Development of diploid embryonic sheep heart cells and sensitivity study of three poxviruses: Lumpy skin disease, Camelpox viruses, and Ecthyma

doi:10.21203/rs.3.rs-2589443/v1

Lumpy skin disease virus (LSDV), Camelpox virus (CPV), and Ecthyma virus (ORFV), the three representative viruses of Poxvirus family, are usually grown on embryonated eggs or primary cells for isolation or production since continuous cell lines are less sensitive. The use of eggs or primary cells presents disadvantages such as availability, potential endogenous contaminants, and limited number of passages. In this study, we developed a diploid cell, originated from an embryonic sheep heart and demonstrated their high prolificity and capability of long-term storage. We also demonstrated the maintain of the diploid cell sensitivity to isolation and growth of three genera of Poxvirus family; Capripoxvirus (LSDV), Orthopoxvirus (CPV) and Parapoxvirus (ORFV).

Developed cell grow with a doubling time of 24h and can reach 40 passages with a satisfactory yield comparable to Lamb primary testis at passage 5. After infection, the titer of each Poxvirus is maintained between 7, 0 and 7,6 log TCID₅₀/ml up to 5 passages, around 6,8 for the three viruses at passages 6 to 25, 6,4 at passage 30 and 5,6 at passage 40. Sensitivity of diploid embryonic heart cells did not decrease after long-term conservation in liquid nitrogen. Results showed a higher sensitivity of those cells comparatively to lamb primary testis, intensively used for Capripoxvirus and parapoxvirus detection and growth or Vero cells for Orthopoxvirus. The study demonstrated the interest of embryonic heart diploid cells for poxvirus isolation and production to avoid constraints of eggs, embryo, or primary cells.

Embryonic heart cells of sheep

Lamb primary testis

Vero cells

Capripoxvirus

Orthopoxvirus

Parapoxvirus

LSDV

CPV

and ORFV

Lumpy skin disease of cattle (LSDV) is one of the most important animal poxvirus infections, caused by a Capripoxvirus, due to its large distribution and serious economic consequences by drop of milk production, weight loss, skin damage and temporary or permanent sterility [1]. Camelpox is a highly contagious disease of camels [2], due to a member of Orthopoxvirus genus causing high mortality of young animals and morbidity in adult camels in Africa, India and Middle East [3] [4]. The World Organization for Animal Health categorizes LSDV and CPV as notifiable diseases [5]. Contagious Ecthyma, also known as contagious pustular dermatitis, or ORF, is an acute dermatitis that can affect sheep, goats, and camels caused by a member of the Parapoxvirus genus, the disease occurs worldwide and threatens human health [6].

The rapid and effective isolation and culture of pathogens is critical for early diagnosis, prevention, and control of pox diseases. Cells that can permit viral replication are important tools for viral disease diagnosis and follow-up studies. In addition, vaccination is the most effective way to control the spread of poxviruses supported by other measures such as stamping out and animal movement restrictions. Historically, vaccine manufacturers have been utilizing primary cells, tissues, fertilized eggs, and even whole organisms as substrates for virus propagation [7]; embryonated chicken eggs (ECEs) were largely used for the isolation of LSDV, CPV and Ecthyma virus[8][9] [10][11].Moreover, this last virus was successfully isolated from primary cells originated from chicken eggs [12]. At present, primary lamb kidney or primary lamb testis cells are the most commonly used for all Capripox genus members and Ecthyma virus production and isolation [13]. Today, vaccine manufacturers are increasingly shifting toward virus growth on continuous cell lines [14]. Currently, Vero cells are the most exploitable cells for the production of pox vaccines [15]. However, these cells provide low titers in comparison with primary cells [16], but those cells present a high risk of viral, fungal, and bacterial contamination, irregular batches, difficult to standardize and limited passage number that can not be stored [17]. Another cell line (OA3.Ts) has been evaluated for Capv propagation, however the titer was low, the growth was slow, and its pestivirus status is unclear [13]. Madin-Darby bovine kidney (MDBK) cells were evaluated for LSDV replication [18], nonetheless the status of this cell line is not confirmed free from BVD [19]. ESH-L is a diploid cell line that has been confirmed to be effective for the detection and diagnosis of CaPVs. In a comparative study, we established that those cells are less permissive than embryonic heart cells of sheep, however, ESH-L were choosen to be most effective alternative to primary cells because it has the advantage of achieveing an important cell passage and the faisability of long-term conservation [16]. The objective of this work is to prepare and purify diploid cells originated from the embryonic heart of sheep (EH) that can reach a significant cell passage and validate it for the propagation and dissemination of parapoxvirus, capripoxviruses and orthopoxvirus.

1. Cell preparation of embryonic heart cells of sheep

Heart primary cells were obtained from three-month-old fetus lamb heart tissue. Heart tissues were first cleaned repeatedly with phosphate buffered saline (PBS), added with 5% Antibiotic- Antimycotic (AB-AM) solution, and placed into a sterile petri dish. The tissue was then cleaned and cut into small pieces with a sterile scalpel and Mayo scissors. Afterwards, the cut pieces were submitted to enzymatic dissociation by trypsin (Wisent Inc., cat n° 325-042-CL) to obtain single cells. The Erlenmeyer flasks of cells were put in a 31°C water bath with gentle agitation. After every 10 minutes, the supernatant was collected in dedicated flasks. Samples of 100 µl were taken to evaluate the number of viable cells by trypan blue (Wisent Inc., cat n° 609-130-EL) before incubation. Cells were then diluted in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent Inc., cat n° 219-015-XK) supplemented with 10% Fetal Bovine Serum (FBS) bovine viral diarrhea virus (BVDV) free then incubated at 37°C with 5% CO₂. The purification step was then done to isolate a pure and homogeneous cell culture. Briefly, the prepared primary heart cell suspension was seeded into culture flasks at a concentration of 2×10⁶ cells/ ml and incubated at 37°C in humidified incubators with 5% CO₂ for 6 h. After incubation, the culture media containing non-adherent cells were discarded and high-glucose DMEM culture medium containing 10% FBS was added to continue purifying the cells. This step was repeated for three passages and then cells were seeded into a culture flask and incubated at 37°C, when the monolayer becomes confluent, the cells sub-cultured continuously up to 40 passages using only 5% of FBS.

2. Cellular evaluation of embryonic heart cells of sheep

2.1. Morphology of embryonic heart cells of sheep

Giemsa staining was performed on the heart lamb cells from the third passage to observe their cell morphology. Briefly, the flask containing cells was rinsed with the full name (PBS) and fixed by ice-cold methanol for 10 minutes. After fixation, the flask was stained with Grunwald for 10 minutes, after rinsing with PBS, cells were stained with diluted Giemsa (1/10) for 30 minutes. Finally, the flask was cleaned with PBS. The stained flask was examined under a microscope to observe the cytology and morphology.

2.2. Cell amplification and storage of embryonic heart cells of sheep

After 24h of incubation, less flasks containing cells were observed by inverse microscope. The medium was replaced with a fresh one and the cells were incubated for daily observation. The cells were trypsinized when the monolayers became confluent. Cells were amplified in order to achieve high cellular concentration; heart primary lambs were trypsinized and centrifuged at 1200 rpm/+4°C for 10 minutes. After the elimination of the supernatant, the cells were suspended in congelation media containing 10% dimethyl sulphoxide (DMSO) (Sigma Aldrich, cat n° D5879-1L) + 20% FBS BVD free + 70% DMEM. The total volume was distributed in cryovials (1ml/cryovial) and replaced in -80 °c for four hours and then in liquid nitrogen for storage until use. After conservation in liquid nitrogen, cell viability was assessed after 24h, 1 week, and 1 month, in order to estimate the possibility of storage of prepared embryonic heart cells. Three thawing were accrued, for each thawing, three vials were thawed separately, and cell viability was evaluated using trypan blue and calculated by the following formula:

% viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100

2.3. Cellular kinetics and doubling time of embryonic heart cells of sheep

Cellular growth kinetic was evaluated by seeding six flasks by 40 000 cell/cm² of primary heart lamb cells and incubated at 37°C with 5% CO2, after every 24h one of the flasks was trypsinized and the cellular yield and cell doubling time were calculated.

Cell doubling time (DT) = ln2/µ, Where µ = ln-Xn - ln-Xn-1 /tn –tn-1.

t: time of trypsination (hours), X: cell number at t

Assays were repeated three times and the results were evaluated by different analysts.

2.4. Hayflick limit of embryonic heart cells of sheep

Three F25 cm² were seeded by 40 000 cell/cm² of embryonic heart cells of sheep and incubated at 37°C with 5% CO2, after four days of incubation the flasks were trypsinized separately and cellular yields were calculated, and the cells were amplified, and another passage were maintained until a cellular yield less than 90 000 cell/cm². The average of three calculated cellular yields were taken into consideration.

3. Study of poxviruses' propagation in embryonic heart cells of sheep

3.1. Virus strains

Three viral strains were used in this study: Echtyma virus isolated from pathological material obtained from sections of tissue from lip lesions in sheep from an infected flock. LSDV Neethling attenuated strain (Capstick. and Coackley, 1961), Camelpox virus CMLV-T8 (El Harrak and Loutfi, 2000).

3.2. Cells

Vero cells (African green Monkey kidney, Cat n◦: CCL-81) and OA3. T cells (Ovine Aries Testis CRL-6546) were provided by ATCC. ESH-L (Embryonic sheepskin) cells were obtained from FLI (Cat N◦: CCLV-RIE 0175). The three cell lines were cultured in Dulbecco’s modified Eagle’s medium (Wisent Inc, cat n◦ 219 − 015-XK), supplemented with 5% FBS for Vero cells and supplemented with 10% FBS for OA3.Ts and ESH-L, and incubated at 37 ◦C with 5% CO2.

Primary lamb testis cells LTS were obtained by castration of a healthy three-month-old male

3.3. Permissivity of embryonic heart cells of sheep to Poxviruses

Four flasks of 5, 10, 15, 20, 25, 30, 35, and 40 passages of embryonic heart cells were seeded by (40 000 cell/cm²). One flask was dedicated for control, and three other flasks were used for viral inoculation. At an MOI of 0,01 of attenuated strains of LSDV, Neethling strain, Echtyma pox, and Camelpox strain.

3.4 Comparison of poxviruses’ propagation and adsorption on embryonic heart cells, primary lamb testis, ESH-L, OA3.Ts, and Vero cells

Three flasks of 25 cm² were seeded by primary heart lamb cells (40 000 cell/cm²), and other three were seeded with primary lamb testis. After three days of incubation, cells were inoculated with virus at an MOI of 0, 01 of attenuated strains of LSDV, Neethling strain, Echtyma virus and Camelpox strain. The titers of the seeded viruses were 6, 3 (log₁₀TCID₅₀/ml) for LSDV, 6, 8 (log₁₀TCID₅₀/ml) for Camelpox virus and, 4, 0 (log₁₀TCID₅₀/ml) for Echtyma virus. After 45 minutes of adsorption, inoculated cells were incubated at 37°C, 5% CO₂ and observed daily for cytopathic effect (CPE) presence by three different analysts. The viruses were harvested when CPE is about 80%. Flask controls were seeded with the same cell concentration for each type of cells and left without any viral infections.

3.5. Poxviruses' adsorption

Four flasks of 25 cm², were seeded with primary heart lamb cells and primary lamb testis (2 flasks/type of cells). One flask of each type of cells were inoculated with attenuated strains of LSDV Neethling strain (MOI 0,01). The other flasks were used as controls.

After 45 min of adsorption, the supernatant containing the virus was eliminated and flasks were revealed by immunoperoxidase assay (IPMA) according to Andy et al. to evaluate viral adsorption on the surface of the two types of cells [20].

4. Statistical analysis

Differences between viruses' titers and cell viability were evaluated for significance by the method of Fisher.

1. Cell preparation of embryonic heart cells of sheep

Figure 1 shows the development of the monolayer of embryonic heart cells of sheep post preparation. After 5 days of preparation, we observed almost six single cells (Fig. A), in 18 days the monolayer becomes confluent and ready to be trypsinized and amplified (Fig.D).

2. Cellular evaluation of embryonic heart cells of sheep

Picture below shows the morphology of the prepared embryonic heart cells. Cell shows the same morphology and display which confirm cell purification (Fig. 1B, C, D)

Cell bodies displayed a large cylindrical and irregular shape. Their cytoplasm spread widely and generally included 3 to 4 protuberances (Fig. 1E, F). The cell nuclei were oval, localized at the center of the cell body, and contained 2 to 3 visible nucleoli. When multiple cells adhered to each other, making the cells appear as a sheet-like monolayer (Fig. 1A).

2.1. Cell viability of Embryonic Heart cells after thawing

Table 1 represents the cell viability of embryonic heart cells after storage in nitrogen liquid, we observe that cells remain viable until one year of storage, which confirm the possibility of their conservation. From 24h post freezing to a month, viability is more than 81% and equal 81% for one year.

2.2. Cellular growth kinetic of embryonic heart cells

The monolayer of embryonic heart cells becomes confluent 72h after seeding with an initial concentration of 40 000 cell/cm², in this point of growth, the cellular yield is maximal (186 000 cell/cm²). After that, cell densities remained stable after 24h of incubation (96h post seeding), and then began to decrease in the following day until they reach a minimal value of 95 000 cell/cm² after 144h post seeding.

2.3. Cellular yields of 40 passages of embryonic heart cells

Embryonic heart cells achieved the 40th passage with a cellular yield of 87 000 cell/cm². From P1 to P20 cellular yields comprise between 180 000 and 150 000 cell/cm², from P21 to P38 cellular yield were between 145 000 and 100 000 cell/cm² (Fig. 3).

3. Comparison of cellular propriety of embryonic heart cells, ESH-L and primary lamb testis

Table 1 explains the cellular proprieties of embryonic heart cells (EH), ESH-L, and primary lamb testis; the monolayer of EH cells requests 72h to be confluent with a cell doubling time of 24h, achieving a maximum passage of 45. As for ESH-L, the monolayer needs 8h more and a cell doubling time of 26h with a cell passage close to EH (P40). LTs cells achieve only P14 with a cell doubling of 32h and 96h for confluence.

Table 1

Cellular proprities of EH, ESH-L, and LTs cells
Cells	Cell doubling time	Time to a confluent monolayer	Maximum passage
EH cells	24h ± 0,6	72h	45 (56 000 cell/cm² )
ESH-L cells	26h ± 0,66	80h	40 (50 000 cell/cm² )
LTs cells	32h	96h	14 (50 000 cell/cm² )

4. Cellular evaluation of embryonic heart cells of sheep

4.1. Pox production on 40 passages of embryonic heart cells

Figure 5 shows the cytopathic effect (CPE) of Lumpy skin disease virus (Fig. 5.C), Camelpox virus (Fig. 5.B), and Echtyma virus (Fig. 5.A). In comparison with cell control (Fig. 5.D), LSDV produce intracytoplasmic inclusion bodies, syncytia, and intranuclear inclusion bodies in cell culture. At the advanced stages of virus propagation, the monolayer is destroyed with infected cells rounding up and detaching from the surface. Camelpox viruses produces multinucleate giant cells or multinucleated syncytia, small holes in the cell sheet caused by the detachment of multinucleate cells. Echtyma viruses’ CPE was characterized by degenerative changes in cells, which caused rounding, nuclear vacuolation, chromatin fragmentation, loss of continuity of the cell monolayer, and intracytoplasmic inclusion bodies.

Poxviruse propagation on embryonic heart cells was evaluated in Table 3, the results were validated statistically after 3 repetitions of the same cells with the same virus (p > 0, 01). As the passage number increased, the virus replication was gradually reduced, in contrast, no significant difference in viral replication among different passages was found from passage 5 to the 20th for LSDV (6, 8 log₁₀TCID₅₀/ml). Moreover, from passage 5 to the 15th passage for Echtyma virus (7, 4 log₁₀TCID₅₀/ml). As for Camelpox virus, the titers were stable from cell passages 1 to 15 (7, 6 log₁₀TCID₅₀/ml).

Table 2

Poxviruse titers on every five passages of embryonic heart cells (EH) of sheep
Cell passage	LSDV titer (log TCID₅₀/ml)	Camelpox virus titer (log TCID₅₀/ml)
1–5	7,0 ± 0,15	7,6 ± 0,1	7,6 ± 0,1
10	6,8 ± 0,05	7,6 ± 0,05	7,4 ± 0,05
15	6,8 ± 0,01	7,6 ± 0,06	7,4 ± 0,06
20	6,8 ± 0,01	6,8 ± 0,05	7,2 ± 0,05
25	6,6 ± 0,1	6,8 ± 0,01	6,8 ± 0,01
30	6,2 ± 0,05	6,6 ± 0,1	6,4 ± 0,15
35	5,9 ± 0,05	6,0 ± 0,01	5,9 ± 0,05
40	5,4 ± 0,1	5,8 ± 0,06	5,6 ± 0,15

3.2. Comparison of pox virus sensitivity in embryonic heart cells and primary lamb testis

Poxviruse propagation on EH cells, primary lamb testis, ESH-L, OA3.Ts, and Vero cells was evaluated in Table 3; the results were validated statistically after 3 repetitions of the same cells with the same virus (p > 0, 01). There is a clear difference between the titer obtained on embryonic heart cells and ESH-L and the titer obtained on other cells. The titers obtained on EH and ESH-L are always higher than 7 (log₁₀TCID₅₀/ml), however on other cells the titer never achieved 7 (log₁₀TCID₅₀/ml). Nevertheless, primary lamb testes provide more replication in comparison with Vero cells and OA3.Ts, where the titers are significantly low, which is explained by the longer time to CPE occurrence and time to 80% CPE. In addition, ESH-L and EH give the same results for LSDV and ORF, as CPV/EH is 0,6 log higher than ESH-L. In addition, their time to CPE occurrence and 80% time were similar.

Table 3

Poxvirus titers in embryonic heart cells, primary lamb testis, ESH-L and Vero cells
	Cells	LSDV	CPV	ORF
CPE occurrence time (h)	EH cells	48h	24h	36h
	ESH-L	48h	24h	36h
	LT cells	60h	48h	54h
	Vero	72h	30h	48h
	OA3.Ts	96h	96h	72h
Time to 80% CPE (h)	EH cells	84h	60h	72h
	ESH-L	84h	60h	72h
	LT cells	108h	72h	96h
	Vero	120h	60h	96h
	OA3.Ts	144h	120h	120h
Virus titers at 80% CPE (log₁₀ TCID₅₀/ml)	EH cells	7,0 /ml ± 0.05	7,6 /ml ± 0.01	7,4 /ml ± 0,05
	ESH-L	7,0 /ml ± 0.05	7,0 /ml ± 0.1	7,4 /ml ± 0,05
	LT cells	6,8 /ml ± 0.1	6,9 /ml ± 0,1	6,2 /ml ± 0,05
	Vero	4,8 /ml ± 0.05	6,0 /ml ± 0,01	5,6 /ml ± 0.10
	OA3.Ts	4,6 /ml ± 0.10	4,6 /ml ± 0,05	5.6 /ml ± 0,05

3.2. Pox virus adsorption

Figure 6 explains LSDV adsorption on embryonic heart and lamb testis cells using IPMA assay. EH control (Fig. 6.A) and primary lamb testis cell control (Fig. 6.D) show no specific staining, in addition the monolayer appears to be clearer than the infected ones. After virus incubation, the EH cells (Fig. 6.B) produced more intense staining than primary lamb testis (Fig. 6.C).

The development of viral vaccine production consists of the optimization of three integrated stages; upstream processing, which is the selection of appropriate producer cell culture, defining optimal growth conditions, and downstream processing; isolation and purification [21]. The first step is considerate as the most critical step in this field, because it defines the highest number of produced viral particles. Primary cells were first used in this area and were most commonly applicable for LSDV vaccine production, however they present many limitations such as low number of passages, their preparation is a heavy process and their storage in liquid nitrogen is not possible. Many of the cells used today have been developed in 1960 and 1970. Established animal cells, such as Vero, Madin Darby canine kidney (MDCK) or chicken embryo fibroblasts (CEFs) are still the main cell lines used for viral vaccine production [22]. Although LSDV vaccine produced on Vero cells has no immunological effect on animals [23]. However, vaccines against Camelpox virus that were produced on Vero cells are potent, safe, and have the potential in controlling the disease, despite the obtained titer is only 10^5,5 log₁₀TCID₅₀/ml [24]. On another hand, these cells were exploited for Ecthyma virus adaptation in cell culture [25]. In addition, Chicken embryo fibroblasts (CEFs) were applied for the preparation of an experimental vaccine against contagious Ecthyma, but the virus titer was very low (10^4.2 log₁₀TCID₅₀/ml) [26].

Our works focuses on the development of diploid cells from the heart of sheep fetus that have the advantages of cell lines such as important cell passage, storage in liquid nitrogen, and repeatable batches and the advantages of primary cells such as producing a high number of viral particles for all types of poxviruses. In recent years, FBS production methods have come under scrutiny because of animal welfare concerns. FBS is harvested from bovine fetuses taken from pregnant cows during slaughter [27]. The common method of harvesting the fetus is by cardiac puncture without any anesthesia. This practice of harvesting FBS is inhumane as it exposes the fetus to pain and/or discomfort [28]. Efforts are now being made to reduce the use of FBS and replace it with synthetic alternatives [29]. Embryonic heart cells of sheep can be grown using only 5 % of FBSlike cell lines, which was not possible for primary cells.

These isolated cells were tested for viability after storage in liquid nitrogen, the number of viable cells in the culture provides an accurate indication of the health of the cell culture. It has been proven that they remain viable (80%) two years after conservation, which means one cell preparation can be enough for more than one year of virus production. Other cellular conditions were studied; cells were demonstrated to have a cell doubling time of 24h and the monolayer became confluent only after 72h cells could be manipulated two times by week, which is benefic in the point of view of time production. Generation of a growth curve can be useful to evaluate the growth characteristics of a cells; Lag phase, which is the initial growth phase of the subculture and re-seeding during which the cell population takes time to recover is about 0h-24h. The cell number remains relatively constant prior to rapid growth. During this phase, the cell replaces elements of the glycocalyx lost during trypsinization, attaches to the substrate, and spreads out. During the spreading process, the cytoskeleton reappears; its reappearance is probably an integral part of the process [30]. Log phase is comprised between 24h and 72h, that is, a period of exponential increase in cell number and growth of the cell population due to continuous division. The length of the log phase depends on the initial seeding density, the growth rate of the cells, and the density at which cell proliferation is inhibited by density. This phase represents the most reproducible form of the culture as the growth fraction viability is high, and the population is at its most uniform [31]. Stationary phase comprise between 72h and 96 h, the culture becomes confluent at the end of the log phase, as growth rates during this phase are reduced, and cell proliferation can cease in some cases due to exhaustion. The cells are in contact with surrounding cells, and the growth surface is occupied. At this stage, the culture enters the stationary phase and the growth fraction falls to between 0% and 10% [32]. Same result was obtained by Guangxiang Wang et al; they found that bovine Sertoli cells grow to a confluent monolayer in 72,16 h in passage 3 among 20 passages [33]. In our study, 45 passages were tested for cellular yields in order to define the optimal passage achieved by the stored cells with a coefficient R² with very strong positive correlation (> 0, 9). Results confirmed that cells at passage 38, 39, and 40 cell division begin to decrease by reaching the Hayflick limit. This is backed by Hayflick that concluded that a cell could complete mitosis or cellular duplication and division only forty to sixty times before undergoing apoptosis and subsequent death [34]. The conclusion held for many cell types, whether they were adult cells or fetal cells. Here appears to be a correlation between the maximum number of passages and aging. This phenomenon is related to telomere length. Repeated mitosis leads to shortening of the telomeres on the DNA of the cell [35]. Rubin et al in 1990 suggested that cellular damage could result from the cells being in an environment that differed from their original environment in the body, or when researchers subjected the cells to laboratory practices [36][37].

The second step of cell characterization is viral testing; the cell substrate should be designed to detect a spectrum of viruses. Appropriate screening tests should be carried out based on the cultivation history of cell lines. The development of a characteristic cytopathogenic effect (CPE) provides an early indication of viral propagation, which is confirmed by our results; three types of poxviruses were replicated on these cells with a specific CPE for each virus. Generally, poxviruses have a double-stranded DNA genome because it encodes its own machinery for genome replication; therefore, its replication occurs in the cytoplasm. Briefly, Ecthyma virus and LSDV produce intracytoplasmic inclusion bodies and a loss of continuity of the cell monolayer, as for Camelpox virus its produce multinucleated syncytia and detachment of multinucleate cells. After finding that these cells are permissive for poxvirus infection, we tested every cell passage (passage 4) for viral sensitivity. Moreover, the findings concerning maximal passage are not enough without viral testing; knowing the maximum cell passage for viral infection is important for the feasibility in industrial scale. Our results showed that no significant difference in viral replication among different passages was found from passage 5 to the 15th passage for Echtyma virus (7,4 log₁₀TCID₅₀/ml). In addition, from passage 5 to the 20th for LSDV (6, 8 log₁₀TCID₅₀/ml), as for Camelpox virus, the titers were stable from cell passages 1 to 15 (7,6 log₁₀TCID₅₀/ml). We can conclude that the passages from 1 to 20 passages could be used for vaccine production, however the passages from 20 to 45 could be employed for laboratory use. Nevertheless, all virus titers obtained on passage 35 were higher than the titers obtained on Vero cells. As for primary lamb testis, the titers were significantly important but always lower than the titers obtained on embryonic primary heat cells of sheep. Ecthyma (6,2 log10 TCID50/ml vs 7,4 log10 TCID50/ml), LSDV (6,8log10 TCID50/ml vs 7,0 log10 TCID50/ml), and CPV (6,0 log10 TCID50/ml vs 7,6 log10 TCID50/ml), beside the virus titer, primary lamb testis cells were recommended to be used until the fifth passage, which is an inconvenient for viral production. In 2021, a study was carried out on the production of lumpy skin disease virus in a novel bioreactor CelCradle™ -500A using LT cells; the system brings many production advantages, but the titers were only comparable with the conventional system that is why an optimization of the process is necessary. Based on our preliminary results, an assay should be done on the replacement of cell substrate with embryonic heart cells of sheep [38] .

ESH-L cells is another cell line that was validated for Capripoxviruses propagation and diagnosis, but Rhazi et al in 2021 confirmed that they give the same titer for LSDV but lower titers for SPPV and GTPV in comparison with heart cells [16]. Another paper was published by the same group later in 2022 proved that ESH-L can be used for the development of an immunoenzymatic quantitative method for sheeppox virus antibody detection, which opens a door for the exploitation of embryonic heart cells for diagnosis of poxviruses [39]. In this study, the EH cells were compared with the other two cell references for poxviruses detection and replication; Vero cells and OA3.Ts, we found out that EH cells are more sensitive to virus infection with a lower time of CPE occurrence and 80%. Which can be a benefit for the virus production process.

Virus life cycle is divided into adsorption, penetration, uncoating, viral genome replication, maturation, and release. To understand the high permissivity of embryonic heart cells, we studied the first step by immunostaining the cells before and after virus adsorption, and we used LT infected cells as a positive control and noninfected embryonic heart cells as a negative control. Figures demonstrate that the infected embryonic heart cells showed more specific staining than infected LT cells. In closing, embryonic heart cells have more specific type-1 membrane glycoprotein receptors for pox viruses is a hypothesis that should be confirmed with specific markings. Furthermore, other studies should be carried out on other steps of viral multiplication.

The use of embryonic heart cells for the culture of poxviruses guarantees viral homogeneity and stability. As well, the use of embryonic heart cells may simplify the production and diagnosis process, reduce the production time and costs, and guarantee batch repeatability. Additionally, it may avoid the risk of contamination with other pathogens that may occur during viral culture from multiple preparations of primary cells. This method thus has great practical significance for industrial scale and large-scale diagnosis.

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Development of diploid embryonic sheep heart cells and sensitivity study of three poxviruses: Lumpy skin disease, Camelpox viruses, and Ecthyma

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

1. Cell preparation of embryonic heart cells of sheep

2. Cellular evaluation of embryonic heart cells of sheep

2.1. Morphology of embryonic heart cells of sheep

2.2. Cell amplification and storage of embryonic heart cells of sheep

2.3. Cellular kinetics and doubling time of embryonic heart cells of sheep

2.4. Hayflick limit of embryonic heart cells of sheep

3. Study of poxviruses' propagation in embryonic heart cells of sheep

3.1. Virus strains

3.2. Cells

3.3. Permissivity of embryonic heart cells of sheep to Poxviruses

3.5. Poxviruses' adsorption

4. Statistical analysis

Results

1. Cell preparation of embryonic heart cells of sheep

2. Cellular evaluation of embryonic heart cells of sheep

2.1. Cell viability of Embryonic Heart cells after thawing

2.2. Cellular growth kinetic of embryonic heart cells

2.3. Cellular yields of 40 passages of embryonic heart cells

3. Comparison of cellular propriety of embryonic heart cells, ESH-L and primary lamb testis

4. Cellular evaluation of embryonic heart cells of sheep

4.1. Pox production on 40 passages of embryonic heart cells

3.2. Comparison of pox virus sensitivity in embryonic heart cells and primary lamb testis

3.2. Pox virus adsorption

Discussion

Conclusion

References

Status:

Journal Publication

Version 1