Elsevier

Biologicals

Volume 70, April 2021, Pages 1-6
Biologicals

Research paper
Adaptation and characterization of Anatid herpesvirus 1 in different permissible cell lines

https://doi.org/10.1016/j.biologicals.2021.02.003Get rights and content

Abstract

Duck viral enteritis is an acute, contagious infection of Anatidae family members. The disease is caused by Anatid herpesvirus 1 (AnHV-1). The infection of AnHV-1 is controlled by vaccination to the flock with chick embryo adapted attenuated vaccine in developed countries. However, its economic impact in developing countries is substantial and there is a need to understand the cell culture spectrum of the virus to produce its vaccine on a mass scale. In the present study, the permissivity of AnHV-1 for different cells was analyzed. The AnHV-1 showed enhanced replication following its serial passage in CEF, DF-1, Vero, MDCK, and QT-35 cells. The characteristic cytopathic effect (CPE) of rounding and clumping of cells were observed in CEF, DF-1, Vero, and QT-35 cell lines. The infectivity and viral replication were highest in CEF, DF-1, Vero, and QT-35 cells. In contrast, the results suggested that MDCK cells are less permissive for AnHV-1 infection with negligible CPE and reduced viral replication. Heterologous cell culture systems other than chicken embryo fibroblasts to adapted live vaccine viruses will provide a system devoid of other avian infectious agents. Moreover, it can be used for the propagation and cultivation of AnHV-1 vaccine strain for developing cell culture-based vaccines with high titer and could be an economical alternative for the existing options.

Introduction

Duck viral enteritis (DVE) is an acute and highly contagious disease of duck, geese, swan, and various other Anatidae family members. The disease is caused by Anatid herpesvirus 1(AnHV-1) [[1], [2], [3]]. AnHV-1 belongs to the family Herpesviridae and subfamily Alphaherpesvirinae [4]. The disease is characterized by vascular damage, tissue hemorrhage, digestive mucosal eruptions, lesions of lymphoid organs, and degenerative changes in parenchymatous organs [[5], [6], [7], [8]]. Waterfowls of all ages are susceptible to the infection. AnHV-1 infection cause high mortality (80–100%) and morbidity in domestic waterfowls and hence, cause an economic burden to the commercial duck industry and possess a threat to wild and migratory waterfowls [3,9]. Recovered waterfowls become the carrier and shed the virus periodically [10]. It has been studied that the virus undergoes latency in trigeminal ganglia (TG) and reactivation of latent AnHV-1 causes further disease outbreaks [11]. The disease is prevalent in many countries and outbreaks have been reported in places such as the USA [6,8] China [3], Bangladesh [12], Egypt [13], Vietnam [14], Poland [15], Germany [16] and India [17].

The primary, diploid, and continuous cell culture systems were used for the production of cell culture-based vaccine [18]. Primary cell culture is acknowledged as the appropriate way since they support a wide range of viruses [15,[19], [20], [21]]. However, primary cells have a limited lifespan and their characteristics may change with subsequent passages [22]. In contrast, continuous cell lines have acquired the ability to proliferate indefinitely and, are more robust and easier to maintain as compared to the primary cells [23,24]. Continuous cell lines are widely used for propagation, adaptation, and attenuation of diverse viruses for the development of cell culture bases vaccines [[24], [25], [26]].

AnHV-1 is known to propagate in primary cell culture of avian origin and a homologous system like cell lines of duck origin, duck embryo fibroblast (DEF-CCL-141) and duck embryo liver (DEL) [[27], [28], [29], [30]]. Vaccination is an extensive approach for the prevention and control of the AnHV-1 infection. In most of the European countries and the USA, both live attenuated and killed vaccines are being used [31,32] against AnHV-1 infection. In India, currently, a chick embryo-adapted live vaccine (Holland strain) is available commercially. However, the risk of contamination by other avian pathogens in the eggs or microbial contaminants during processing has obstructed vaccine supplies and depends on a continuous supply of egg which could be limited in the period of pandemic and extreme conditions of weather [33]. To date, AnHV-1 has been propagated in primary cell lines of avian origin. Recently, the adaptation of the virulent strain of AnHV-1 has been studied in the Vero cell line [34]. AnHV-1 remains highly cell-associated which require rigorous freeze-thaw cycle to rupture cell membrane and the limited study of its propagation in continuous cell line make it difficult to work with.

In the present work, we conducted a preliminary study to propagate the vaccine strain of AnHV-1 in four different continuous cell lines along with primary cells by studying characteristic CPE, viral infectivity and replication kinetics, and its gene expression.

Section snippets

Cells and virus

The African green monkey kidney (Vero), Japanese quail fibrosarcoma cell line (QT-35), and Madin-Darby Canine Kidney (MDCK) cell lines were procured from the National Centre for Cell Science, Pune, India. Continuous chicken fibroblast cell line (DF-1) was procured from ATCC (Manassas, VA, USA). The primary chicken embryo fibroblast cell (CEF) was made from nine days old embryonated chicken egg as per standard protocol [35]. All the cells were maintained in Dulbecco's Modified Eagle Medium

Passage and adaptation of AnHV-1 in different cell line

AnHV-1 infected CEF, DF-1 Vero, QT-35 and MDCK showed no characteristic CPE during initial passages. From the 5th passage onwards gradual development of CPE was observed in CEF, DF-1, Vero, and QT-35 cell line whereas no visible CPE was observed in MDCK cells. Following 48 h of infection, the CEF and DF-1 cells started showing CPE and a prominent bulging and aggregation with clusters of affected cells were evident as early as 3 days post-infection (dpi) followed by the clumping of rounded cells

Discussion

Cell culture is reported as one of the methods for isolation of AnHV-1. Primary duck embryo fibroblast (DEF) and primary Muscovy duck embryo fibroblasts (MDEF) has been reported to be used in AnHV-1 isolation. Besides, Muscovy duck embryo liver (MDEL) cells were thought to be even more sensitive for virus isolation [28,38,39]. Primary virus isolation can also be done by inoculating the virus into the chorioallantoic membrane of 9–14 days old embryonated duck and chicken eggs [8]. However,

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgment

The poultry virus research in our laboratory is currently supported by the Department of biotechnology, India grants BT/562/NE/U-Excel/2016 and BT/PR24308/NER/95/644/2017.

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