Assay of Several Inactivation Steps on West Nile Virus and H7N1 Highly Pathogenic Avian Influenza Virus Suspensions

Objectives: In any research laboratory, precautionary measures must be taken in order to reduce or eliminate the potential risk of accidental infection by biosafety level 3 (BSL3) pathogens, as Highly Pathogenic Avian Influenza Virus (HPAI) or West Nile Virus (WNV). Appropriate virus inactivation procedures have to be set up to allow necessary further processing of specimens outside BSL3 facilities. Methods: To study the elimination of WN and HPAI virus infectivity, the effect of different chemical and physical inactivation procedures on viral suspensions were investigated. A proper cell culture assay for each virus was performed to verify several treatments, which are commonly performed preceding transfer of materials outside of biocontainment, still allowing further investigations like sequencing or genome amplification. Results: Chemical inactivation with AVL buffer (Qiagen), Trizol® Reagent or Phenol chloroform: isoamylic alcohol treatment, as well as physical treatment (heat at two temperatures and three contact times) reduced viral infectivity in the viral suspension below the detection limit. Conclusion: Thermal treatments, but also Trizol® Reagent and AVL buffer (Qiagen), are suitable to produce noninfectious specimens for further use in molecular biology techniques. MDCK cells (Madin Darby Canine Kidney) were used for infection with highly pathogenic H7N1 avian influenza (HPAI) virus (A/Instituto Zooprofilattico Sperimentale della Lombardi e dell ́Emilia Romagna, Brescia, Italy). Cells were grown in 96-well plates using Earle’s MEM supplemented with 2 mM L-Glutamine (Biochrom AG), 10% Foetal calf serum (FCS, Euroclone Ref. ECS0180L), and Penicillin/Streptomycin/ Nystatin (100 U/100 μl/100 U per ml, Biochrom AG and SIGMA, respectively). Biosafety Biosafety


Introduction
Over the last few years, there has been an important increase in research on viral emerging diseases. This has led to rise in the numbers of BSL3 facilities handling those emerging viruses (Avian Influenza Virus, SARS Coronavirus, and West Nile Virus). However, the set up and maintenance of huge BSL3 areas is expensive. If proper inactivation procedures are evaluated and performed in a standardized way, the BSL3 areas could be smaller, and especially devoted to initial handling and inactivation treatment of a BSL3 pathogen, while further technical processing (immunology, molecular biology) could be performed on the bench of a BSL3 area or indeed, areas of lower biosafety level (BSL2).
West Nile virus (WN virus), belonging to the genus Flavivirus in the Flaviviridae family, are spherical with a lipid envelope, 40-60 nm in diameter, enclosing a single linear positive sense RNA molecule [1]. This virus can cause human diseases as meningitis and encephalitis and for its handling and propagation, a BSL3 environment is mandatory.
HPAI viruses belong to Orthomyxoviridae family. Virions are pleomorphic, 80-120 nm in diameter, enclosing a negative strand RNA genome constituted by 8 segments [1]. They can cause a severe human disease and also require containment level 3.
Swine Vesicular Disease Virus (SVDV) is a species in the genus Enterovirus, in the Picornaviridae family. It is a non-enveloped virus, with icosahedral capsids of 22-30 nm in diameter surrounding a RNA genome [1]. They were included in these studies as a representative of non-enveloped viruses.
Thus, the objective of the present study was to verify the ability of several treatments on the reduction of infectious titres of WN and HPAI viral suspensions.
The threshold standards of efficacy (>4 log 10 R) to be achieved has been taken from the guidelines of viral safety standard of blood derivative products [2,3], an issue that became especially important with the rise of AIDS, as well as with episodes of transmission of hepatitis C virus to recipients. In case of total inactivation, a further use of those treated materials in research assays on the bench could be allowed. and Penicillin/Streptomycin/Nystatin (100 U/100 µg/40 U per ml, from Biochrom AG and SIGMA, respectively), were used for swine vesicular disease virus (SVDV; UK-72 strain, kindly provided by Institute for Animal Health, Pirbright, UK). SVDV was included as a model of nonenveloped virus, with a higher tenacity to inactivation treatments than enveloped viruses [4][5][6].

Inactivation treatments
Chemical treatments: The following treatments were evaluated: viral suspension mixed with Phenol:chloroform:isoamylic alcohol (1:9, v:v) for 10 minutes; a mixture of Trizol ® reagent and viral stock (9:1, v:v) for 10 minutes and a mixture of AVL buffer and viral stock (9:1, v:v), also for 10 minutes. Immediately after treatment, treated and nontreated samples were serially diluted and titrated.
Heat treatments: Viral stocks diluted 1/10 in PBS were heated at 70°C for 5 or 15 minutes or at 100°C for 8 minutes, by using a thermoblock (VLM Bio1, VLM GmbH, Germany). Treated samples were transferred to an ice bath immediately after the treatment. Next, samples were diluted and titrated with the non-treated samples.

Experimental assays
The initial titres of all viral suspensions were up to 7 log 10 Tissue Culture Infectious Dose or TCID 50 /ml. For all viruses, two different viral stocks were used. All viral suspensions (WNV, H7N1 and SVDV) were obtained from supernatants of the corresponding infected cell cultures (Vero, MDCK and IBR-2, respectively). All samples (non-treated and treated after the corresponding contact times) were 10-fold serially diluted with PBS and titrated twice in the corresponding cell lines grown in 96 well-plates, using at least eight wells for each dilution. All treatments were performed in duplicate and samples titrated twice. and Emmons RW [7], and expressed as log 10 TCID 50 /ml. In all cases, cytotoxicity assays were performed in order to ascertain the first non cytotoxic dilution. In those assays, reaction mixtures in which viral suspensions were replaced by the corresponding volumes of cell culture medium were 5-fold serially diluted with PBS, and titrated twice in the corresponding cell lines grown in 96 well-plates using 8 replicates by dilution. Any destruction of cell monolayers should be assigned to toxicity, as viruses (and its hypothetical cytopathic effects) were not present in the reaction mixture. Only the first non cytotoxic dilution of the reaction mixtures was assayed to look for residual infectivity.
The reduction factor (RF) was calculated as the difference in the quotient of the infectious titre before (initial titre or before treatment, BT) and after incubation of the virus with the reagent, or thermal treatment (remaining or residual virus, after treatment, AT), expressed on a log 10 scale.

Results
Results of the different inactivation steps are depicted in Table  1. Initial virus titres in all assays were currently higher than 6.5 log 10 TCID50/ml (with the sole exception of one HPAI trial). With respect to thermal treatment, Reduction Factors (RF) up to 4.5 log 10 was achieved in all cases, regardless of the virus type or the assayed temperature. As Phenol: chloroform: isoamylic alcohol, AVL or Trizol ® resulted in higher toxicity for the aforementioned cell lines; the final RF achieved was lower, between 2.5 and 4.5 log 10 . Moreover, in most of the cases, total inactivation (or inactivation below the detection threshold of the experimental assay) was reached. The symbol "≥" before each figure of viral titres at RF column indicated that the threshold of detection has been achieved, the first non cytotoxic dilution able to be assayed was completely free of viruses.

Discussion
When assaying viral inactivation, two different approaches can be followed: to assay a defined step from a well-known and standardized procedure, or to include an extra step to assure an additional viral inactivation. In our case, the approaches evaluated in this study were chosen because they represent component parts of widely accepted protocols (or commercial kits) in molecular biology. All data are expressed as log 10  When symbol ≤ appears before a viral titre at AT column, it means that all wells of the first non cytotoxic dilution of the reaction mixture were free of viruses, CPE was not observed; however, the inactivation could be higher. Phenol: chloroform: isoamylic alcohol is an old and well-known step in many in-house nucleic acid (NA) extraction procedures. AVL lysis buffer is included in a commercial nucleic acid extraction kit (QIAamp Viral RNA Kit, Qiagen) and it contains guanidine thiocyanate. Also, Trizol ® treatment is in the basis of several in house NA extraction procedures. Finally, the thermal treatments are also part of standardized protocols (70°C for 5 minutes in Nucleospin ® RNA virus kit or 10-15 minutes in Nucleospin ® Blood kit, both from Macherey-Nagel, Germany) and 100°C treatments for 8 minutes is classically used in our facility in bacterial NA extraction protocols for further processing outside.

WNV H7N1 AIV SVDV
SVDV was chosen as a control of non-enveloped virus, opposite to BSL3 enveloped viruses as HPAI virus or WNV. As a general rule, non-enveloped viruses are more resistant than enveloped viruses to physical and chemical inactivation treatments [4][5][6]. In the conditions, no significant differences in inactivation between SVDV and WNV or HPAI virus were observed, probably due to the strength of the applied procedures.
Inactivation of WNV in a detergent-containing buffer has been previously demonstrated [8]. Reductions of 3 log 10 WNV pfu's, by treatment with AVL or Trizol have been previously described [9]. As those effects have been observed for several other enveloped viruses (bunyavirus, alphavirus, filovirus), it could be generalized for the majority of enveloped viruses (as Avian Influenza). In this context, the inactivation results (but also the cytotoxicity results; cell death was reported until 1/100 dilution for AVL and 1/100-1/1000 for Trizol) for HPAI virus, are in total agreement with previous studies [9].
As expected, thermal treatments gave a strong safety margin in terms of robust inactivation of all tested temperatures and viruses. Classically, pasteurization treatments have given excellent results in terms of viral infectivity reduction [10,11]. These deleterious effects of higher temperature have been previously reported for BSL2 viruses as Hepatitis A virus, Reovirus, Bovine Parvovirus, Pseudorabies virus [12], or Encephalomyocarditis virus, but also for BSL3 viruses [13][14][15][16]. Frankfurt-1 strain of SARS Coronavirus suffered 4-5 log 10 reduction (log 10 R) in infectious titres, when spiked in several plasma products and submitted to 60°C for 1 hour [17]. WNV is easily inactivated by pasteurization (60°C for 10 hours) [18,19]. With respect to HPAI virus, effective inactivation at 70°C to 73.9°C for less than 1 s has been reported [16]. Indeed, around 4 log 10 R has been recorded at the end of ramp-up from room temperature to 70°C (lasting 40 seconds) in thermoblock for two HPAI isolates [15], and predicted D-values for H5N1 in chicken meat at 70°C have been reported as short as 0.3-0.5 s [19]. In the given conditions, all trials fulfilled the general commitment of 4 log 10 R, but one single trial (70°C for 15 minutes) failed in the gold standard rule: no infectivity in final samples. The explanation for this could be related to inadequate mixing of the viral suspension, air bubbles or viral aggregation. Incomplete poliovirus inactivation, depending on mixing has been reported [20,21]. Air bubbles poorly conduct heat, and viral aggregation is an old and well-known explanation, when deviations in normal inactivation slopes were reported.
Since, the efficacy of the aforementioned inactivation procedures for the assayed viruses has been verified, the question arises whether these methods are suitable also for other BSL3 pathogens be handled in the facility in the near future (Rift Valley fever virus), but also for non human pathogenic viruses with a well know profile of resistance against inactivation treatments (as canine, bovine or porcine parvovirus), that could be used as model viruses [22,23].
In conclusion, different inactivation procedures (sometimes adapted from steps of commercial kits) for BSL3 pathogens (WNV and HPAIV) were evaluated. The RF obtained allows the use of treated samples in subsequent molecular biology assays on the bench. In fact, nowadays those inactivation procedures have been implemented in the laboratory with good reproducibility and accuracy.