- Email: [email protected]
Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity
Accepted Manuscript Title: Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity Authors: Shyam Singh Dahiya, Sachin Kumar, Sharat Chandra Mehta, Raghvendar Singh, Kashi Nath, Shirish D. Narnaware, Fateh Chand Tuteja PII: DOI: Reference:
S0001-706X(17)30058-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.03.011 ACTROP 4237
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
16-1-2017 11-3-2017 11-3-2017
Please cite this article as: Dahiya, Shyam Singh, Kumar, Sachin, Mehta, Sharat Chandra, Singh, Raghvendar, Nath, Kashi, Narnaware, Shirish D., Tuteja, Fateh Chand, Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity
Shyam Singh Dahiya*1, Sachin Kumar2, Sharat Chandra Mehta1, Raghvendar Singh1, Kashi Nath1, Shirish D. Narnaware1,, Fateh Chand Tuteja1
1
National Research Center on Camel, Jorbeer, Bikaner, Rajasthan -334001 (India)
2
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam-
781039 (India)
*
Corresponding author:
Contact number: +91 151 2230183 Email address: [email protected]
Abstract: Camelpox is an important viral disease of camels, which may produce mild skin lesions or severe systemic infections. Camelpox virus (CMLV) isolates retrieved from an incidence of camelpox in camels at Bikaner, India were characterized on the basis of genotype and pathotype. Histopathological examination of the CMLV scab revealed intracytoplasmic-eosinophilic inclusion bodies. The phylogenetic analysis of all eight CMLV isolates for C18L gene nucleotide sequence revealed its clustering with its strains M-96 from Kazakhstan and CMS from Iran. The study will help 1
to understand the transmission chain, pathobiology, and epidemiology of circulating CMLV strains. The full genome sequencing of some of the exemplary samples of CMLV is recommended in order to plan and implement a suitable control strategy.
Keywords: Camelpox; isolation; PCR; Histopathology; Phylogenetic
Short communication: Camelpox has been recognized as one of the very important skin disease of camels (Salem et al., 2008). Except the introduced dromedary camel in Australia and tylopods (llama and related species) in South America (Mosadeghhesari et al., 2014), camelpox affects both dromedary as well as Bactrian camels (Kaaden, 2002). Camelpox is of significant concern since it may cause high morbidity and relatively high mortality in the young camels. In addition, loss of condition and drop in the milk production are other serious concerns affecting the economy and livelihood of the camel owners (Azwai et al., 1996). The disease is marked by fever, enlarged lymph nodes and skin lesions. Camelpox infection may lead to abortion in pregnant females (Anonymous, 2008). The disease can manifest itself either in the form of mild skin infection or the moderate to severe systemic infections depending upon the virus strain and the immune status of the animal (Kaaden, 2002). Camelpox has been reported from many countries where camel are reared (Al-Zi'abi et al., 2007). Camelpox outbreaks have been reported in the Middle East, in Asia and in the southern parts of Russia and India. The disease is endemic in these countries and sporadic outbreaks occur usually during the rainy season (Anonymous, 2008). There are incidences (unplublished) of camelpox both within as well as in the 2
adjacent areas of Bikaner district of Rajasthan, India. Recently, camelpox has also been reported from Ethiopia and Iran (Gelaye et al., 2016; Mosadeghhesari et al., 2014). Camelpox is caused by Orthopoxvirus cameli virus (CMLV), which belongs to the genus Orthopoxvirus within the family Poxviridae. CMLV is most closely related to the variola virus, the aetiological agent of smallpox (Anonymous, 2008). CMLV may get transmitted directly from the infected camels or indirectly via the contaminated secretion and excretion (Ramyar and Hessami, 1972). The arthropods have been reported to transmit CMLV among camels (Pfeffer et al., 1996). In general, Camelpox can be diagnosed based upon the clinical lesion (Kaaden, 2002) . However, it may be misdiagnosed with other pox like diseases. Among available techniques, polymerase chain reaction (PCR) offers a rapid way of diagnostics for CMLV (Khalafalla et al., 2015). At present, CMLV infection is diagnosed based on the amplification of various genes viz. A-type inclusion body protein (ATIP), the hemagglutinin (HA), the ankyrin repeat protein (C18L) or the DNA polymerase (DNA pol) genes (Meyer et al., 1994; Meyer et al., 1997; Ropp et al., 1995). Antigenically related ATIPs have been denoted to several orthopoxviruses (OPV) and its sequence comparison revealed the conserved N terminus of ATIPs among cowpox, vaccinia, variola, camelpox and mousepox virus. The restriction enzyme analyses and blot hybridizations disclosed the existence of defined deletions and thus its proficiency for diagnostic differentiation among OPVs (Meyer et al., 1994). Among poxviruses, only OPVs produce an HA which upon digestion with TaqI, HhaI, and RsaI, provides sufficient differentiation to identify species, certain subspecies and strains (Ropp et al., 1995). Likewise, DNA pol gene is group specific and most conserved non-structural protein among OPV (Sahay, 2006). A single-plex C18L gene for CMLV and a duplex-PCR comprising of C18L along with DNA pol gene have been reported to diagnose OPV and CMLV simultaneously (Balamurugan et al., 2009). The immunomodulatory genes of CMLV like 6L gene (also known as Golgi anti-apoptotic protein-GAAP) has been utilized for camelpox characterization (Nagarajan et al., 2013). In addition, schlafen-like protein and open reading frame (ORF) 11R proteins of CMLV is very similar to the 3
vaccinia virus epidermal growth factor have also been explored for its characterization (Nagarajan et al., 2013). Camelpox can be controlled by vaccination. At present, Ducapox- a live attenuated vaccine produced in South Africa and an inactivated adjuvant vaccine produced in Morocco are commercially available to vaccinate camel calf as early as six months (Khalafalla, 2003). Both the vaccines have been tested satisfactorily for their safety, potency and immunogenicity for humoral as well as cellular immune response against challenge (Khalafalla, 2003). The inactivated adjuvant vaccine requires revaccination at eight weeks post primary immunization whereas live attenuated vaccine provides protection for at least one year (Khalafalla, 2003). However, a booster vaccination is recommended for young animals to avoid interference by maternal antibodies. The animals must be vaccinated annually in case of inactivated vaccines. Although, different laboratories have characterized and confirmed CMLV in India, any report of its incidence/outbreak has not been reported recently. In the present study, a Camelpox incidence has been reported in dromedarian camels from the Rajasthan province in India. An organized camel farm in the Rajasthan/Bikaner province which lies in the Thar Desert of India recorded the outbreak of camelpox. The geographical location is characterized by high temperature and tropical climatic condition. The camels were maintained under a semi-intensive system of management. In December, 2014, camels of age ranging from 1 to 13 years of either sex were reported to the veterinary clinics with high fever and skin lesion in the form of pox-like scabs (Figure 1a). The scabs were collected in 10 % formal saline for histopathological examination. The formalin fixed tissue samples were embedded in paraffin, cut into 4–5-μm sections and stained with hematoxylin and eosin stain using the standard procedure. Total genomic DNA was extracted from collected scab samples (Table 1a) using PureLink® Genomic DNA Kit (Invitrogen, USA). The genomic DNA was used to amplify the C18L, ATIP, and HA genes of CMLV by specific primers designed from available gene sequences from GenBank (GenBank accession number: AF438165, AY009089). The 243 bp PCR amplified product 4
of all the eight isolates were purified and sequenced by dideoxy method (ABI Biosciences, USA). The nucleotide sequences and the deduced amino acid sequences of the C18L gene was analyzed and compared with available GenBank sequences. The phylogenetic analysis of the nucleotide sequence of the CMLV isolated from Rajasthan/Bikaner was done by the maximum likelihood statistical method using molecular evolutionary genetics analysis software (MEGA 6) (Tamura et al., 2013). In the present study, histopathological examination of the collected scab samples after haematoxylin and eosin staining revealed intracytoplasmic-eosinophilic inclusion bodies (Figure 1b). The histopathological examination showed characteristic cytoplasmic swelling, vacuolation and ballooning of the keratinocytes of the outer stratum spinosum of the epidermis (Duraffour et al., 2011). It has been proposed that intracytoplasmic eosinophilic inclusion bodies may be considered as characteristic for poxvirus infection (Anonymous, 2008). Our histopathological finding of intracytoplasmic inclusion bodies suggested the poxvirus infection in the Bikaner isolates. All the eight isolates showed positive amplification for C18L, ATIP, and HA genes of CMPV. The topoisomerase gene specific primers specific for contagious ecthyma was used as a negative control for differential diagnosis (Nagarajan et al., 2011). However, only C18L gene sequence from all the eight isolates were sequenced (Figure 1c), analyzed and submitted to GenBank (GenBank accession numbers KX889096- KX889103). All the eight isolates showed 100% nucleotide identity with CMLV isolates from India (GenBank accession numbers: GQ465931, EF592574, EF205282), Israel (GenBank accession numbers: KX770279, KX770280, KX770282), CMLV strain 0408151v (GenBank accession number: KP768318), CMLV strain M-96 (GenBank accession number: AF438165) and CMLV strain CMS (GenBank accession number: AY009089). Bikaner isolates of CMLV showed 90% nucleotide identity with vaccinia virus strain Tashkent clone TKT4 (GenBank accession number: KM044310). For different poxviruses viz. various strains of Vaccinia virus, Cowpox virus, CMLV isolates vary from 89-98% at nucleotide. 5
At amino acid level, all eight isolates showed 100% identity with C18L-like protein gene of CMLV (GenBank accession number: ACV88143) while minimum identity of 62-75% with Ankyrin repeat-containing protein of Racoon poxvirus (GenBank accession number: YP_009143317) (Table 1b). A total of 50, C18L gene sequences submitted to GenBank were used for phylogenetic analysis of CMLV isolated from Bikaner (Figure 2). The phylogenetic analysis revealed that all eight isolates from Bikaner clustered with CMLV strains 0408151v, M-96 and CMS (Nagarajan et al., 2013). The serum samples collected from the infected camels could not be analyzed for antibody titre which further emphasizes the lack of availability of any sero-diagnostic kit against CMLV. Although young animals and pregnant animals are more susceptible to camelpox infection, however, in the present study, camels aged from 10-12 years have found to be infected with CMLV (Duraffour et al., 2007). Recently, camelpox outbreak has been reported from Iran and showed 100% identity with CMLV strains CMS and M-96 (Mosadeghhesari et al., 2014). Our study also revealed that CMLV isolates from Bikaner/India clutch together with CMS and M-96 strains. Although the 243 bp sequence of C18L gene specific for CMLV has been exploited for specific and rapid detection of camelpox among orthopoxviruses, the partial sequence may not carry enough information to highlight the variation among Bikaner isolates of CMLV. In order to comprehend the variation at molecular level, full genome sequencing of some of the local isolates is required. As the majority of the camel farmers in India are nomadic and so do not practice intensive or semi-intensive care system for camels, besides there is lack of diagnostic facilities at many of the remote areas along their tracks. This accounts for non-reporting and proper documentation of morbidity and mortality data for many of the camel’s infection especially camelpox. Difficulty in the diagnosis and control measures for emerging and re-emerging diseases in the developing countries has supported several viral outbreaks in humans and animals (M et al., 2015). In arid regions, camels provide backbone to the livelihood of the human populations. Any loss of animal, body condition or 6
reduction in milk yield due to camelpox has serious economic impacts (Duraffour et al., 2011). In spite of the regular report of CMLV infection, there is paucity of its strain information circulating in India. Although, live attenuated and inactivated adjuvant vaccines are available for camelpox, there are no reports of camelpox vaccination in India (Dahiya et al., 2016). In this context, the significance of a strict vigilance to address any new CMLV outbreak cannot be overemphasized (Dahiya et al., 2016). From the time when CMLV had been used as an alternative to vaccinia virus (VACV) (Hafez et al., 1992), report for the sequence resemblance of CMLV to variola virus and recently the first human report from India, a sufficient interest has been shown by various researchers as evident by the publications from across the globe (Bera et al., 2011; Duraffour et al., 2011; Gubser and Smith, 2002; Khalafalla, 2003; Mosadeghhesari et al., 2014). Our study contributes to this growing interest in CMLV as a fresh proclaim from India. However no such human case was observed during the present study. A focused vigilance is required to address any focal incidence of CMLV in order to better understand its transmission chain, pathobiology and epidemiology in India. Moreover, it is mandatory to plan a control strategy to avoid any major outbreak of CMLV both in camels and/or in human. Although we do not know much about the existing strains of CMLV, there might be a circulating one, which behaves entirely different than what is known.
Conflict of interests The authors have declared that no competing interests exist.
Acknowledgements The authors thank the Council, ICAR, New Delhi, India to support the camelpox research.
7
References Al-Zi'abi, O., Nishikawa, H., Meyer, H., 2007. The first outbreak of camelpox in Syria. J Vet Med Sci 69, 541-543. Anonymous, 2008. Camelpox, in: OIE (Ed.). OIE terrestrial manual, Paris, France, pp. 1-14. Azwai, S.M., Carter, S.D., Woldehiwet, Z., Wernery, U., 1996. Serology of Orthopoxvirus cameli infection in dromedary camels: analysis by ELISA and western blotting. Comp Immunol Microbiol Infect Dis 19, 65-78. Balamurugan, V., Bhanuprakash, V., Hosamani, M., Jayappa, K.D., Venkatesan, G., Chauhan, B., Singh, R.K., 2009. A polymerase chain reaction strategy for the diagnosis of camelpox. J Vet Diagn Invest 21, 231-237. Bera, B.C., Shanmugasundaram, K., Barua, S., Venkatesan, G., Virmani, N., Riyesh, T., Gulati, B.R., Bhanuprakash, V., Vaid, R.K., Kakker, N.K., Malik, P., Bansal, M., Gadvi, S., Singh, R.V., Yadav, V., Sardarilal, Nagarajan, G., Balamurugan, V., Hosamani, M., Pathak, K.M., Singh, R.K., 2011. Zoonotic cases of camelpox infection in India. Vet Microbiol 152, 29-38. Dahiya, S.S., Kumar, S., Mehta, S.C., Narnaware, S.D., Singh, R., Tuteja, F.C., 2016. Camelpox: A brief review on its epidemiology, current status and challenges. Acta Trop 158, 32-38. Duraffour, S., Meyer, H., Andrei, G., Snoeck, R., 2011. Camelpox virus. Antiviral Res 92, 167-186. Duraffour, S., Snoeck, R., de Vos, R., van Den Oord, J.J., Crance, J.M., Garin, D., Hruby, D.E., Jordan, R., De Clercq, E., Andrei, G., 2007. Activity of the anti-orthopoxvirus compound ST-246 against vaccinia, cowpox and camelpox viruses in cell monolayers and organotypic raft cultures. Antivir Ther 12, 1205-1216. Gelaye, E., Achenbach, J.E., Ayelet, G., Jenberie, S., Yami, M., Grabherr, R., Loitsch, A., Diallo, A., Lamien, C.E., 2016. Genetic characterization of poxviruses in Camelus dromedarius in Ethiopia, 2011-2014. Antiviral Res 134, 17-25. Gubser, C., Smith, G.L., 2002. The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox. J Gen Virol 83, 855-872. Hafez, S.M., al-Sukayran, A., dela Cruz, D., Mazloum, K.S., al-Bokmy, A.M., al-Mukayel, A., Amjad, A.M., 1992. Development of a live cell culture camelpox vaccine. Vaccine 10, 533-539. Kaaden, W.U., 2002. Camel pox. In: Infectious Diseases in Camelids, Second Edition ed, Blackwell Science Berlin, Germany, pp. 176-185. Khalafalla, A.I., Al-Busada, K.A., El-Sabagh, I.M., 2015. Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels. Virol J 12, 102. Khalafalla, A.I., El Dirdiri, G.A, 2003. Laboratory and field investigations of a live attenuated and an inactivated camelpox vaccine. J. Camel Pract. Res 10, 191-200. M, P., R, Y., S, P., C, S., G, S., RK, S., 2015. Camelpox and buffalopox: Two emerging and re-emerging orthopox viral diseases of India. Adv. Anim. Vet. Sci. 3, 527-541. Meyer, H., Pfeffer, M., Rziha, H.J., 1994. Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of Orthopoxvirus species by polymerase chain reaction. J Gen Virol 75 ( Pt 8), 1975-1981. Meyer, H., Ropp, S.L., Esposito, J.J., 1997. Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses. J Virol Methods 64, 217-221. Mosadeghhesari, M., Oryan, A., Zibaee, S., Varshovi, H.R., 2014. Molecular investigation and cultivation of camelpox virus in Iran. Arch Virol 159, 3005-3011. Nagarajan, G., Swami, S.K., Dahiya, S.S., Sivakumar, G., Narnaware, S.D., Tuteja, F.C., Patil, N.V., 2011. Sequence analysis of topoisomerase gene of pseudocowpox virus isolates from camels (Camelus dromedarius). Virus Res 158, 277-280. Nagarajan, G., Swami, S.K., Dahiya, S.S., Sivakumar, G., Yadav, V.K., Tuteja, F.C., Narnaware, S.D., Patil, N.V., 2013. Phylogenetic analysis of immunomodulatory protein genes of camelpoxvirus obtained from India. Comp Immunol Microbiol Infect Dis 36, 415-424. Pfeffer, M., Meyer, H., Wernery, U., Kaaden, O.R., 1996. Comparison of camelpox viruses isolated in Dubai. Vet Microbiol 49, 135-146. Ramyar, H., Hessami, M., 1972. Isolation, cultivation and characterization of camel pox virus. Zentralbl Veterinarmed B 19, 182-189. 8
Ropp, S.L., Jin, Q., Knight, J.C., Massung, R.F., Esposito, J.J., 1995. PCR strategy for identification and differentiation of small pox and other orthopoxviruses. J Clin Microbiol 33, 2069-2076. Sahay, B., 2006. Evaluation of gene silencing by RNA interference (RNAi) in control of animal virus infections, Virology. IVRI India, Izatnagar. Salem, S.A.H., Omayma, A.S., Nahed, A.M., Arafa, A.A., 2008. Isolation and Molecular Characterization of Camel Pox Virus. J. Comp. Path. Clinic. Path 2, 306-318. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 2725-2729.
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Figure Legends:
Figure 1a. Camelpox specific lesions over the body of dromedary camel.
Figure 1b. Histopathological examination of camelpox scab: intracytoplasmic- eosinophilic inclusion bodies are marked with arrow. Figure 1c. PCR amplification for C18L gene; lane 1-8: CMLV/Bikaner isolates, M: DNA size marker100 bp ladder
10
Figure 2. Phylogenetic analysis of camelpox isolates from Bikaner using C18L gene sequences. A total of 50 sequences were taken from GenBank. The phylogenetic tree was constructed by maximum likelihood method using Tamura-Nei model in Mega 6 program including 500 replica of bootstrap.
11
Table 1a: Details of CMLV isolates from Bikaner, India
S. No.
Age
Sex
Breed
Clinical history (Pregnancy/lactation/past diseases or concurrent infection with pox)
(Years) 01
4-5
M
Mewari
Severly affected, scabs over complete body
02
9-10
M
Mewari
Mange, Severly affected, scabs over complete body
03
5-6
F
Jaisalmeri
No clinical history
04
5-6
F
Kachchhi
Mange, parturition once
05
5-6
F
Bikaneri
Parturition once
06
11-12
M
Mewari
Mange
07
9-10
M
Mewari
Mange
08
2-3
M
Kachchhi
No clinical history
Table 1b: Genbank accession number of each camelox virus (CMLV) isolate along with their percent identity at nucleotide and amino acid level.
12
Sr. No.
Isolate name/ GenBank accession number
% Nucleotide identity
% Amino acid identity
Maximum
Minimum
Maximum
Minimum
01
Camelpox virus(NRCC_CMLV1)/ KX889096
100 (KP768318, AF43816, AY009089)
92(JN654979)
100(ACV88143)
67(YP_009143317)
02
Camelpox virus(NRCC_CMLV2)/ KX889097
100 (KP768318, AF438165,
90(KM044310)
100 (ACV88143)
73 (YP_009143317)
03
Camelpox virus(NRCC_CMLV3)/ KX889098
100 (KP768318, AF43816, AY009089)
90 (KM044310)
100 (ACV88143)
73 (YP_009143317)
04
Camelpox virus(NRCC_CMLV4)/ KX889099
100 (KP768318, AF438165, AY009089)
90 (KM044310)
100 (ACV88143)
74 (YP_009143317)
05
Camelpox virus(NRCC_CMLV5)/ KX889100
100 (KP768318, AF438165, AY009089)
90 (KM044310)
100 (ACV88143)
62 (YP_009143317)
06
Camelpox virus(NRCC_CMLV6)/ KX889101
100 (KP768318, AF438165, AY009089
90(KM044310)
100 (ACV88143
75 (YP_009143317)
07
Camelpox virus(NRCC_CMLV7)/ KX889102
100 (KP768318, AF438165, AY009089)
90 (KM044310)
100 (ACV88143)
75 (YP_009143317)
08
Camelpox virus(NRCC_CMLV8)/ KX889103
100 (KP768318, AF43816, AY009089)
90 (KM044310)
100 (ACV88143)
75 (YP_009143317)
KP768318: CMLV strain 0408151v, UK AF438165: CMLV M-96, Kazakhstan AY009089: CMLV CMS, Iran KM044310: Vaccinia virus strain Tashkent clone TKT4, Canada JN654979: Vaccinia virus strain Dryvax clone DPP12, complete genome, Canada ACV88143:Partial C18L-like protein, India YP_009143317: Ankyrin repeat-containing protein [Raccoonpox virus], USA
13
S0001-706X(17)30058-X http://dx.doi.org/doi:10.1016/j.actatropica.2017.03.011 ACTROP 4237
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
16-1-2017 11-3-2017 11-3-2017
Please cite this article as: Dahiya, Shyam Singh, Kumar, Sachin, Mehta, Sharat Chandra, Singh, Raghvendar, Nath, Kashi, Narnaware, Shirish D., Tuteja, Fateh Chand, Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity
Shyam Singh Dahiya*1, Sachin Kumar2, Sharat Chandra Mehta1, Raghvendar Singh1, Kashi Nath1, Shirish D. Narnaware1,, Fateh Chand Tuteja1
1
National Research Center on Camel, Jorbeer, Bikaner, Rajasthan -334001 (India)
2
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam-
781039 (India)
*
Corresponding author:
Contact number: +91 151 2230183 Email address: [email protected]
Abstract: Camelpox is an important viral disease of camels, which may produce mild skin lesions or severe systemic infections. Camelpox virus (CMLV) isolates retrieved from an incidence of camelpox in camels at Bikaner, India were characterized on the basis of genotype and pathotype. Histopathological examination of the CMLV scab revealed intracytoplasmic-eosinophilic inclusion bodies. The phylogenetic analysis of all eight CMLV isolates for C18L gene nucleotide sequence revealed its clustering with its strains M-96 from Kazakhstan and CMS from Iran. The study will help 1
to understand the transmission chain, pathobiology, and epidemiology of circulating CMLV strains. The full genome sequencing of some of the exemplary samples of CMLV is recommended in order to plan and implement a suitable control strategy.
Keywords: Camelpox; isolation; PCR; Histopathology; Phylogenetic
Short communication: Camelpox has been recognized as one of the very important skin disease of camels (Salem et al., 2008). Except the introduced dromedary camel in Australia and tylopods (llama and related species) in South America (Mosadeghhesari et al., 2014), camelpox affects both dromedary as well as Bactrian camels (Kaaden, 2002). Camelpox is of significant concern since it may cause high morbidity and relatively high mortality in the young camels. In addition, loss of condition and drop in the milk production are other serious concerns affecting the economy and livelihood of the camel owners (Azwai et al., 1996). The disease is marked by fever, enlarged lymph nodes and skin lesions. Camelpox infection may lead to abortion in pregnant females (Anonymous, 2008). The disease can manifest itself either in the form of mild skin infection or the moderate to severe systemic infections depending upon the virus strain and the immune status of the animal (Kaaden, 2002). Camelpox has been reported from many countries where camel are reared (Al-Zi'abi et al., 2007). Camelpox outbreaks have been reported in the Middle East, in Asia and in the southern parts of Russia and India. The disease is endemic in these countries and sporadic outbreaks occur usually during the rainy season (Anonymous, 2008). There are incidences (unplublished) of camelpox both within as well as in the 2
adjacent areas of Bikaner district of Rajasthan, India. Recently, camelpox has also been reported from Ethiopia and Iran (Gelaye et al., 2016; Mosadeghhesari et al., 2014). Camelpox is caused by Orthopoxvirus cameli virus (CMLV), which belongs to the genus Orthopoxvirus within the family Poxviridae. CMLV is most closely related to the variola virus, the aetiological agent of smallpox (Anonymous, 2008). CMLV may get transmitted directly from the infected camels or indirectly via the contaminated secretion and excretion (Ramyar and Hessami, 1972). The arthropods have been reported to transmit CMLV among camels (Pfeffer et al., 1996). In general, Camelpox can be diagnosed based upon the clinical lesion (Kaaden, 2002) . However, it may be misdiagnosed with other pox like diseases. Among available techniques, polymerase chain reaction (PCR) offers a rapid way of diagnostics for CMLV (Khalafalla et al., 2015). At present, CMLV infection is diagnosed based on the amplification of various genes viz. A-type inclusion body protein (ATIP), the hemagglutinin (HA), the ankyrin repeat protein (C18L) or the DNA polymerase (DNA pol) genes (Meyer et al., 1994; Meyer et al., 1997; Ropp et al., 1995). Antigenically related ATIPs have been denoted to several orthopoxviruses (OPV) and its sequence comparison revealed the conserved N terminus of ATIPs among cowpox, vaccinia, variola, camelpox and mousepox virus. The restriction enzyme analyses and blot hybridizations disclosed the existence of defined deletions and thus its proficiency for diagnostic differentiation among OPVs (Meyer et al., 1994). Among poxviruses, only OPVs produce an HA which upon digestion with TaqI, HhaI, and RsaI, provides sufficient differentiation to identify species, certain subspecies and strains (Ropp et al., 1995). Likewise, DNA pol gene is group specific and most conserved non-structural protein among OPV (Sahay, 2006). A single-plex C18L gene for CMLV and a duplex-PCR comprising of C18L along with DNA pol gene have been reported to diagnose OPV and CMLV simultaneously (Balamurugan et al., 2009). The immunomodulatory genes of CMLV like 6L gene (also known as Golgi anti-apoptotic protein-GAAP) has been utilized for camelpox characterization (Nagarajan et al., 2013). In addition, schlafen-like protein and open reading frame (ORF) 11R proteins of CMLV is very similar to the 3
vaccinia virus epidermal growth factor have also been explored for its characterization (Nagarajan et al., 2013). Camelpox can be controlled by vaccination. At present, Ducapox- a live attenuated vaccine produced in South Africa and an inactivated adjuvant vaccine produced in Morocco are commercially available to vaccinate camel calf as early as six months (Khalafalla, 2003). Both the vaccines have been tested satisfactorily for their safety, potency and immunogenicity for humoral as well as cellular immune response against challenge (Khalafalla, 2003). The inactivated adjuvant vaccine requires revaccination at eight weeks post primary immunization whereas live attenuated vaccine provides protection for at least one year (Khalafalla, 2003). However, a booster vaccination is recommended for young animals to avoid interference by maternal antibodies. The animals must be vaccinated annually in case of inactivated vaccines. Although, different laboratories have characterized and confirmed CMLV in India, any report of its incidence/outbreak has not been reported recently. In the present study, a Camelpox incidence has been reported in dromedarian camels from the Rajasthan province in India. An organized camel farm in the Rajasthan/Bikaner province which lies in the Thar Desert of India recorded the outbreak of camelpox. The geographical location is characterized by high temperature and tropical climatic condition. The camels were maintained under a semi-intensive system of management. In December, 2014, camels of age ranging from 1 to 13 years of either sex were reported to the veterinary clinics with high fever and skin lesion in the form of pox-like scabs (Figure 1a). The scabs were collected in 10 % formal saline for histopathological examination. The formalin fixed tissue samples were embedded in paraffin, cut into 4–5-μm sections and stained with hematoxylin and eosin stain using the standard procedure. Total genomic DNA was extracted from collected scab samples (Table 1a) using PureLink® Genomic DNA Kit (Invitrogen, USA). The genomic DNA was used to amplify the C18L, ATIP, and HA genes of CMLV by specific primers designed from available gene sequences from GenBank (GenBank accession number: AF438165, AY009089). The 243 bp PCR amplified product 4
of all the eight isolates were purified and sequenced by dideoxy method (ABI Biosciences, USA). The nucleotide sequences and the deduced amino acid sequences of the C18L gene was analyzed and compared with available GenBank sequences. The phylogenetic analysis of the nucleotide sequence of the CMLV isolated from Rajasthan/Bikaner was done by the maximum likelihood statistical method using molecular evolutionary genetics analysis software (MEGA 6) (Tamura et al., 2013). In the present study, histopathological examination of the collected scab samples after haematoxylin and eosin staining revealed intracytoplasmic-eosinophilic inclusion bodies (Figure 1b). The histopathological examination showed characteristic cytoplasmic swelling, vacuolation and ballooning of the keratinocytes of the outer stratum spinosum of the epidermis (Duraffour et al., 2011). It has been proposed that intracytoplasmic eosinophilic inclusion bodies may be considered as characteristic for poxvirus infection (Anonymous, 2008). Our histopathological finding of intracytoplasmic inclusion bodies suggested the poxvirus infection in the Bikaner isolates. All the eight isolates showed positive amplification for C18L, ATIP, and HA genes of CMPV. The topoisomerase gene specific primers specific for contagious ecthyma was used as a negative control for differential diagnosis (Nagarajan et al., 2011). However, only C18L gene sequence from all the eight isolates were sequenced (Figure 1c), analyzed and submitted to GenBank (GenBank accession numbers KX889096- KX889103). All the eight isolates showed 100% nucleotide identity with CMLV isolates from India (GenBank accession numbers: GQ465931, EF592574, EF205282), Israel (GenBank accession numbers: KX770279, KX770280, KX770282), CMLV strain 0408151v (GenBank accession number: KP768318), CMLV strain M-96 (GenBank accession number: AF438165) and CMLV strain CMS (GenBank accession number: AY009089). Bikaner isolates of CMLV showed 90% nucleotide identity with vaccinia virus strain Tashkent clone TKT4 (GenBank accession number: KM044310). For different poxviruses viz. various strains of Vaccinia virus, Cowpox virus, CMLV isolates vary from 89-98% at nucleotide. 5
At amino acid level, all eight isolates showed 100% identity with C18L-like protein gene of CMLV (GenBank accession number: ACV88143) while minimum identity of 62-75% with Ankyrin repeat-containing protein of Racoon poxvirus (GenBank accession number: YP_009143317) (Table 1b). A total of 50, C18L gene sequences submitted to GenBank were used for phylogenetic analysis of CMLV isolated from Bikaner (Figure 2). The phylogenetic analysis revealed that all eight isolates from Bikaner clustered with CMLV strains 0408151v, M-96 and CMS (Nagarajan et al., 2013). The serum samples collected from the infected camels could not be analyzed for antibody titre which further emphasizes the lack of availability of any sero-diagnostic kit against CMLV. Although young animals and pregnant animals are more susceptible to camelpox infection, however, in the present study, camels aged from 10-12 years have found to be infected with CMLV (Duraffour et al., 2007). Recently, camelpox outbreak has been reported from Iran and showed 100% identity with CMLV strains CMS and M-96 (Mosadeghhesari et al., 2014). Our study also revealed that CMLV isolates from Bikaner/India clutch together with CMS and M-96 strains. Although the 243 bp sequence of C18L gene specific for CMLV has been exploited for specific and rapid detection of camelpox among orthopoxviruses, the partial sequence may not carry enough information to highlight the variation among Bikaner isolates of CMLV. In order to comprehend the variation at molecular level, full genome sequencing of some of the local isolates is required. As the majority of the camel farmers in India are nomadic and so do not practice intensive or semi-intensive care system for camels, besides there is lack of diagnostic facilities at many of the remote areas along their tracks. This accounts for non-reporting and proper documentation of morbidity and mortality data for many of the camel’s infection especially camelpox. Difficulty in the diagnosis and control measures for emerging and re-emerging diseases in the developing countries has supported several viral outbreaks in humans and animals (M et al., 2015). In arid regions, camels provide backbone to the livelihood of the human populations. Any loss of animal, body condition or 6
reduction in milk yield due to camelpox has serious economic impacts (Duraffour et al., 2011). In spite of the regular report of CMLV infection, there is paucity of its strain information circulating in India. Although, live attenuated and inactivated adjuvant vaccines are available for camelpox, there are no reports of camelpox vaccination in India (Dahiya et al., 2016). In this context, the significance of a strict vigilance to address any new CMLV outbreak cannot be overemphasized (Dahiya et al., 2016). From the time when CMLV had been used as an alternative to vaccinia virus (VACV) (Hafez et al., 1992), report for the sequence resemblance of CMLV to variola virus and recently the first human report from India, a sufficient interest has been shown by various researchers as evident by the publications from across the globe (Bera et al., 2011; Duraffour et al., 2011; Gubser and Smith, 2002; Khalafalla, 2003; Mosadeghhesari et al., 2014). Our study contributes to this growing interest in CMLV as a fresh proclaim from India. However no such human case was observed during the present study. A focused vigilance is required to address any focal incidence of CMLV in order to better understand its transmission chain, pathobiology and epidemiology in India. Moreover, it is mandatory to plan a control strategy to avoid any major outbreak of CMLV both in camels and/or in human. Although we do not know much about the existing strains of CMLV, there might be a circulating one, which behaves entirely different than what is known.
Conflict of interests The authors have declared that no competing interests exist.
Acknowledgements The authors thank the Council, ICAR, New Delhi, India to support the camelpox research.
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References Al-Zi'abi, O., Nishikawa, H., Meyer, H., 2007. The first outbreak of camelpox in Syria. J Vet Med Sci 69, 541-543. Anonymous, 2008. Camelpox, in: OIE (Ed.). OIE terrestrial manual, Paris, France, pp. 1-14. Azwai, S.M., Carter, S.D., Woldehiwet, Z., Wernery, U., 1996. Serology of Orthopoxvirus cameli infection in dromedary camels: analysis by ELISA and western blotting. Comp Immunol Microbiol Infect Dis 19, 65-78. Balamurugan, V., Bhanuprakash, V., Hosamani, M., Jayappa, K.D., Venkatesan, G., Chauhan, B., Singh, R.K., 2009. A polymerase chain reaction strategy for the diagnosis of camelpox. J Vet Diagn Invest 21, 231-237. Bera, B.C., Shanmugasundaram, K., Barua, S., Venkatesan, G., Virmani, N., Riyesh, T., Gulati, B.R., Bhanuprakash, V., Vaid, R.K., Kakker, N.K., Malik, P., Bansal, M., Gadvi, S., Singh, R.V., Yadav, V., Sardarilal, Nagarajan, G., Balamurugan, V., Hosamani, M., Pathak, K.M., Singh, R.K., 2011. Zoonotic cases of camelpox infection in India. Vet Microbiol 152, 29-38. Dahiya, S.S., Kumar, S., Mehta, S.C., Narnaware, S.D., Singh, R., Tuteja, F.C., 2016. Camelpox: A brief review on its epidemiology, current status and challenges. Acta Trop 158, 32-38. Duraffour, S., Meyer, H., Andrei, G., Snoeck, R., 2011. Camelpox virus. Antiviral Res 92, 167-186. Duraffour, S., Snoeck, R., de Vos, R., van Den Oord, J.J., Crance, J.M., Garin, D., Hruby, D.E., Jordan, R., De Clercq, E., Andrei, G., 2007. Activity of the anti-orthopoxvirus compound ST-246 against vaccinia, cowpox and camelpox viruses in cell monolayers and organotypic raft cultures. Antivir Ther 12, 1205-1216. Gelaye, E., Achenbach, J.E., Ayelet, G., Jenberie, S., Yami, M., Grabherr, R., Loitsch, A., Diallo, A., Lamien, C.E., 2016. Genetic characterization of poxviruses in Camelus dromedarius in Ethiopia, 2011-2014. Antiviral Res 134, 17-25. Gubser, C., Smith, G.L., 2002. The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox. J Gen Virol 83, 855-872. Hafez, S.M., al-Sukayran, A., dela Cruz, D., Mazloum, K.S., al-Bokmy, A.M., al-Mukayel, A., Amjad, A.M., 1992. Development of a live cell culture camelpox vaccine. Vaccine 10, 533-539. Kaaden, W.U., 2002. Camel pox. In: Infectious Diseases in Camelids, Second Edition ed, Blackwell Science Berlin, Germany, pp. 176-185. Khalafalla, A.I., Al-Busada, K.A., El-Sabagh, I.M., 2015. Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels. Virol J 12, 102. Khalafalla, A.I., El Dirdiri, G.A, 2003. Laboratory and field investigations of a live attenuated and an inactivated camelpox vaccine. J. Camel Pract. Res 10, 191-200. M, P., R, Y., S, P., C, S., G, S., RK, S., 2015. Camelpox and buffalopox: Two emerging and re-emerging orthopox viral diseases of India. Adv. Anim. Vet. Sci. 3, 527-541. Meyer, H., Pfeffer, M., Rziha, H.J., 1994. Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of Orthopoxvirus species by polymerase chain reaction. J Gen Virol 75 ( Pt 8), 1975-1981. Meyer, H., Ropp, S.L., Esposito, J.J., 1997. Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses. J Virol Methods 64, 217-221. Mosadeghhesari, M., Oryan, A., Zibaee, S., Varshovi, H.R., 2014. Molecular investigation and cultivation of camelpox virus in Iran. Arch Virol 159, 3005-3011. Nagarajan, G., Swami, S.K., Dahiya, S.S., Sivakumar, G., Narnaware, S.D., Tuteja, F.C., Patil, N.V., 2011. Sequence analysis of topoisomerase gene of pseudocowpox virus isolates from camels (Camelus dromedarius). Virus Res 158, 277-280. Nagarajan, G., Swami, S.K., Dahiya, S.S., Sivakumar, G., Yadav, V.K., Tuteja, F.C., Narnaware, S.D., Patil, N.V., 2013. Phylogenetic analysis of immunomodulatory protein genes of camelpoxvirus obtained from India. Comp Immunol Microbiol Infect Dis 36, 415-424. Pfeffer, M., Meyer, H., Wernery, U., Kaaden, O.R., 1996. Comparison of camelpox viruses isolated in Dubai. Vet Microbiol 49, 135-146. Ramyar, H., Hessami, M., 1972. Isolation, cultivation and characterization of camel pox virus. Zentralbl Veterinarmed B 19, 182-189. 8
Ropp, S.L., Jin, Q., Knight, J.C., Massung, R.F., Esposito, J.J., 1995. PCR strategy for identification and differentiation of small pox and other orthopoxviruses. J Clin Microbiol 33, 2069-2076. Sahay, B., 2006. Evaluation of gene silencing by RNA interference (RNAi) in control of animal virus infections, Virology. IVRI India, Izatnagar. Salem, S.A.H., Omayma, A.S., Nahed, A.M., Arafa, A.A., 2008. Isolation and Molecular Characterization of Camel Pox Virus. J. Comp. Path. Clinic. Path 2, 306-318. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 2725-2729.
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Figure Legends:
Figure 1a. Camelpox specific lesions over the body of dromedary camel.
Figure 1b. Histopathological examination of camelpox scab: intracytoplasmic- eosinophilic inclusion bodies are marked with arrow. Figure 1c. PCR amplification for C18L gene; lane 1-8: CMLV/Bikaner isolates, M: DNA size marker100 bp ladder
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Figure 2. Phylogenetic analysis of camelpox isolates from Bikaner using C18L gene sequences. A total of 50 sequences were taken from GenBank. The phylogenetic tree was constructed by maximum likelihood method using Tamura-Nei model in Mega 6 program including 500 replica of bootstrap.
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Table 1a: Details of CMLV isolates from Bikaner, India
S. No.
Age
Sex
Breed
Clinical history (Pregnancy/lactation/past diseases or concurrent infection with pox)
(Years) 01
4-5
M
Mewari
Severly affected, scabs over complete body
02
9-10
M
Mewari
Mange, Severly affected, scabs over complete body
03
5-6
F
Jaisalmeri
No clinical history
04
5-6
F
Kachchhi
Mange, parturition once
05
5-6
F
Bikaneri
Parturition once
06
11-12
M
Mewari
Mange
07
9-10
M
Mewari
Mange
08
2-3
M
Kachchhi
No clinical history
Table 1b: Genbank accession number of each camelox virus (CMLV) isolate along with their percent identity at nucleotide and amino acid level.
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Sr. No.
Isolate name/ GenBank accession number
% Nucleotide identity
% Amino acid identity
Maximum
Minimum
Maximum
Minimum
01
Camelpox virus(NRCC_CMLV1)/ KX889096
100 (KP768318, AF43816, AY009089)
92(JN654979)
100(ACV88143)
67(YP_009143317)
02
Camelpox virus(NRCC_CMLV2)/ KX889097
100 (KP768318, AF438165,
90(KM044310)
100 (ACV88143)
73 (YP_009143317)
03
Camelpox virus(NRCC_CMLV3)/ KX889098
100 (KP768318, AF43816, AY009089)
90 (KM044310)
100 (ACV88143)
73 (YP_009143317)
04
Camelpox virus(NRCC_CMLV4)/ KX889099
100 (KP768318, AF438165, AY009089)
90 (KM044310)
100 (ACV88143)
74 (YP_009143317)
05
Camelpox virus(NRCC_CMLV5)/ KX889100
100 (KP768318, AF438165, AY009089)
90 (KM044310)
100 (ACV88143)
62 (YP_009143317)
06
Camelpox virus(NRCC_CMLV6)/ KX889101
100 (KP768318, AF438165, AY009089
90(KM044310)
100 (ACV88143
75 (YP_009143317)
07
Camelpox virus(NRCC_CMLV7)/ KX889102
100 (KP768318, AF438165, AY009089)
90 (KM044310)
100 (ACV88143)
75 (YP_009143317)
08
Camelpox virus(NRCC_CMLV8)/ KX889103
100 (KP768318, AF43816, AY009089)
90 (KM044310)
100 (ACV88143)
75 (YP_009143317)
KP768318: CMLV strain 0408151v, UK AF438165: CMLV M-96, Kazakhstan AY009089: CMLV CMS, Iran KM044310: Vaccinia virus strain Tashkent clone TKT4, Canada JN654979: Vaccinia virus strain Dryvax clone DPP12, complete genome, Canada ACV88143:Partial C18L-like protein, India YP_009143317: Ankyrin repeat-containing protein [Raccoonpox virus], USA
13