can expedite the disinfection process (8), but at -20°C, rapid freezing, the organic load on surfaces, and pathogen resistance can make the disinfection process ineffective (9,19). In the current study, the Virkon solution supplemented with 2030%20PG, 2020%20MeOH, or 2020%20CaCl2 resulted in the full inactivation of 2006. H6N2 AIV’s EID50 was 0 log10 in 5 minutes at ?20°C. These findings are consistent with earlier research that found that antifreeze agents added to oxidizing disinfectants, such as Virkon, could effectively kill AIV and the Newcastle disease virus at subfreezing temperatures (9,10). %20In the current study, the AIV was reduced by approximately 6% log10% EID50% within 5% of a minute with the addition of either 2020% MeOH or 2020% CaCl2%, but only by 4% log10% with the addition of 2030% PG% (20) When the Accel concentration was increased from 5% to 6. With the addition of any of the 3% antifreeze agents, a 25% a-log10 reduction within 5 minutes was achieved (%)20
The information displayed is the average quantity of virus found on three separate sets of test or control disks used in repeated tests. To identify significant variations in the quantity of virus recovered from disks incubated at the same temperature for the same contact time, one-way analysis of variance (15) was employed. P was chosen as the crucial threshold for significance. 05.