Isolation and phylogenetic analysis of an orf virus from sheep in Makhdoom, India.

Orf (contagious ecthyma) is an exanthematic disease caused by a parapoxvirus and occurs primarily in sheep and goats with zoonotic implications. In the present investigation, an orf outbreak in the Muzzaffarnagari sheep flock at the Central Institute for Research on Goats (CIRG), Makhdoom, Mathura, Uttar Pradesh, India, was investigated. Primary goat testes cell culture was used for isolation of the orf virus (ORFV) for the first time. The identity of the virus was confirmed by amplification and sequence analysis of the major envelope glycoprotein (B2L) gene and named ORFV/sheep/India/2012/CIRG. On phylogenetic analysis of B2L protein gene, it clustered with the ORFV strains from China suggesting distinct ORFV strains are circulating in India. On comparison of nucleotide and deduced amino acid sequence analysis (n = 63), a unique 126S residue was observed in ORFV/sheep/India/2012/CIRG. On further sequence analysis (B2L) of different ORFV strains (n = 63), some conserved amino acid residues were identified as host-specific (sheep, human, camel, takin, and musk ox) and have been summarized.

Concurrent Resolution of Chronic Diarrhea Likely Due to Crohn's Disease and Infection with Mycobacterium avium paratuberculosis.

Examination of samples of stool from a 61-year-old male patient, presenting with the clinical symptoms of Crohn's disease (CD), revealed massive shedding of acid fast bacilli with the morphology of Mycobacterium avium paratuberculosis (MAP), the causative agent of Johne's disease in cattle. MAP was cultured from the stool. Biotyping of the bacterium isolated from cultures of stool demonstrated, it was the Indian Bison biotype of MAP, the dominant biotype infecting livestock and humans in India. Based on this finding and because the patient was unresponsive to standard therapy used in India to treat patients with gastrointestinal inflammatory disorders, the patient was placed on a regimen of multi-antibiotic therapy, currently used to treat tuberculosis and CD. After 1 year of treatment, the patient's health was restored, concurrent with cessation of shedding of MAP in his stool. This patient is the first case shown to shed MAP from the stool who was cured of infection with antibiotics and who was concurrently cured of clinical signs of CD.

Complexities in Isolation and Purification of Multiple Viruses from Mixed Viral Infections: Viral Interference, Persistence and Exclusion.

Successful purification of multiple viruses from mixed infections remains a challenge. In this study, we investigated peste des petits ruminants virus (PPRV) and foot-and-mouth disease virus (FMDV) mixed infection in goats. Rather than in a single cell type, cytopathic effect (CPE) of the virus was observed in cocultured Vero/BHK-21 cells at 6th blind passage (BP). PPRV, but not FMDV could be purified from the virus mixture by plaque assay. Viral RNA (mixture) transfection in BHK-21 cells produced FMDV but not PPRV virions, a strategy which we have successfully employed for the first time to eliminate the negative-stranded RNA virus from the virus mixture. FMDV phenotypes, such as replication competent but noncytolytic, cytolytic but defective in plaque formation and, cytolytic but defective in both plaque formation and standard FMDV genome were observed respectively, at passage level BP8, BP15 and BP19 and hence complicated virus isolation in the cell culture system. Mixed infection was not found to induce any significant antigenic and genetic diversity in both PPRV and FMDV. Further, we for the first time demonstrated the viral interference between PPRV and FMDV. Prior transfection of PPRV RNA, but not Newcastle disease virus (NDV) and rotavirus RNA resulted in reduced FMDV replication in BHK-21 cells suggesting that the PPRV RNA-induced interference was specifically directed against FMDV. On long-term coinfection of some acute pathogenic viruses (all possible combinations of PPRV, FMDV, NDV and buffalopox virus) in Vero cells, in most cases, one of the coinfecting viruses was excluded at passage level 5 suggesting that the long-term coinfection may modify viral persistence. To the best of our knowledge, this is the first documented evidence describing a natural mixed infection of FMDV and PPRV. The study not only provides simple and reliable methodologies for isolation and purification of two epidemiologically and economically important groups of viruses, but could also help in establishing better guidelines for trading animals that could transmit further infections and epidemics in disease free nations.

Isolation, identification and characterization of a Peste des Petits Ruminants virus from an outbreak in Nanakpur, India.

A Peste des Petits Ruminants virus (PPRV) was isolated from an outbreak that occurred in sheep and goats in Nanakpur village of Mathura District in Uttar Pradesh (India). Based on hemagglutination of chicken red blood cells (rbcs), cytopathic effect similar to that caused by the Morbilliviruses in Vero cells, and amplification and sequence analysis of the viral nucleoprotein (N) gene, the identity of the virus was confirmed as PPRV and named PPRV/C. hircus-tc/India/2012/Nanakpur1 (in short PPRV/Nkp1/2012). However, based on its poor neutralization with monoclonal antibodies, escape detection by commercial ELISA, and unsuccessful amplification of the hemagglutinin (H) and the fusion (F) genes by several pairs of published PCR primers it was concluded that the PPRV/Nkp1/2012 may not be closely related to lineage type IV PPR viruses believed to be present in the Indian subcontinent. A plaque assay for titration of the PPRV was developed for the first time. The virus was plaque purified and its growth characteristics were studied in the African green monkey kidney (Vero) cells and baby hamster kidney (BHK-21) cells. In a one-step growth curve analysis it was concluded that the duration of the PPRV life cycle is 6-8h, an uncharacterized part of PPRV replication. These findings provide information for devising control strategies against PPR in India by choosing a homologous candidate vaccine prototype.