Lumpy Skin Virus ; Herpesviridae Inevitable Synergism


 Background: Lumpy skin virus causes huge economic losses in farm animals. Herpesviridae are opportunistic and showed synergistic role but worsen disease symptoms. By using TEM & negative staining one avoid the problematic concerns of differential diagnosis.Materials and Methods: Cutaneous crusts collected from infected cattle, samples examined directly by the electron microscopy, then confirmed by the Conventional Polymerase Chain Reaction and partially sequenced by ORF103 gene.Results: This study reveals that the source of infection was not related to the vaccines. It turns out to be a natural infection by Lumpy skin disease virus isolate Evros / GR/15 and 99% identity. A comparison of the gene sequence of the vaccine against the virus showed significant divergence between them. Negative staining electron microscopy reveals Lumpy skin disease virus & Herpes viruses in different sizes and forms. Herpesviridae members were contaminating LSD lesions as TEM reveals the presence of different herpes types. Conclusions: LSD virus of cattle showed virulent nature in natural outbreak. However, the herpes viruses invade infected tissues and propagate inside cells of lumps that revealed its opportunistic behavior. Consequently; Antivirals against Herpesviridae would theoretically relief lumpy skin disease symptoms and help in control efforts.


Background
Lumpy Skin Disease (LSD) is the pox disease of cattle, is the most threat to cattle industry. Lumpy Skin Disease Virus (LSDV) belongs to genus Capripoxvirus [Poxviridae], which as poxvirus have linear double-stranded DNA genome, enveloped, large, epithelia-tropic. This virus has caused multiple epidemics in different places in Egypt, throughout the year and is still present. Lumpy Skin Disease was rst recognized in Zambia in 1929. However, the rst recorded infections in Egypt were in 1988, in Europe in Cyprus in 2014, Greece in 2015, and Balkan countries in 2016 [1][2][3][4][5][6][7]. The poxvirus contains A+T-rich genome, more than 100 polypeptides, arranged in core, lateral body, membrane, and envelope. The membranes are lipoprotein bilayer which surrounds; a core and lateral bodies. The outer surface is studded with randomly arranged surface tubule elements. The intracellular mature virion has the nucleoprotein core and the lateral bodies enclosed by inner membrane, and outer membranes [1]. However, the enveloped virion is fully matured virus and covered by smooth capsule formed mainly of lipoprotein and glycoproteins. The enveloped virus contains a distinct glycoprotein; these virus antigens present in the envelope are capable of eliciting immunity [3]. Pathogenesis of LSDV in cattle; primarily the LSD virus propagated inside the epithelium of the skin prickle cell layer causing hyperplasia which leads to hyperkeratosis and parakeratosis. The endothelial cells of blood vessels infected by the virus suffer necrosis which leads to thrombus formation with subsequent infarction. LSDV has the a nity to produce pox lesions in various internal organs, skin and the mucous membranes.
Pathogenesis of that disease showed its destructive behaviors towards affected cells, as LSDV cause necrosis to blood vessels' endothelium and smooth muscles surrounding blood vessel' walls. Conjoint of ischemia of tissues and direct necrotic effects on affected skin cells leads to the typical pathological gure of lumps [8,9].
Herpes viruses are ubiquitous in nature, which causes a wide variety of diseases. The Herpes virus mature virions are enveloped, ds-DNA genome, with speci c TEM morphology. The electron microscopic studies on the morphology of herpes viruses, the negative staining method of Brenner & Horne (1956) revealed the morphological detail which has become so characteristic of the group [10]. Epstein (1962a) has described the envelope as a triple-layered membranous structure indistinguishable from host cell membrane [11]. Epstein (1962b) showed dense core region contained DNA and type of xation and embedding affected core morphology [12]. It could be postulated that poxviruses able to exerts their effect by the presence of the indispensable herpes virus's coinfections. [13][14][15][16].
Lumpy Skin Virus continues spreading from Eastern Europe to Asia. However, LSD was rst reported in Asia and the Paci c region in 2019 in northwest China, Bangladesh and India. During 2020, LSD has continued its spread across continental Asia; south and south East Asia con rming outbreaks. LSD recorded in Bangladesh, India, Nepal and Sri Lanka in 2019. In South Asia, the disease spread through Northeastern Thailand to China, Chinese Taipei, Hong Kong, Bhutan, Myanmar and Vietnam [17][18][19][20][21].

Materials And Methods
Live Attenuated Vaccine-Vaccine Batch 18024 Live attenuated vaccine (for cattle and buffalo's poxviruses), produced by (VSVRI) of Egypt used in the present work, for comparing it by the circulating LSDV in Egypt.

Samples Preparation
Tissue specimens collected from infected cattle and obtained by detaching skin crusts and kept in sterile tubes and kept at 4°C until the speci c procedures [22].

Electron Microscopic Examinations
Transmission EM The procedures were performed at Cairo University, faculty of agriculture. The Direct Electron Microscope & Negative Staining is a rapid method.

Test procedures
Direct Electron Microscopy (DEM): The original biological specimens were re-suspended in 50 µL of Phosphate-Buffered Saline (PBS) pH 7.2. One drop of the suspension was put on EM grid and submitted to negative staining technique with 2% Potassium Phosphotungstate (PTK) pH 6.4. The grids were examined and the viruses were documented in a Philips EM400-T electron microscope operating in 80 kV. All samples examined by NS-DEM, and the procedures were performed according to [10,23].

Polymerase Chain Reaction (PCR) Assays
The conventional method and partial genome sequencing for

Symptoms
Cattle suffering Lumpy Skin Disease showed enlarged lymph nodes; skin nodules in different stages. New nodules showing subcutaneous swellings erupted beneath the old ones, bleeding from old lesions was seen as a result of animal movements. Prolonged lesions lead to emaciation ( Figure 1).

1-Lumpy skin virus
Forms of LSDV referring to: • Extracellular Enveloped Virion (EEV) transmitted outside for disseminating the infection.
• Virulent Virus (VV); new form indicating virulence behavior and aggressive outbreak.
The study ndings:

EEV form
The TEM-negative staining picture shows a number of viruses in poor condition with cracking marks in their viral cover. They are relatively different in size and form, 138 nm-220 nm, but are united in their condition. Perhaps the shape of the picture is not indicative of its reality, but compared to other images came this judgment (Figures 2, 3). The photos showed Herpes viruses surrounding the LSD virus and attached to its outer cover (envelope). Herpes viruses were collected in very large numbers and appeared in the adherent examination, although the test was performed on a solution, which means that the Herpes virus is well integrated with the LSD virus. The images show a large number of viruses of relatively different sizes but all are covered by the smooth viral membranes ( Figure 4). The LSD viruses appear as a smooth brick shapes ( Figures 5, 6).

M form
The image of the virus is clear and is covered with arranged structures in the form of overlapping short tubules of about (100 nm x 160 nm) ( Figure 7). The picture shows a number of viruses partially covered by the internal viral membranes and surrounded by another incomplete membrane. Some viruses look relatively dark and surrounded by a light-colored membrane, it is surrounded by a light-colored membrane that may be composed of materials that do not absorb the dye. There is some variation in the size of viruses (111 nm-221 nm) ( Figure 8). The picture shows a number of viruses partially covered by the internal viral membranes and surrounded by another incomplete membrane. The picture shows well the internal structure of the virus and the ex-act cover consisting of short, intermittent tubes revolving around a center axis; it is surrounded by a thick, incomplete membrane, as if the virus is in the shape of a horseshoe ( Figure 9).

VV form
A virion showing a component inside the virus appeared cone-shaped. Half of the virus is exposed and the back is given as a funnel.  Figure 14). This virus is surrounded by a transparent membrane showing the components it contains. The composition inside the virus appears to be quite similar to the nutshells. Inside the gure shows a dark color while the frame that draws the shape is light colored. This form of LSDV indicates that it is very virulent; it takes off its covers in preparation for new host [15,16]. Herpesviridae

NS-DEM analysis
The TEM, negative staining images shows herpes virus as a tennis ball (core) in the middle of a halo (envelope). This is because the membranes surrounding it break easily to give this distinctive shape of 23 nm x 77 nm (Figures 17-21 [25][26][27]. The electron microscope ndings are in accordance with Weiss (1968) who mentioned that lumpy skin disease virus consist of a complex interwoven network of strands each with an approximate and presenting an irregular surface structure. A regular arrangement of "teeth" along the margins of some particles was evident. Preparations negatively stained at pH 8.5 showed a predominance of particles with multilayered capsules. These particles did not show the thread-like surface structure but consisted of a homogeneous granular mass enveloped by a capsule. The capsule appeared to have an irregular outer membrane, a thicker central part and a distinct inner membrane [2].
It has recorded that Poxvirus outer and inner membranes are lipoprotein bilayer which surrounding the two lateral bodies and the viral core. The non-enveloped virus showed the surface short tubular arrangements, giving the mature LSD virus its textured surface (Figures 7-9). The mature virion composed of a nucleoprotein core and associated lateral bodies enclosed by smooth inner membranes and outer tubular lipoprotein coat (intracellular) and some virions wrapped by the envelope (extracellular) (Figures 5,6) as additional lipid bilayer that giving the poxvirus the smooth brick-shaped appearance [1,11]. Negative staining EM of the mature virion of LSD virus show viral membrane encloses a biconcave or cylindrical core that supposed containing the viral DNA genome and the organized nucleoprotein complex (Figures 10, 11). Two lateral bodies present in the concave region between the core wall and inner membranes ( Figure 15, 16), the nucleoprotein complex might be cylindrical, folded along the long axis ( Figure 9) arranged linearly [15,16]. A comparison of the gene sequence of the vaccine (sheep pox virus) against the LSD virus showed signi cant divergence between them.
LSD virus detected in multiple form and size but is genetically identical and has distinct antigenicity. LSDV impair the innate immune system, so that it is di cult to eradicate. The only way for successful control of disease is the e cient preventive measures because vaccination against showed unusual failure, as many evidences was reporting this fact. For examples, Croatia has a special experience regarding LSD in 2016, for the rst-time vaccination of the entire cattle population was conducted without an index case. The presence of vaccine viral particles was detected in milk, skin nodules; blood and nasal swabs in seven from total of eight herds. The presence of virus genome was detected in ve cows from 10 up to 21-day post-vaccination [27].
The virus was successfully isolated on cell culture from 10 up to 21-day post-vaccination from three animals [26]. However researchers in Jordan reported adverse reactions following vaccination, the geographical areas enrolled in their study were free of the disease and away from the outbreak governorates.  Figure 1 Cattle suffering Lumpy Skin Disease: showed enlarged lymph nodes, the skin nodules were in different stages, some were sloughed taking a piece of ear cartilages to the ground. New nodules showing sub-cutaneous swellings erupted beneath the old ones. Some bleeding from old lesions was seen as a result of animal movements. Emaciation was observed, (sit fast) lesions were also present between different stages of skin lesions. [Fayoum Gov. August, 2018].                   TEM, negative staining picture shows two herpes viruses as a tennis balls (cores) in the middle of a halo (envelope). This is because the membranes surrounding them break easily to give this distinctive shape [NS-DEM 200000X].

Figure 21
The TEM, negative staining picture shows herpes viruses in different sizes appear as a tennis balls (cores) in the middle of a halo (envelope). This is because the membranes surrounding them break easily to give this distinctive shape. The image shows herpes virus in large numbers and active state [NS-DEM 150000X].

Figure 22
The TEM, negative staining picture shows naked and enveloped herpes virus of aver-age sizes of 78.5 nm-81 nm. The core of enveloped herpes virus measure about 33 nm. [NS-DEM 100000X]. Figure 23