MNK1 inhibitor as an antiviral agent suppresses buffalopox virus protein synthesis.

A small molecule chemical inhibitor CGP57380 that blocks activation of MAPK interacting kinase 1 (MNK1) was found to significantly suppress buffalopox virus (BPXV) replication. BPXV infection was shown to induce MNK1 activation. Depletion of MNK1 by small interfering RNA (siRNA), blocking activation of extracellular regulated kinase (ERK, an upstream activator of MNK1) and disruption of eIF4E/eIF4G interaction (downstream substrate of MNK1 which plays a central role in cap-dependent translation initiation), resulted in reduced BPXV replication, suggesting that ERK/MNK1/eIF4E signaling is a prerequisite for BPXV replication. With the help of time-of-addition and virus step-specific assays, CGP57380 treatment was shown to decrease synthesis of viral genome (DNA). Disruption of ERK/MNK1/eIF4E signaling resulted in reduced synthesis of viral proteins, suggesting that BPXV utilizes cap-dependent mechanism of translation initiation. Therefore, we concluded that decreased synthesis of viral genome in presence of MNK1 inhibitor is the result of reduced synthesis of viral proteins. Furthermore, BPXV was sequentially passaged (P = 40) in presence of CGP57380 or vehicle control (DMSO). As compared to P0 and P40-control viruses, P40-CGP57380 virus replicated at significantly higher (∼10-fold) titers in presence of CGP57380, although a complete resistance could not be achieved. In a BPXV egg infection model, CGP57380 was found to prevent development of pock lesions on chorioallantoic membrane (CAM) as well as associated mortality of the embryonated chicken eggs. We for the first time demonstrated in vitro and in ovo antiviral efficacy of CGP57380 against BPXV and identified that ERK/MNK1 signaling is a prerequisite for synthesis of viral proteins. Our study also describes a rare report about generation of drug-resistant viral variants against a host-targeting antiviral agent.

Role of MAPK/MNK1 signaling in virus replication.

Viruses are obligate intracellular parasites; they heavily depend on the host cell machinery to effectively replicate and produce new progeny virus particles. Following viral infection, diverse cell signaling pathways are initiated by the cells, with the major goal of establishing an antiviral state. However, viruses have been shown to exploit cellular signaling pathways for their own effective replication. Genome-wide siRNA screens have also identified numerous host factors that either support (proviral) or inhibit (antiviral) virus replication. Some of the host factors might be dispensable for the host but may be critical for virus replication; therefore such cellular factors may serve as targets for development of antiviral therapeutics. Mitogen activated protein kinase (MAPK) is a major cell signaling pathway that is known to be activated by diverse group of viruses. MAPK interacting kinase 1 (MNK1) has been shown to regulate both cap-dependent and internal ribosomal entry sites (IRES)-mediated mRNA translation. In this review we have discuss the role of MAPK in virus replication, particularly the role of MNK1 in replication and translation of viral genome.

Temporal variations in patterns of Escherichia coli strain diversity and antimicrobial resistance in the migrant Egyptian vulture.

Aims: Multiple antimicrobial resistance in Escherichia coli of wild vertebrates is a global concern with scarce assessments on the subject from developing countries that have high human-wild species interactions. We studied the ecology of E. coli in a wintering population of Egyptian Vultures in India to understand temporal changes in both E. coli strains and patterns of antimicrobial resistance. Methods and Results: We ribotyped E. coli strains and assessed antimicrobial resistance from wintering vultures at a highly synanthropic carcass dump in north-west India. Both E. coli occurence (90.32%) and resistance to multiple antimicrobials (71.43%) were very high. Clear temporal patterns were apparent. Diversity of strains changed and homogenized at the end of the Vultures' wintering period, while the resistance pattern showed significantly difference inter-annually, as well as between arrival and departing individuals within a wintering cycle. Significance of study: The carcass dump environment altered both E. coli strains and multiple antimicrobial resistance in migratory Egyptian Vultures within a season. Long-distance migratory species could therefore disseminate resistant E. coli strains across broad geographical scales rendering regional mitigation strategies to control multiple antimicrobial resistance in bacteria ineffective.

Typing of Campylobacter jejuni isolated from poultry on the basis of flaA-RFLP by various restriction enzymes.

RFLP analysis of the flagellin (flaA) gene was compared using three different restriction endonucleases i.e DdeI, HinfI and DpnII to determine the genetic diversity among 43 Campylobacter jejuni isolates of poultry origin from the same geographical area. flaA gene was amplified in all the isolates and RFLP analysis showed variations. Dde-based RFLP was found most efficient in discriminating C. jejuni isolates by generating 15 different Dde-RFLP patterns with discriminatory index (D.I) of 0.9258 whereas DpnII produced seven Dpn-RFLP patterns (D.I .= 0.8427). While HinfI enzyme produced only six Hinf-RFLP patterns (D.I .= 0.6977). The discrimination of Dpn-RFLP was comparable to discrimination given by Dde-RFLP analysis, which is generally used to study flaA gene RFLP.

Identification and in silico analysis of cattle DExH/D box RNA helicases.

The helicases are motor proteins participating in a range of nucleic acid metabolisms. RNA helicase families are characterized by the presence of conserved motifs. This article reports a comprehensive in silico analysis of Bos taurus DExH/D helicase members. Bovine helicases were identified using the helicase domain sequences including 38 DDX (DEAD box) and 16 DHX (DEAH box) members. Signature motifs were used for the validation of these proteins. Putative sub cellular localization and phylogenetic relationship for these RNA helicases were established. Comparative analysis of these proteins with human DDX and DHX members was carried out. These bovine helicase have been assigned putative physiological functions. Present study of cattle DExH/D helicase will provides an invaluable source for the detailed biochemical and physiological research on these members.

Isolation, serotype diversity and antibiogram of Salmonella enterica isolated from different species of poultry in India

Objective:To study the occurrence and serotype diversity of Salmonella isolates in different species of poultry (chicken, emu and duck) and determine their resistance pattern against various antibiotics of different classes. Methods:About 507 samples comprising 202 caecal contents and 305 fecal samples from chicken, emu and duck were processed for isolation of Salmonella enterica. Salmonellae were isolated and detected by standard protocol of ISO 6579 Amendment 1:Annex D. Genetic confirmation was also made by using 16S rRNA genus specific PCR. Serotype specific PCR was also done to detect the most common serovars viz. Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Gallinarum. All obtained isolates were subjected to a set of 25 antibiotics to study their antibiogram by using Baeur-Kirby disk diffusion method. Results:Out of 507 samples processed, 32 isolates of Salmonella enterica (18 from caecal contents and 14 from faecal samples) were obtained, of which 24 belonged to 6 different serovars, 6 were untypeable and 2 were rough strains. Salmonella Enteritidis was the most predominant serotype (9), followed by Salmonella Typhimurium (5), Salmonella Virchow (4), Salmonella Gallinarum (3), Salmonella Reading (2) and Salmonella Altona (1). Antibiotic resistance pattern was maximum (100%) to oxacillin, penicillin and clindamycin, followed by ampicillin (68.75%), tetracycline (65.62%), nalidixic acid (56.25%) and colistin (46.87%). High sensitivity of isolates was recorded for chloramphenicol (96.87%) followed by meropenem (84.37%). Conclusions:Occurrence of high proportion of serovars in our study which can cause serious gastroenteritis in humans is a matter of concern. Salmonella Altona has been detected for the first time in India from poultry. This serotype is known to cause serious outbreaks of gastroenteritis in humans. Multidrug resistant isolates were recovered at high percentage which can be attributed to non-judicious use of antibiotics both in prophylaxis and treatment regimen. This observation draws serious attention as poultry serves as an important source of transmission of these multidrug resistant Salmonella serovars to humans.

Peste des petits ruminants virus infection of small ruminants: a comprehensive review.

Peste des petits ruminants (PPR) is caused by a Morbillivirus that belongs to the family Paramyxoviridae. PPR is an acute, highly contagious and fatal disease primarily affecting goats and sheep, whereas cattle undergo sub-clinical infection. With morbidity and mortality rates that can be as high as 90%, PPR is classified as an OIE (Office International des Epizooties)-listed disease. Considering the importance of sheep and goats in the livelihood of the poor and marginal farmers in Africa and South Asia, PPR is an important concern for food security and poverty alleviation. PPR virus (PPRV) and rinderpest virus (RPV) are closely related Morbilliviruses. Rinderpest has been globally eradicated by mass vaccination. Though a live attenuated vaccine is available against PPR for immunoprophylaxis, due to its instability in subtropical climate (thermo-sensitivity), unavailability of required doses and insufficient coverage (herd immunity), the disease control program has not been a great success. Further, emerging evidence of poor cross neutralization between vaccine strain and PPRV strains currently circulating in the field has raised concerns about the protective efficacy of the existing PPR vaccines. This review summarizes the recent advancement in PPRV replication, its pathogenesis, immune response to vaccine and disease control. Attempts have also been made to highlight the current trends in understanding the host susceptibility and resistance to PPR.

Comparative efficacy of ethanol and isopropanol against feline calicivirus, a norovirus surrogate.

BACKGROUND: Improper disinfection of environmental surfaces contaminated by the feces or vomitus of infected patients is believed to be a major cause of the spread of noroviruses (NoV) in close institutional settings. Although several disinfectants are available, the search for safe and effective disinfectant continues. Because alcohol and alcohol-based products have been used as antiseptics and their efficacy against several enveloped viruses has been documented, we wanted to determine their efficacy against nonenveloped calicivirus. METHODS: Feline calicivirus (FCV) was used as a surrogate for NoVs, using the carrier test. We evaluated the virucidal efficacy of various concentrations of ethanol and isopropanol against FCV, dried on an inanimate, nonporous contact surface for contact times of 1, 3, and 10 minutes. The virus was eluted after alcohol treatment and titrated in feline kidney cells. Percentage virus inactivation was calculated by comparing these titers with those obtained with virus eluted from controls. RESULTS: Ethanol at 70% and 90% and isopropanol at 40% to 60% concentrations were found to be the most effective, killing 99% of FCV within a short contact time of 1 minute. CONCLUSION: Isopropanol was more efficacious than ethanol at 40% to 60% concentrations, suggesting that the use of an appropriate concentration of isopropanol or ethanol would help in controlling the transmission of NoVs from environmental contact surfaces.

Survival on uncommon fomites of feline calicivirus, a surrogate of noroviruses.

BACKGROUND: Norovirus (NoV) transmission occurs mainly through food and fomites. Contaminated human fingers can transfer the virus to inanimate objects, which may then spread the virus to susceptible persons. However, no information is available on the survival of NoVs on fomites, which may be of importance in the transmission of NoVs in institutional settings such as hospitals and nursing homes. METHODS: In the absence of any in vitro cultivation system for NoVs, feline calicivirus (FCV) was used as a surrogate. Several fomites such as computer mouse, keyboard keys, telephone wire, telephone receiver, telephone buttons, and brass disks representing faucets and door handle surfaces were artificially contaminated with known amounts of FCV. Samples were taken at regular time intervals, and virus was titrated in feline kidney cells to determine its survival on these surfaces. RESULTS: Survivability of FCV varied with fomite type. The virus survived for up to 3 days on telephone buttons and receivers, for 1 or 2 days on computer mouse, and for 8 to 12 hours on keyboard keys and brass. The time for 90% virus reduction was <4 hours on computer keys, mouse, brass, and telephone wire; 4 to 8 hours on telephone receiver; and 12 to 24 hours on telephone buttons. CONCLUSION: The results of this study confirm that FCV (and perhaps NoV) can survive on fomites such as computers, telephones, and faucets and may be transmitted to humans using these contaminated materials. This may necessitate regular cleaning or disinfection of these items, especially in hospitals and nursing homes and after known outbreaks of NoVs.

Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.

Effect of temperature on the survival of F-specific RNA coliphage, feline calicivirus, and Escherichia coli in chlorinated water.

We compared the survival of F-specific RNA coliphage MS2, feline calicivirus, and E. coli in normal tap water and in tap water treated to an initial concentration of 50 ppm free chlorine and held at 4 degrees C, 25 degrees C, or 37 degrees C for up to 28 days. Our aim was to determine which of these two organisms (coliphage or E. coli) was better at indicating norovirus survival under the conditions of the experiment. There was a relatively rapid decline of FCV and E. coli in 50 ppm chlorine treated water and both organisms were undetectable within one day irrespective of the temperature. In contrast, FRNA phage survived for 7 to 14 days in 50 ppm chlorine treated water at all temperatures. All organisms survived for 28 days in tap water at 4 degrees C, but FCV was undetectable on day 21 and day 7 at 25 degrees C and 37 degrees C, respectively. Greater survival of FRNA phage compared to E. coli in 50 ppm chlorine treated water suggests that these organisms should be further investigated as indicators of norovirus in depurated shellfish, sanitized produce, and treated wastewater which are all subject to high-level chlorine treatment.

The effect of pooling sera on the detection of avian pneumovirus antibodies using an enzyme-linked immunosorbent assay test.

Pooling of samples is a cost-effective approach to estimate disease prevalence and to identify infected individuals. The objective of this study was to evaluate the use of serum pools for the detection of avian pneumovirus infection in turkey flocks by enzyme-linked immunosorbent assay, so that a minimum number of tests can be performed without compromising the sensitivity and specificity of the test. A total of 900 field samples were tested; 20 samples from each of 45 flocks. All samples were tested individually followed by pool testing in groups of 3, 4, 5, and 7 samples each. The number of positive pools for a given pool size was positively associated with the number of positive samples. In a separate experiment, the effect of dilution was examined by pooling 1 positive sample with different numbers of negative samples to form pools of sizes 2-7. These laboratory results were analyzed and integrated into a simulation model aimed at evaluating cost-efficient testing procedures. The probability of detecting an infected flock depended on prevalence of infection, size of serum pool, and the cutoff value used for optical density difference. At a theoretical prevalence of 20%, the probability of detecting an infected flock was 0.93 and 0.86 for a pool of 2 and 7, respectively. The probability of detecting positive flocks increased with increased prevalence and decreased cutoff. Pooling of samples represented a significant reduction in the cost of testing, suggesting that pooling is more advantageous and cost effective than testing individual samples.

Occurrence of Escherichia coli, noroviruses, and F-specific coliphages in fresh market-ready produce.

Forty samples of fresh produce collected from retail food establishments were examined to determine the occurrence of Escherichia coli, F-specific coliphages, and noroviruses. An additional six samples were collected from a restaurant undergoing investigation for a norovirus outbreak. Nineteen (48%) of the retail samples and all outbreak samples were preprocessed (cut, shredded, chopped, or peeled) at or before the point of purchase. Reverse transcription-PCR, with the use of primers JV 12 and JV 13, failed to detect norovirus RNA in any of the samples. All six outbreak samples and 13 (33%) retail samples were positive for F-specific coliphages (odds ratio undefined, P = 0.003). Processed retail samples appeared more likely to contain F-specific coliphages than unprocessed samples (odds ratio 3.8; 95% confidence interval 0.8 to 20.0). Only two (5.0%) retail samples were positive for E. coli; outbreak samples were not tested for E. coli. The results of this preliminary survey suggest that F-specific coliphages could be useful conservative indicators of fecal contamination of produce and its associated virological risks. Large-scale surveys should be conducted to confirm these findings.