FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION (See Section 10 and Rule 13)
Title of invention:
A method for concentration of viruses
Applicant
Indian Institute of Technology Bombay, an autonomous research and educational institution established in India by a special Act of the Parliament of the Republic of India under the Indian Institute of Technology Act 1961, Powai, Mumbai 400076, Maharashtra, India
Inventors
Suparna Mukherji, Pisharody Lakshmi Krishnakumar, Sumathi Suresh, all Indian nationals having its address at Indian Institute of Technology Bombay, Department of Centre for Environmental Science and Engineering, Powai, Mumbai 400076, Maharashtra, India
Preamble to the description
The following specification particularly describes the invention and the
manner in which it is to be performed

FIELD OF THE INVENTION
[001] The present invention relates to a method for concentration of viruses and more particularly, to a packed-bed adsorbent column for simultaneously concentrating various classes of viruses.
BACKGROUND OF THE INVENTION
[002] Increased anthropogenic activities have resulted in the contamination of water bodies by microorganisms, such as pathogenic bacteria and enteric viruses. These microorganisms, especially the enteric viruses cause diseases like diarrhea, gastroenteritis and are also capable of causing respiratory tract infections, hepatitis, conjunctivitis, and paralysis. Further, contamination of drinking water resources can also result in epidemics. For instance, drinking water containing 100 copy/L of an enteric virus may prove to be fatal if consumed. Hence, it is essential to ascertain presence and subsequently determine the concentration of enteric viruses in various natural water sources in order to deploy any kind of control measures.
[003] Viruses, however, have different growth conditions and behavioral patterns, due to which detection of a particular type of virus may be time consuming, cost intensive and tedious. In view of this, various concentration techniques have been developed over the years based on particle sizes and surface properties of the viruses. The most widely used primary concentration techniques are adsorption elution method and ultrafiltration method. The adsorption elution method involves use of electropositive or electronegative filters or cartridges. The electropositive filters are reported to clog easily, and recovery of the viruses from

these filters is highly variable. Further, the electronegative filters need
preconditioning by acidification of the water/wastewater sample since the viruses
generally carry negative charge at neutral pH. This makes acidification, a
cumbersome process for large volumes of water. On the other hand, the
ultrafiltration method which is based on size exclusion principle was found to be
time consuming and expensive.
[004] Anion exchange resins, such as Amberlite has been explored as an
adsorbent for concentrating viruses. However, the reproducibility and the
applicability of the method for subsequent quantification based on both molecular
and culture based methods have not been explored adequately.
[005] JP2004129548 discloses DEAE cellulose filled fluidized bed column for
the concentration of small spherical round viruses from lean wastewater samples.
However, since DEAE cellulose is a powdered adsorbent with gelatinous nature,
it cannot be employed in a packed bed column. Thus, the method becomes energy
intensive as DEAE cellulose columns cannot be operated in down flow mode
under gravity and require high velocity for fluidization.
[006] Further, the existing techniques are known to show variable recovery rates,
while some techniques are cost intensive and time consuming.
[007] Hence, there is a need in art for an adsorption elution process that
efficiently concentrates different classes or varieties of viruses efficiently for
subsequent quantification and addresses the aforementioned problems.

SUMMARY OF THE INVENTION
[008] Accordingly, the present invention in one aspect provides a method for
concentrating viruses dilutely contained in water comprising the following steps:
preparing an adsorbent column comprising DEAE immobilized on silica gel;
passing the water through the column at a predetermined flow rate; passing a
complementary eluent for recovering the virus adsorbed on the column.
[009] The present invention, in another aspect, provides a kit for increasing the
concentration of a virus dilutely contained in water, the kit comprising:
an adsorbent column comprising of DEAE immobilized on silica gel and a
complementary eluent for eluting the viruses from the adsorbent.
BRIEF DESCRIPTION OF THE DRAWINGS
[010] Reference will be made to embodiments of the invention, examples of which may be illustrated in accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
Figure 1 shows FTIR spectra for silanized silica gel before and after DEAE functionalization in accordance with an embodiment of the invention.
Figure 2 shows breakthrough profiles of SUSP2 for columns packed with DEAE silica gel and silanized silica gel in accordance with an embodiment of the invention.
Figure 3 shows a SUSP2 elution profile from the DEAE silica gel packed column using ES-2 eluent in accordance with an embodiment of the invention.

Figure 4 shows Breakthrough profiles of MS2 for column packed with DEAE silica gel and silanized silica gel in accordance with an embodiment of the invention.
Figures 5a and 5b show MS2 elution profiles for a DEAE silica gel column using a) EM-7 and b) FM-3 eluent in accordance with an embodiment of the invention.
Figures 6a, 6b and 6c show Standard curve for qPCR based quantification of a) SUSP2 b) MS2 c) Rotavirus in accordance with an embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[011] The present invention discloses a method for concentrating viruses, particularly enteric viruses and coliphages, dilutely contained in water and is useful for simultaneously concentrating multiple classes of viruses from a water source. Moreover, the method is capable of concentrating even very low concentration of viruses with high recovery rate and excellent reproducibility. [012] In an aspect of the present invention, the method comprises passing the source water through an adsorbent column comprising N, N-diethyl-ethanolamine (DEAE) immobilized on silica gel. The DEAE and silica gel is in a ratio in the range of 0.5:1 to 2:1 (v/w), preferably in the ratio of 1:1 (v/w). [013] Further, the particles of DEAE silica gel adsorbent have diameter in the range of 75 µm to 150 µm. In a preferred embodiment, the diameter of the

particles of the DEAE silica gel adsorbent is of 100-200 mesh size as specified by
the manufacturer.
[014] The water is passed through the adsorbent column at a predetermined flow
rate of 5 mL/min and 50 mL/min. In a preferred embodiment, the flow rate of
water used is 50 mL/min. As a result of passing the water through the adsorbent
column, the virus is adsorbed onto the adsorbent.
[015] Subsequently, the adsorbed viruses are eluted using an eluent
complementary to the virus being concentrated. Table 1 shows the various eluents
screened for desorption of coliphages from structurally similar adsorbents and the
optimized eluents were tested for elution of various viruses from DEAE silica gel
and were utilized in this invention.

MS2 SUSP2
EM-1 0.1 % Triton X 100 +1M NaCl + 0.05 M KH2PO4 (pH 9.2) ES-1 1 M NaCl + 2% Tween 80 + 0.05 M KH2PO4 (pH 9.2)
EM-2 0.1 % Tween 80 + 1 M NaCl + 0.05 M KH2PO4 (pH 9.2) ES-2 3 % Tween 80 + 1.5 M NaC1 + 0.05 M KH2PO4 (pH 9.2)
EM-3 1 % NaPP + Phosphate buffer +-glycine ES-3 1.5 M NaCl + 2 % Tween 80 + 0.05 M KH2PO4 (pH 9.2)
EM-4 1 % Tween 80 + 1 M NaCl + 0.05 M KH2PO4 (pH 9.2) ES^ 2 M NaCl + 3 % Tween 80 + 0.05 M KH2PO4 (pH 9.2)
EM-5 2 % Tween 80 + 1 M NaCl + 0.05 M KH2PO4 (pH 9.2) ES-5 3 % Triton X 100+ 1.5 M NaCl + 0.05 M KH2PO4 (pH 9.2)
EM-6 3 % Tween 80 + 1 M NaCl + 0.05 M KH2PO4 (pH 9.2) ES-0 0.1 % Tween 80 + 0.5 M EDTA (pH
7.2)
EM-7 2 % Tween 80 + 1.5 M NaCl + 0.05 M KH2PO4 (pH 9.2) ES-7 2 M NaNO3+ 3 % Tween 80 + 0.05 M KH2PO4
EMS 1 % Tween 80 + 1 % NaPP+ 0.05 M KH2PO4 (pH 9.2) ES-8 1% Tween 80 + 1% NaPP + 0.05 M KH2P04
FM-1 Glycine 3X broth (pH 10.2) ES-9 Glycine 3X broth (2 M NaCl,pH 10.2) + 3% Tween 80
FM-2 Glycine 3X broth (pH 10.2) 2% Tween 80 FS-1 Glvcine 3X broth (pH 10.2), 1.5 M NaC1 + 2% Tween 80 - KH2PO4
FM-3 Glycine 3X broth (pH 10.2, 1.5 M NaCl) + 2% Tween 80 FS-2 NanS:Oj + 0.1% Tween 80 (pH 9.2)
FM-4 Glycine 3X broth (pH 10.2, 1.5 M
NaCl, 3% BE) + 2 % Tween 80 FS-3 Glycine 3X broth (pH 10.2) + 2 % Tween 80
FM-5 Glycine 3X broth (pH 10.2, 2 M NaCl) + 2% Tween 80 FS-4 Glvcine 3X broth (pH 10.2, 1.5M NaCl) + 2% Tween 80
FS-5 Glvcine 3X Broth (pH 10.2, 1.5 M NaCl) + 3% Tween 80
FS-6 Glvcine 3X Broth (pH 10.2, 2 M NaCl) + 3% Tween 80
FS-7 Glycine 3X Broth (pH 10.2, 1.5 M NaCl, 3% BE) + 3% Tween 80
EM and ES eluents were used for eluting MS2 and SUSP2 from DEAE cellulose, respectively; FM and FS eluents were used for eluting MS2 and SUSP2 from FaRHA, respectively.
Table 1 : Table 1 shows the various eluents screened for desorption of coliphages from structurally similar adsorbents and the optimized eluents tested for elution of various viruses from DEAE - silica gel

[016] The eluted viruses were then concentrated using further concentration
processes including, ultracentrifugation and polyethylene glycol precipitation but
is not limited to the aforementioned methods.
[017] In another aspect, the invention relates to a kit for increasing the
concentration of a virus dilutely contained in water using the method as described
herein above, the kit comprising:
an adsorbent column comprising of DEAE immobilized on silica gel and a
complementary eluent for eluting the viruses from the adsorbent.
Examples
The following experimental examples are illustrative of the invention but not limitative of the scope thereof:
Materials and Methods Development of sample virus stock
[018] MS2 coliphage and bacterial host E.coli C3000 were procured. Further, SUSP2, which is a somatic coliphage, was isolated from a water source. The SUSP2 and the MS2 coliphages were propagated by allowing them to infect the E.coli C3000 in the exponential growth phase (absorbance at 600 nm of 0.4-0.8) for 6 h at 37°C under shaking condition (100 rpm). The bacterium was removed by centrifugation at 6000 g for 10 min at 4°C and the supernatant was filtered through 0.22 μm filters. The filtrate was subjected to ultracentrifugation at 25,000 rpm for 2 h at 4°C. After removing the supernatant, the pellet was re-suspended in

a buffer solution. The so obtained virus stock was stored at 4°C and was periodically enumerated using a double layer agar method.
Preparation of adsorbent DEAE silica gel
[019] The method used was adapted from Kundu and Roy (1989). Briefly, 10 g of silica gel (mesh size 200-400) was dried under vacuum for 30 min. The dried silica gel was added to a mixture containing 100 mL of 10% y-glycidoxypropyltrimethoxysilane in toluene and 10 ml of N, N diethyl-ethanolamine (DEAE) and incubated for 20 h at 45°C. Hydrolysis and condensation of the coupling agent, y-glycidoxypropyltrimethoxysilane facilitated the functionalization of silica gel with DEAE. The reaction mixture was allowed to cool to room temperature. The unbound silane was removed by washing with methanol followed by water and conversion to chloride form was achieved using dilute hydrochloric acid. After drying under vacuum, the DEAE silica gel was stored at room temperature. Silanized silica gel was prepared similarly by contacting 10 g of dried silica gel with 100 mL of 10% y-glycidoxypropyltrimethoxysilane in toluene for 20 h at 45°C and subsequently subjecting the adsorbent to similar post treatment steps.
Characterization of the DEAE Silica gel adsorbent
[020] FTIR analysis of the silica gel before and after functionalization with the DEAE was performed using a 3000 Hyperion Microscope with Vertex 80 FTIR System. The spectra of the DEAE silica gel was compared with that of activated

silica gel for identifying the unique functional groups on the adsorbent and their role on virus adsorption.
Column studies with distilled water spiked with coliphages
[021] Column studies were conducted in a column with diameter 2.2 cm at a flow rate of 50 mL/min. Water sample (10 L) spiked with varying concentration of the SUSP2 and the MS2 was passed through the DEAE silica gel column of 10 cm packing depth in the packed bed mode. Samples were collected from the column outlet at periodic intervals and were analyzed by either plaque assay or qPCR. Eluent used for recovery of the SUSP2 was 3% Tween 80 + 1.5 M NaCl + 0.05 M KH2PO4 at pH 9.2 (ES-2). For MS2, the eluent comprised of glycine 3 X broth (1.5 M NaCl, pH 10.2) and 2% Tween 80 (FM-3). The eluents were previously optimized for maximum recovery of coliphages from the adsorbent. In order to check the role of silica gel on virus adsorption, column studies were also conducted with columns containing silanized silica gel with no DEAE immobilized on it. The efficacy of regeneration of the DEAE silica gel adsorbent was tested for 4 cycles of adsorption and regeneration. All the experiments were conducted in duplicate, and the error bars depict standard error.
Column studies with lake water
[022] Lake water samples were collected from various lakes. The water samples were passed through the DEAE silica gel column (10 cm) at a flow-rate of 50 mL/min. Elution was subsequently performed using 50 mL of the ES-2, followed by 50 mL of the FM-3 eluent. Secondary concentration was done by polyethylene

glycol (PEG) precipitation. Briefly, 15% PEG 6000 and 2% NaCl was added to the primary concentrate and incubated at 4°C for 20 h. Thereafter, the suspension was centrifuged at 10,000 g at 4°C and the pellet was re-suspended in 1 mL of SM buffer. The Lake concentrate was further subjected to qPCR after isolation. Genomic material was isolated from the lake concentrate and subsequently reverse transcription was performed before quantification of the copy number in the concentrate using qPCR. The same volume of the lake water was concentrated using only ultracentrifuge for primary concentration and its recovery was compared to that obtained for the DEAE column.
Results
Adsorbent Characterization
[023] Immobilization of the DEAE on silanized silica gel is expected to increase the amide and Si-O-Si groups on the DEAE silica gel. The FTIR spectrum for the silica gel before and after the treatment was analyzed and a significant increase in peaks corresponding to Si-O-Si, and NH group was observed. The FTIR spectra and the wavelength corresponding to the peaks are shown in Figure 1.
Column studies for somatic coliphage ‘SUSP2’ spiked in distilled water
[024] SUSP2 spiked distilled water was passed through a column packed with DEAE silica gel (2.2 cm ID, 10 cm packing depth) at a flow rate of 50 mL/min. Loading was conducted at three different initial virus concentration in duplicate (six columns) and thereafter the virus loaded on the adsorbent was eluted by passing 300 mL of an optimized eluent (ES-2) at 50 mL/min. Breakthrough

profiles of the column during loading of SUSP2 at initial concentration of 104 and 105 PFU/L are shown in Figure 2. The column was found to reach saturation within 100 min of operation. The recovery of the virus from the column was also relatively high. The cumulative elution profile from a column loaded with influent SUSP2 concentration of 103 PFU/L for 3 h is shown in Figure 3. Rapid elution was observed, such that the count of SUSP2 recovered remained constant beyond 2 min. Table 2 herein below, summarizes the recovery of viruses from DEAE silica gel packed column. The columns were loaded by passing 10 L of SUSP2 spiked distilled water through the column. Percentage recovery of coliphage on elution was computed by mass balance. The ratio of number of viruses in the eluent to the number of viruses adsorbed on the adsorbent, DEAE silica gel, was measured to quantify the recovery efficiency. Maximum recovery of 95.26% was achieved for the DEAE silica gel column loaded with 105 PFU/L of SUSP2. For initial SUSP2 concentration 104 PFU/L and 103 PFU/L, the recovery achieved was 92.45% and 93.75%, respectively.

Co
(PFU/L) Volume
of water
(L)
Passed
at 50
mL/min Cumulative
number of
cells
adsorbed
(PFU) Cumulative
number of cells
eluted
(PFU) % Recovery
3 3.5 x 10 10 3.5 × 10 4 3.28 × 10 4 93.75
4 1.0 x10 10 4.80 × 10 4 4.43 × 10 4 92.45
4.6 × 10 5 10 1.78 × 10 6 1.69 × 10 6 95.26
Co = Concentration of virus in the influent

Table 2: Recovery of SUSP2 from DEAE silica gel column using ES-2 eluent
applied at Q= 50 mL/min
[025] Compared to methods such as ultracentrifugation, where 10 L volume of water containing virus can be concentrated to 1 mL in 100 h, in this method it was possible to concentrate virus from 10 L of water within 3 h and 5 min. Thus, greater concentration factor (102) could be achieved within a shorter duration compared to ultracentrifugation. Studies on SUSP2 are not reported in the literature as it is a relatively new virus. Also, for adsorption-elution studies conducted for concentration of viruses from large volumes, the recovery is reported to vary from virus to virus and it is also dependent on the adsorbent used. Control studies conducted with silanized silica gel with identical conditions as the DEAE silica gel column suggested that silanized silica gel alone was not capable of adsorbing SUSP2 as effectively as DEAE silica gel. However, some interaction between silica gel and the coliphage (SUSP2) was observed which is evident from Figure 2.
Column studies for F specific coliphage ‘MS2’ spiked in distilled water
[026] Flow-through studies were conducted for MS2 on DEAE silica gel column (depth: 10 cm, diameter: 2.2 cm) at a flow rate of 50 mL/min. The water sample (10 L) containing, 105 PFU/L MS2, was passed through the column and it was observed that the column did not reach its exhaustion even after 200 min (Ct/Co: 0.033) of operation (Figure 4). No coliphage was observed in the effluent for 200 min when 104 PFU/L spiked distilled water was passed through the DEAE silica

gel packed column (Figure 4). However, columns with silanized silica gel reached saturation in less than 50 min.
[027] Elution was done using two eluents, EM-7 (1.5 M NaCl + 0.05 M KH2PO4 + 2% Tween 80, pH 9.2) and FM-3 (Table 1). While FM-3 (Glycine 3X broth + 1.5 M NaCl + 2% Tween 80, pH 10.2) could elute almost all the virus (100%) adsorbed on the column, elution with EM-7 showed poor recovery of virus. Cumulative elution profile for MS2 elution for eluents EM-7 and FM-3 are shown in Figure 5a-b. The FM-3 eluent was earlier optimized for desorption of MS2 from Moringa oleifera (seed protein) functionalized rice husk ash while the EM-7 eluent was optimized for DEAE cellulose. Thus, possibly silica in the DEAE silica gel may also be interacting with the virus during adsorption. In order to confirm the same, a flow through study was conducted in a column packed with activated silica gel alone. Some interaction of MS2 with silanized silica gel is evident from Figure 4. The DEAE silica gel column could be easily regenerated using FM-3 eluent and it was observed that the adsorbent did not lose its efficiency even after four rounds of adsorption and elution. Recovery of MS2 from DEAE silica gel column with FM-3 eluent at varying concentrations of MS2 is shown in Table 3 herein below. Thus, it can be inferred that coliphages adsorb effectively on DEAE silica gel and can be recovered from the column using eluents for the target virus of interest.

Co
(PFU/L) Volume
of water
(L)
Passed
at 50 Cumulative
number of
cells
adsorbed
(PFU) Cumulative
number of
cells eluted
(PFU) % Recovery

mL/min
2.25 × 10 3 10 2.02 × 10 4 1.99 × 10 4 98.27
2.82 × 10 4 10 2.54 × 10 5 2.48 × 10 5 97.54
2.61 × 10 5 10 2.31 × 10 6 2.41 × 10 6 104.19
Table 3: Recovery of MS2 into eluent FM-3 from DEAE silica gel column (Q= 50 mL/min, depth= 10 cm)
Column studies for lake water samples
[028] Water samples from various lakes were collected in the month of August, 2019. Lake water (4.8 L) collected from 8 different locations of a same lake was mixed to obtain a composite sample. The water collected was pre-filtered prior to concentration using the DEAE silica gel packed bed column (DEAE silica gel). Water samples were also collected from a relatively smaller lake, 3 L of water from 5 different sampling locations were collected in September, 2019, mixed to form a composite sample, and pretreated as mentioned for the previous lake composite sample. The concentration of SUSP2, MS2 and Rotavirus A (RVA) in the concentrated sample was quantified through qPCR. The standard curve correlating Ct value and Log (copy number) for each virus tested is shown in Figure 6. Concentration using the DEAE silica gel column using the adsorption elution method was compared to that using ultracentrifugation. It can be observed that the performance of both methods were comparable for concentration of virus from lake water.

[029] Table 4 summarizes the virus quantified by DEAE silica gel column and ultracentrifugation as primary concentration method over the monsoon season for samples collected from the two lakes. Monsoon was chosen as the season for checking the efficiency of DEAE silica gel column, since water samples collected in this season are expected to have the lowest viral load. Thus, if the DEAE silica gel column is capable of concentrating the virus when present at such low concentration, it would be effective for virus concentration in all the seasons. It can be observed from Table 5 that DEAE silica gel column was capable of detecting viruses even when the concentration was less than 1 copy/L. Even when the viruses were not detectable based on concentration by ultracentrifugation as the primary concentration method, DEAE silica gel column was capable of capturing the viruses. Also, the time required for concentration of viruses from water samples were much higher in the ultracentrifugation method compared to that using the DEAE silica gel column. Virus concentration from water sample (10 L) requires ~100 h for primary concentration by ultracentrifugation. In contrast, ~3-4 h is required for concentration by the DEAE silica gel packed column as developed in the present invention.

August October- September- October-
Lake 1 Lake 1 Lake 2 Lake 2
Virus (Copies/L) (Copies/L) (Copies/L) (Copies/L)

DEAE UC DEAE UC DEAE UC DEAE UC
silica
gel
column silica gel column silica gel column silica
gel
column 0
49
4
SUSP2 < 1 <1 0 0 <1 0 0

MS2 0 4.5 13.5 8.5 105 0 64

RVA 3152 795 112 0 0 0 14

Table 4: Comparison of primary concentration methods for analysis of virus in lake water samples: Adsorption elution method based on a packed bed of DEAE silica gel and ultracentrifugation (UC) method
[030] Advantageously, the DEAE silica gel adsorbent of the present invention shows good recovery for both coliphage SUSP2 and MS2 at concentrations as low as 10 PFU/L with eluents ES-2 and FM-3, respectively. It was found that the DEAE silica gel column was capable of concentrating even very low concentration of all the three viruses (SUSP2, MS2 and RVA). The recovery rate was comparable to that with ultracentrifuge and the method required lower processing time and could handle larger volumes of water unlike concentration based on ultracentrifugation. Additionally, DEAE silica gel is used in a packed bed column due to its large particle size, and its inert nature under ambient conditions.
[031] While the present invention has been described with respect to certain embodiments, it will be apparent to those skilled in the art that various changes and modification may be made without departing from the scope of the invention as defined in the following claims.

We Claim:
1. A method for concentrating viruses dilutely contained in water
comprising the following steps: preparing a packed bed adsorbent
column comprising DEAE immobilized on silica gel; passing the
water through the column at a predetermined flow rate; eluting
with a complementary eluent for recovering the virus adsorbed on
the column and further concentrating the virus eluted in the eluent.
2. The method as claimed in claim 1, wherein the DEAE - silica gel
is in a ratio in the range of 0.5:1 to 2:1 v/w.
3. The method as claimed in claim 2, wherein the DEAE - silica gel is in a ratio of 1:1 v/w.
4. The method as claimed in claim 1, wherein the DEAE - silica gel has particle size in the range of 75 µm to 150 µm .
5. The method as claimed in claim 1, wherein predetermined flow rate of water is 50 mL/min
6. The method as claimed in claim 1, wherein the process includes secondary concentration methods.
7. The method as claimed in claim 1, wherein the further concentration methods are selected from ultracentrifugation, polyethylene glycol precipitation, sodium chloride (NaCl)

precipitation, organic flocculation, aluminum hydroxide
precipitation-hydroextraction.
8. A kit for increasing the concentration of a virus dilutely contained
in water by the method as claimed in claim 1, the kit comprising: an adsorbent column comprising DEAE immobilized on silica gel and a complementary eluent for eluting the viruses from the adsorbent.