Comparative efficacy of different Carbopol concentrations as a stabilizer of live attenuated sheep pox virus vaccine against lumpy skin disease

In Egypt, the lyophilized live attenuated sheep pox virus vaccine has been used for the vaccination of cattle against lumpy skin disease virus to control its economic impact on livestock industry. In this endeavor, we validate the efficacy of Carbopol® as a stabilizer and adjuvant to enhance immunogenicity of such a heterologous sheep pox virus vaccine against lumpy skin disease. Lyophilization of sheep pox virus vaccine stabilized with Carbopol® produced better physical and antigenic properties than freeze-drying with lactalbumin/sucrose stabilizer; this was manifested by superior disc uniformity, thermo-stability at 37oC, and less reduction in virus titer. Immunization of calves' groups with variable sheep pox vaccine doses containing different Carbopol® concentrations revealed that 103.5 TCID50 of sheep pox virus vaccine enclosing 0.5 percent Carbopol® is the field dose of choice. Moreover, it induced protective serum neutralizing index of 2.5 and a ELISA S/P ratio of 36, by the 4th week post vaccination. Besides, the inclusion of 0.5 percent Carbopol® in formulation of the sheep pox virus vaccine was safe in bovines and enhanced cellular immune response to lumpy skin disease virus, as evidenced by increased T cell proliferation. Hence, it is recommended to use Carbopol® as 0.5 percent in preparation of live attenuated sheep pox virus vaccine to confer better protection against lumpy skin disease virus infection(AU)

En Egipto, la vacuna atenuada liofilizada contra el virus de la viruela ovina ha sido utilizado para la vacunación del ganado, contra el virus de la dermatosis nodular contagiosa, para controlar su impacto económico en la industria ganadera. En este trabajo, validamos la eficacia del Carbopol®, como estabilizador y adyuvante, para mejorar la inmunogenicidad de dicha vacuna heteróloga contra la dermatosis nodular contagiosa. La liofilización de la vacuna contra el virus de la viruela ovina estabilizada con Carbopol®, resultó en mejores propiedades físicas y antigénicas que la liofilización con el estabilizador de lactoalbúmina/sacarosa; lo anterior se manifestó en la uniformidad superior del disco, la termoestabilidad a 37°C y la menor reducción del título del virus. La inmunización de grupos de terneros con dosis variables de vacuna contra el virus de la viruela ovina, que contenían diferentes concentraciones de Carbopol®, reveló que la dosis de campo de elección fue 103,5 TCID50 de la vacuna contra el virus de la viruela ovina conteniendo 0,5 por ciento de Carbopol®, la que indujo un índice de neutralización sérica protectora de 2,5 y una relación S/P de ELISA de 36 a la cuarta semana después de la vacunación. Además, la inclusión de Carbopol® al 0,5 por ciento en la formulación de la vacuna contra el virus de la viruela ovina fue segura en los bovinos y potenció la respuesta inmunitaria celular contra el virus de la dermatosis nodular contagiosa, como lo demuestra el aumento de la proliferación de células T. Por lo tanto, se recomienda el uso de Carbopol® al 0,5 por ciento en la preparación de la vacuna viva atenuada contra el virus de la viruela ovina para conferir una mejor protección contra la infección por el virus de la dermatosis nodular contagiosa(AU)

Genome-wide expression analysis reveal host genes involved in immediate-early infections of different sheeppox virus strains.

This study explored the transcriptome of lamb testis cells infected with sheeppox virus (SPPV) wild strain (WS) and vaccine strain (VS) at an immediate-early time. Most of the differentially expressed genes (DEGs) and differentially expressed highly connected (DEHC) gene network were found to be involved in SPPV-VS infection compared to SPPV-WS. Further, the signaling pathways were mostly involved in SPPV-VS infection than SPPV-WS. SPPV modulates the expression of several important host proteins such as CD40, FAS, ITGß1, ITGα1, Pak1, Pak2, CD14, ILK leading to viral attachment and entry; immune-related DEGs such as MAPK, JNK, ERK, NFKB, IKB, PI3K, STAT which provide optimal cellular condition for early viral protein expression; and FOXO3, ATF, CDKNA1, TCF, SRF, BDNF which help in inducing apoptosis and MPTP, BAD and Tp53 inhibits apoptosis or cell death at the immediate-early time. The results captured the specific genes and enabled to understand distinct pathogenic mechanisms employed by VS and WS of SPPV.

Experimental infection of indigenous North African goats with goatpox virus.

BACKGROUND: Goatpox is a viral disease caused by infection with goatpox virus (GTPV) of the genus Capripoxvirus, Poxviridae family. Capripoxviruses cause serious disease to livestock and contribute to huge economic losses. Goatpox and sheeppox are endemic to Africa, particularly north of the Equator, the Middle East and many parts of Asia. GTPV and sheeppox virus are considered host-specific; however, both strains can cause clinical disease in either goats or sheep with more severe disease in the homologous species and mild or sub-clinical infection in the other. Goatpox has never been reported in Morocco, Algeria or Tunisia despite the huge population of goats living in proximity with sheep in those countries. To evaluate the susceptibility and pathogenicity of indigenous North African goats to GTPV infection, we experimentally inoculated eight locally bred goats with a virulent Vietnamese isolate of GTPV. Two uninfected goats were kept as controls. Clinical examination was carried out daily and blood was sampled for virology and for investigating the antibody response. After necropsy, tissues were collected and assessed for viral DNA using real-time PCR. RESULTS: Following the experimental infection, all inoculated goats displayed clinical signs characteristic of goatpox including varying degrees of hyperthermia, loss of appetite, inactivity and cutaneous lesions. The infection severely affected three of the infected animals while moderate to mild disease was noticed in the remaining goats. A high antibody response was developed. High viral DNA loads were detected in skin crusts and nodules, and subcutaneous tissue at the injection site with cycle threshold (Ct) values ranging from 14.6 to 22.9, while lower viral loads were found in liver and lung (Ct = 35.7 and 35.1). The results confirmed subcutaneous tropism of the virus. CONCLUSION: Clinical signs of goatpox were reproduced in indigenous North African goats and confirmed a high susceptibility of the North African goat breed to GTPV infection. A clinical scoring system is proposed that can be applied in GTPV vaccine efficacy studies.

Extended sequencing of vaccine and wild-type capripoxvirus isolates provides insights into genes modulating virulence and host range.

The genus Capripoxvirus in the subfamily Chordopoxvirinae, family Poxviridae, comprises sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which cause the eponymous diseases across parts of Africa, the Middle East and Asia. These diseases cause significant economic losses and can have a devastating impact on the livelihoods and food security of small farm holders. So far, only live classically attenuated SPPV, GTPV and LSDV vaccines are commercially available and the history, safety and efficacy of many have not been well established. Here, we report 13 new capripoxvirus genome sequences, including the hairpin telomeres, from both pathogenic field isolates and vaccine strains. We have also updated the genome annotations to incorporate recent advances in our understanding of poxvirus biology. These new genomes and genes grouped phenetically with other previously sequenced capripoxvirus strains, and these new alignments collectively identified several recurring alterations in genes thought to modulate virulence and host range. In particular, some of the many large capripoxvirus ankyrin and kelch-like proteins are commonly mutated in vaccine strains, while the variola virus B22R-like gene homolog has also been disrupted in many vaccine isolates. Among these vaccine isolates, frameshift mutations are especially common and clearly present a risk of reversion to wild type in vaccines bearing these mutations. A consistent pattern of gene inactivation from LSDV to GTPV and then SPPV is also observed, much like the pattern of gene loss in orthopoxviruses, but, rather surprisingly, the overall genome size of ~150 kbp remains relatively constant. These data provide new insights into the evolution of capripoxviruses and the determinants of pathogenicity and host range. They will find application in the development of new vaccines with better safety, efficacy and trade profiles.

Genetic studies of terminal regions of vaccine and field isolates of capripoxviruses.

Sheeppox and goatpox are two of the most important diseases associated with significant economic loss and impact on animal trade. In spite of the use of vaccines, outbreaks are being reported on several occasions. Therefore, deciphering the host specificity and virulence of sheeppox virus (SPPV) and goatpox virus (GTPV) is important in developing effective vaccines. It is opined that genes located in the terminal regions play a major role in determining host range and/or virulence. In the present study, nine isolates (6 GTPV and 3 SPPV; included both vaccine and virulent viruses) were genetically characterized by targeting 11 genes (7 host-range and 4 virulence genes) which are located in the terminal regions of capripoxviruses. In the genetic analyses, it was observed that there are several nucleotide and amino acid signatures which are specific for either SPPV or GTPV. However, surprisingly, none of the 11 genes could be able to differentiate the vaccine and field viruses of GTPV and SPPV. Our study indicates that the genes of the terminal regions may have a role in determining the host-specificity but the involvemet in determinatin of virulence/attenuation is not certain at least for the isolates used in the current study. Therefore, it is likely that some other genes located in terminal/central regions may also play a role in determination of virulence and pathogenesis which needs to be confirmed by whole-genome sequencing of several vaccine and virulent viruses.

Construction of an attenuated goatpox virus AV41 strain by deleting the TK gene and ORF8-18.

Goatpox virus (GTPV) is prevalent in goats and is associated with high mortality. This virus causes fever, skin nodules, lesions in the respiratory and lymph node enlargement. Considering the safety risks and side effects of vaccination with attenuated live GPTV vaccine strain AV41, an attenuated goatpox virus (GTPV-TK-ORF), was constructed by deleting non-essential gene fragments without affecting replication and related to the virulence and immunomodulatory functions of the goatpox virus AV41 strain (GTPV-AV41) using homologous recombination and the Cre (Cyclization Recombination Enzyme)/Loxp system. The results of both in vivo and in vitro experiments demonstrated that GTPV-TK-ORF was safer than wild type GTPV-AV41, possessed satisfactory immunogenicity, and could protect goats from a virulent GTPV-AV40 infection. Moreover, the IFN-γ, GTPV-specific antibody, and neutralizing antibody levels in the GTPV-TK-ORF-immunized group were significantly higher than that in the normal saline control group following immunization (P < 0.01). Thus, GTPV-TK-ORF may be used as a potential novel vaccine and viral vector with good safety and immunogenicity.

The 135 Gene of Goatpox Virus Encodes an Inhibitor of NF-κB and Apoptosis and May Serve as an Improved Insertion Site To Generate Vectored Live Vaccine.

Goatpox virus (GTPV) is an important member of the Capripoxvirus genus of the Poxviridae Capripoxviruses have large and complex DNA genomes encoding many unknown proteins that may contribute to virulence. We identified that the 135 open reading frame of GTPV is an early gene that encodes an ∼18-kDa protein that is nonessential for viral replication in cells. This protein functioned as an inhibitor of NF-κB activation and apoptosis and is similar to the N1L protein of vaccinia virus. In the natural host, sheep, deletion of the 135 gene from the GTPV live vaccine strain AV41 resulted in less attenuation than that induced by deletion of the tk gene, a well-defined nonessential gene in the poxvirus genome. Using the 135 gene as the insertion site, a recombinant AV41 strain expressing hemagglutinin of peste des petits ruminants virus (PPRV) was generated and elicited stronger neutralization antibody responses than those obtained using the traditional tk gene as the insertion site. These results suggest that the 135 gene of GTPV encodes an immunomodulatory protein to suppress host innate immunity and may serve as an optimized insertion site to generate capripoxvirus-vectored live dual vaccines.IMPORTANCE Capripoxviruses are etiological agents of important diseases in sheep, goats, and cattle. There are rare reports about viral protein function related to capripoxviruses. In the present study, we found that the 135 protein of GTPV plays an important role in inhibition of innate immunity and apoptosis in host cells. Use of the 135 gene as the insertion site to generate a vectored vaccine resulted in stronger adaptive immune responses than those obtained using the tk locus as the insertion site. As capripoxviruses are promising virus-vectored vaccines against many important diseases in small ruminants and cattle, the 135 gene may serve as an improved insertion site to generate recombinant capripoxvirus-vectored live dual vaccines.

Serum Biochemistry of Lumpy Skin Disease Virus-Infected Cattle.

Lumpy skin disease is an economically important poxvirus disease of cattle. Vaccination is the main method of control but sporadic outbreaks have been reported in Turkey. This study was carried out to determine the changes in serum biochemical values of cattle naturally infected with lumpy skin disease virus (LSDV). For this study, blood samples in EDTA, serum samples, and nodular skin lesions were obtained from clinically infected animals (n = 15) whereas blood samples in EDTA and serum samples were collected from healthy animals (n = 15). A quantitative real-time PCR method was used to detect Capripoxvirus (CaPV) DNA in clinical samples. A real-time PCR high-resolution melt assay was performed to genotype CaPVs. Serum cardiac, hepatic, and renal damage markers and lipid metabolism products were measured by autoanalyzer. LSDV nucleic acid was detected in all samples which were obtained from clinically infected cattle. The results of serum biochemical analysis showed that aspartate aminotransferase, alkaline phosphatase, total protein, and creatinine concentrations were markedly increased in serum from infected animals. However, there were no significant differences in the other biochemical parameters evaluated. The results of the current study suggest that liver and kidney failures occur during LSDV infection. These findings may help in developing effective treatment strategies in LSDV infection.

Capripox disease in Ethiopia: Genetic differences between field isolates and vaccine strain, and implications for vaccination failure.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) of the genus Capripoxvirus (CaPV) cause capripox disease in sheep, goats and cattle, respectively. These viruses are not strictly host-specific and their geographical distribution is complex. In Ethiopia, where sheep, goats and cattle are all affected, a live attenuated vaccine strain (KS1-O180) is used for immunization of both small ruminants and cattle. Although occurrences of the disease in vaccinated cattle are frequently reported, information on the circulating isolates and their relation to the vaccine strain in use are still missing. The present study addressed the parameters associated with vaccination failure in Ethiopia. Retrospective outbreak data were compiled and isolates collected from thirteen outbreaks in small ruminants and cattle at various geographical locations and years were analyzed and compared to the vaccine strain. Isolates of GTPV and LSDV genotypes were responsible for the capripox outbreaks in small ruminants and cattle, respectively, while SPPV was absent. Pathogenic isolates collected from vaccinated cattle were identical to those from the non-vaccinated ones. The vaccine strain, genetically distinct from the outbreak isolates, was not responsible for these outbreaks. This study shows capripox to be highly significant in Ethiopia due to low performance of the local vaccine and insufficient vaccination coverage. The development of new, more efficient vaccine strains, a GTPV strain for small ruminants and a LSDV for cattle, is needed to promote the acceptance by farmers, thus contribute to better control of CaPVs in Ethiopia.

Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a distinct set of mammalian proapoptotic proteins.

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.

Identification of an intergenic region that is not essential for replication of goatpox virus.

A recombinant goatpox virus was constructed in which an enhanced green fluorescent protein gene was inserted under the control of the 11K late promoter, a guanine phosphoribosyltransferase gene was inserted under the control of the 7.5K early/late promoter, and exogenous genes were inserted into an intergenic region between loci gp_24 and gp_24.5 of the recombinant genome. Analysis of protein expression showed that LT cells infected with only the recombinant virus produced specific fluorescence. A comparative growth assay demonstrated the stability of the recombinant virus at the replication level. These results suggest that the intergenic region is not essential for replication of goatpox virus.

Yemen and Vietnam capripoxviruses demonstrate a distinct host preference for goats compared with sheep.

Sheeppox and goatpox are caused by viruses that are members of the genus Capripoxvirus, and globally result in significant production losses. To improve the understanding of disease pathogenesis and evaluate host species preferences, sheep and goats were inoculated either with a capripoxvirus isolate from Yemen or from a recent outbreak in Vietnam. Blood, swabs and tissues were collected at various time points following experimental challenge and assessed for viral DNA content using real-time PCR and infectivity using virus isolation. The Yemen isolate was considerably more pathogenic in goats with 100 % mortality and morbidity compared with sheep with 0 % mortality and 100 % morbidity. The Vietnam isolate was also more pathogenic in goats with 100 % morbidity and an estimated 33 % mortality rate compared with mild morbidity and a 0 % mortality rate in sheep. Higher viral titres were observed in nasal, oral and conjunctival swabs from goats inoculated with either the Yemen or Vietnam isolate compared with those collected from sheep. Although the highest viral titres were detected in primary and secondary skin lesions in sheep and goats, the severity of clinical disease observed in each species varied according to the inoculum used. Whereas both the Yemen and Vietnam isolates clearly caused more severe disease in goats, the Yemen isolate was also moderately pathogenic in sheep. The Vietnam isolate, in contrast, caused only very mild disease in sheep. Limited DNA sequencing revealed ORF 074 of the Vietnam isolate to be identical to that of several goatpox virus isolates from China, suggesting a possible Chinese origin.

Cell culture adapted sheeppox virus as a challenge virus for potency testing of sheeppox vaccine.

Sheeppox virus from an outbreak of sheeppox that occurred in Srinagar (Jammu and Kashmir, India) in 2000 was isolated by inoculation of susceptible sheep and further re-isolated in cell culture. The field virus, adapted to grow in lamb testes culture, was evaluated for its potential use as challenge virus in potency testing of sheeppox vaccine currently in use. The virus (passage 6) produced severe disease in susceptible sheep when inoculated subcutaneously with a dose of 106.2 TCID50. The virus identity was confirmed by PCR, sequencing of P32 gene and species-specific signature residues identified in deduced aa sequence of the gene. The virus was successfully evaluated for its virulence using two batches of sheep pox vaccines. Use of this field virus enables consistent potency experiments of sheeppox vaccines avoiding use of animals for its propagation and titration.

Capripoxviruses: an emerging worldwide threat to sheep, goats and cattle.

Capripoxviruses are the cause of sheeppox, goatpox and lumpy skin disease (LSD) of cattle. These diseases are of great economic significance to farmers in regions in which they are endemic and are a major constraint to international trade in livestock and their products. Although the distribution of capripoxviruses is considerably reduced from what it was even 50 years ago, they are now expanding their territory, with recent outbreaks of sheeppox or goatpox in Vietnam, Mongolia and Greece, and outbreaks of LSD in Ethiopia, Egypt and Israel. Increased legal and illegal trade in live animals provides the potential for further spread, with, for instance, the possibility of LSD becoming firmly established in Asia. This review briefly summarizes what is known about capripoxviruses, including their impact on livestock production, their geographic range, host-specificity, clinical disease, transmission and genomics, and considers current developments in diagnostic tests and vaccines. Capripoxviruses have the potential to become emerging disease threats because of global climate change and changes in patterns of trade in animals and animal products. They also could be used as economic bioterrorism agents.

Capripoxvirus tissue tropism and shedding: A quantitative study in experimentally infected sheep and goats.

Sheeppox virus and goatpox virus cause systemic disease in sheep and goats that is often associated with high morbidity and high mortality. To increase understanding of the pathogenesis of these diseases, we undertook quantitative time-course studies in sheep and goats following intradermal inoculation of Nigerian sheeppox virus or Indian goatpox virus in their respective homologous hosts. Viremia, determined by virus isolation and real-time PCR, cleared within 2 to 3 weeks post inoculation. Peak shedding of viral DNA and infectious virus in nasal, conjunctival and oral secretions occurred between 10 and 14 days post inoculation, and persisted at low levels for up to an additional 3 to 6 weeks. Although gross lesions developed in multiple organ systems, highest viral titers were detected in skin and in discrete sites within oronasal tissues and gastrointestinal tract. The temporal distribution of infectious virus and viral DNA in tissues suggests an underlying pathogenesis that is similar to smallpox and monkeypox where greatest viral replication occurs in the skin. Our data demonstrate that capripoxvirus infections in sheep and goats provide additional and convenient models which are suitable not only for evaluation of poxvirus-specific vaccine concepts and therapeutics, but also study of poxvirus-host interactions.

Sheeppox virus kelch-like gene SPPV-019 affects virus virulence.

Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host range, including three genes with similarity to kelch-like genes of other poxviruses and eukaryotes. Here, a mutant SPPV with a deletion in the SPPV-019 kelch-like gene, DeltaKLP, was derived from the pathogenic strain SPPV-SA. DeltaKLP exhibited in vitro growth characteristics similar to those of SPPV-SA and revertant virus (RvKLP). DeltaKLP-infected cells exhibited a reduction in Ca(2+)-independent cell adhesion, suggesting that SPPV-019 may modulate cellular adhesion. When inoculated in sheep by the intranasal or intradermal routes, DeltaKLP was markedly attenuated, since all DeltaKLP-infected lambs survived infection. In contrast, SPPV-SA and RvKLP induced mortality approaching 100%. Lambs inoculated with DeltaKLP exhibited marked reduction or delay in fever response, gross lesions, viremia, and virus shedding compared to parental and revertant viruses. Together, these findings indicate that SPPV-019 is a significant SPPV virulence determinant in sheep.

A Vero cell-attenuated Goatpox virus provides protection against virulent virus challenge.

An Indian isolate of Goatpox virus (GTPV) was adapted and propagated in Vero cells for development of an attenuated virus. The virus was initially passaged in primary lamb testes cells and subsequently in Vero cells. At the 55th passage, the virus showed evidence of attenuation when tested for safety in seronegative goats. At this stage, the virus was found to be completely non-pathogenic. The virus was passaged further and the 60th passage was used for testing its immunogenicity in goats. The latter were inoculated with 10, 100 and 1000 TCID50 of the attenuated virus by intradermal (i.d.) route and challenged after 28 days with virulent GTPV. The attenuated virus produced no adverse reaction even at the highest dose and conferred complete protection even at the lowest dose against challenge with a high dose (2 x 10(6) of 50% skin-reactive dose SRD50) of virulent virus. Increased levels of virus-specific serum antibodies could be demonstrated by both indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test in all the immunized goats. No horizontal transmission of the virus from the immunized to in-contact animals took place. Our results suggest that this attenuated virus could be a safe, immunogenic and potent candidate for developing a vaccine against goatpox.

Long term immunity in African cattle vaccinated with a recombinant capripox-rinderpest virus vaccine.

Cattle were vaccinated with a recombinant capripox-rinderpest vaccine designed to protect cattle from infection with either rinderpest virus (RPV) or lumpy skin disease virus (LSDV). Vaccination did not induce any adverse clinical responses or show evidence of transmission of the vaccine virus to in-contact control animals. Approximately 50% of the cattle were solidly protected from challenge with a lethal dose of virulent RPV 2 years after vaccination while at 3 years approx. 30% were fully protected. In the case of LSDV, all of 4 vaccinated cattle challenged with virulent LSDV at 2 years were completely protected from clinical disease while 2 of 5 vaccinated cattle were completely protected at 3 years. The recombinant vaccine showed no loss of potency when stored lyophylized at 4 degrees C for up to 1 year. These results indicate that capripoxvirus is a suitable vector for the development of safe, effective and stable recombinant vaccines for cattle.