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Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.
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Aedes , Infecções por Alphavirus , Alphavirus , Arbovírus , Vírus Chikungunya , Animais , Camundongos , Humanos , Aedes/genética , Alphavirus/genética , Vírus Chikungunya/genética , Mosquitos Vetores/genética , Glicoproteínas , Imunoglobulinas , Proteínas de MembranaRESUMO
Obesity has been identified as an independent risk factor for severe outcomes in humans with coronavirus disease 2019 (COVID-19) and other infectious diseases. Here, we established a mouse model of COVID-19 using the murine betacoronavirus, mouse hepatitis virus 1 (MHV-1). C57BL/6 and C3H/HeJ mice exposed to MHV-1 developed mild and severe disease, respectively. Obese C57BL/6 mice developed clinical manifestations similar to those of lean controls. In contrast, all obese C3H/HeJ mice succumbed by 8 days postinfection, compared to a 50% mortality rate in lean controls. Notably, both lean and obese C3H/HeJ mice exposed to MHV-1 developed lung lesions consistent with severe human COVID-19, with marked evidence of diffuse alveolar damage (DAD). To identify early predictive biomarkers of worsened disease outcomes in obese C3H/HeJ mice, we sequenced RNA from whole blood 2 days postinfection and assessed changes in gene and pathway expression. Many pathways uniquely altered in obese C3H/HeJ mice postinfection aligned with those found in humans with severe COVID-19. Furthermore, we observed altered gene expression related to the unfolded protein response and lipid metabolism in infected obese mice compared to their lean counterparts, suggesting a role in the severity of disease outcomes. This study presents a novel model for studying COVID-19 and elucidating the mechanisms underlying severe disease outcomes in obese and other hosts.
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COVID-19 , Vírus da Hepatite Murina , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos C3H , Vírus da Hepatite Murina/genética , COVID-19/complicações , Obesidade/complicações , Perfilação da Expressão GênicaRESUMO
Chikungunya virus (CHIKV) is an alphavirus that has re-emerged globally over the last two decades and has the potential to become endemic in the United States due to the presence of competent mosquito vectors, Aedes aegypti and Aedes albopictus. CHIK disease is characterised by fever, rash, and joint pain, and causes chronic debilitating joint pain and swelling in >50% of infected individuals. Given the disease severity caused by CHIKV and the global presence of vectors to facilitate its spread, strategies to reduce viral transmission are desperately needed; however, the human biological factors driving CHIKV transmission are poorly understood. Towards that end, we have previously shown that mosquitoes fed on alphavirus-infected obese mice have reduced infection and transmission rates compared to those fed on infected lean mice despite similar viremia in lean and obese mice. One of the many host factors that increase in obese hosts is insulin, which was previously shown to impact the infection of mosquitoes by several flaviviruses. However, insulin's impact on alphavirus infection of live mosquitoes is unknown and whether insulin influences mosquito-borne virus transmission has not been tested. To test this, we exposed A. aegypti mosquitoes to bloodmeals with CHIKV in the presence or absence of physiologically relevant levels of insulin and found that insulin significantly lowered both infection and transmission rates. RNA sequencing analysis on mosquito midguts isolated at 1-day-post-infectious-bloodmeal (dpbm) showed enrichment in genes in the Toll immune pathway in the presence of insulin, which was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We then sought to determine if the Toll pathway plays a role in CHIKV infection of Ae. aegypti mosquitoes; therefore, we knocked down Myd88, a critical immune adaptor molecule for the Toll pathway, in live mosquitoes, and found increased CHIKV infection compared to the mock knockdown control group. Overall, these data demonstrate that insulin reduces CHIKV transmission by Ae. aegypti and activates the Toll pathway in mosquitoes, suggesting that conditions resulting in higher serum insulin concentrations may reduce alphavirus transmission. Finally, these studies suggest that strategies to activate insulin or Toll signalling in mosquitoes may be an effective control strategy against medically relevant alphaviruses.
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Aedes , Vírus Chikungunya , Animais , Humanos , Camundongos , Vírus Chikungunya/genética , Aedes/fisiologia , Insulina , Camundongos Obesos , ArtralgiaRESUMO
Climate change is one of the biggest challenges of the twenty-first century for sustainable agricultural production. Several reports highlighted the need for better agricultural practices and use of eco-friendly methods for sustainable crop production under such situations. In this context, Trichoderma species could be a model fungus to sustain crop productivity. Currently, these are widely used as inoculants for biocontrol, biofertilization, and phytostimulation. They are reported to improve photosynthetic efficiency, enhance nutrient uptake and increase nitrogen use efficiency in crops. Moreover, they can be used to produce bio-energy, facilitate plants for adaptation and mitigate adverse effect of climate change. The technological advancement in high throughput DNA sequencing and biotechnology provided deep insight into the complex and diverse biotic interactions established in nature by Trichoderma spp. and efforts are being made to translate this knowledge to enhance crop growth, resistance to disease and tolerance to abiotic stresses under field conditions. The discovery of several traits and genes that are involved in the beneficial effects of Trichoderma spp. has resulted in better understanding of the performance of bioinoculants in the field, and will lead to more efficient use of these strains and possibly to their improvement by genetic modification. The present mini-review is an effort to elucidate the molecular basis of plant growth promotion and defence activation by Trichoderma spp. to garner broad perspectives regarding their functioning and applicability for climate resilient agriculture.
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Agricultura/métodos , Mudança Climática , Produtos Agrícolas , Trichoderma/fisiologia , Adaptação Fisiológica , Biotecnologia/métodos , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/imunologia , Fertilizantes , Alimentos , Nitrogênio , Desenvolvimento Vegetal , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Solo/química , Microbiologia do Solo , Estresse Fisiológico , Trichoderma/classificação , Trichoderma/genética , Trichoderma/patogenicidadeRESUMO
Alphaviruses are vector-borne, medically relevant, positive-stranded RNA viruses that cause disease in animals and humans worldwide. Of this group, chikungunya virus (CHIKV) is the most significant human pathogen, responsible for generating millions of infections leading to severe febrile illness and debilitating chronic joint pain. Currently, there are limited treatments to protect against alphavirus disease; thus, there is a tremendous need to generate safe and effective vaccines. Live-attenuated vaccines (LAVs) are cost-effective and potent immunization strategies capable of generating long-term protection in a single dose. However, LAVs often produce systemic viral replication, which can lead to unwanted post-vaccination side effects and pose a risk of reversion to a pathogenic phenotype and transmission to mosquitoes. Here, we utilized a chimeric infectious clone of CHIKV engineered with the domain C of the E2 gene of Semliki Forest virus (SFV) to express IFNγ and IL-21-two potent antiviral and immunomodulatory cytokines-in order to improve the LAV's attenuation while maintaining immunogenicity. The IFNγ- and IL-21-expressing vaccine candidates were stable during passage and significantly attenuated post-vaccination, as mice experienced reduced footpad swelling with minimal systemic replication and dissemination capacity compared to the parental vaccine. Additionally, these candidates provided complete protection to mice challenged with WT CHIKV. Our dual attenuation strategy represents an innovative way to generate safe and effective alphavirus vaccines that could be applied to other viruses.
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Pervasive SARS-CoV-2 infections in humans have led to multiple transmission events to animals. While SARS-CoV-2 has a potential broad wildlife host range, most documented infections have been in captive animals and a single wildlife species, the white-tailed deer. The full extent of SARS-CoV-2 exposure among wildlife communities and the factors that influence wildlife transmission risk remain unknown. We sampled 23 species of wildlife for SARS-CoV-2 and examined the effects of urbanization and human use on seropositivity. Here, we document positive detections of SARS-CoV-2 RNA in six species, including the deer mouse, Virginia opossum, raccoon, groundhog, Eastern cottontail, and Eastern red bat between May 2022-September 2023 across Virginia and Washington, D.C., USA. In addition, we found that sites with high human activity had three times higher seroprevalence than low human-use areas. We obtained SARS-CoV-2 genomic sequences from nine individuals of six species which were assigned to seven Pango lineages of the Omicron variant. The close match to variants circulating in humans at the time suggests at least seven recent human-to-animal transmission events. Our data support that exposure to SARS-CoV-2 has been widespread in wildlife communities and suggests that areas with high human activity may serve as points of contact for cross-species transmission.
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Animais Selvagens , COVID-19 , SARS-CoV-2 , Animais , COVID-19/transmissão , COVID-19/epidemiologia , COVID-19/virologia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Animais Selvagens/virologia , Humanos , Estudos Soroepidemiológicos , Filogenia , Quirópteros/virologia , Virginia/epidemiologia , Guaxinins/virologia , District of Columbia/epidemiologia , Cervos/virologia , Genoma Viral , Urbanização , Anticorpos Antivirais/sangue , RNA Viral/genéticaRESUMO
Single-cell RNA sequencing (scRNA-seq) requires the preparation of a highly viable single-cell suspension to get reliable sequencing results. Here, we present a protocol for isolating mouse footpad leukocytes while maintaining high viability. We describe steps for footpad collection, enzymatic tissue dissociation, leukocyte isolation and purification, and cell fixation and preservation. We then detail combinatorial barcoding, library preparation, scRNA-seq, and data analysis. Cells can be used to generate a complete molecular atlas at the single cell level.
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Leucócitos , Animais , Camundongos , Citometria de Fluxo/métodos , Separação Celular/métodos , Análise de Sequência de RNA/métodosRESUMO
Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.
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Mahamrutyunjaya rasa (MHR), an Ayurvedic formulation, used as cardiotonic, contains potentially toxic compounds like aconitine, which are detoxified during preparation using traditional methods. Comparative toxicological evaluation of laboratory prepared formulation (F1) and two marketed formulations (F2 and F3) were performed based on their effects on viability of H9c2 cells and after single oral dose administration in mice. Cardioprotective effect of formulations at 25 and 50 mg/kg doses were studied in isoproterenol- (ISO-) induced myocardial infarcted rats. F1 and F2 did not affect the cell viability, while F3 decreased the cell viability in concentration and time-dependent manner. Rats administered with ISO showed significant increase in the serum levels of glutamate oxaloacetate transaminase, alkaline phosphotase, creatinine kinase isoenzymes, lactate dehydrogenase, and uric acid, while F1 and F2 treatment showed significant reduction in the same. F3 showed further increase in the serum levels of enzymes and uric acid in ISO-challenged rats. High pressure liquid chromatographic analysis of formulations showed higher concentration of aconitine in F3. Study shows that F1 and F2 possess cardioprotective property with higher safety, while formulation F3 cannot be used as cardioprotective due to its cytotoxic effects. Thus, proper quality assessment methods are required during preparation of traditional formulations.
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SARS-CoV-2 emerged in 2019 as a devastating viral pathogen with no available preventative or treatment to control what led to the current global pandemic. The continued spread of the virus and increasing death toll necessitate the development of effective antiviral treatments to combat this virus. To this end, we evaluated a new class of organometallic complexes as potential antivirals. Our findings demonstrate that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(1,3-dicyclohexylimidazol-2-ylidene)Cl2 (complex 2) and Cp*Rh(dipivaloylmethanato)Cl (complex 4), have direct virucidal activity against SARS-CoV-2. Subsequent in vitro testing suggests that complex 4 is the more stable and effective complex and demonstrates that both 2 and 4 have low toxicity in Vero E6 and Calu-3 cells. The results presented here highlight the potential application of organometallic complexes as antivirals and support further investigation into their activity.
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Antivirais/farmacologia , Compostos Organometálicos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Compostos Organometálicos/química , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.
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COVID-19/epidemiologia , Modelos Animais de Doenças , Obesidade/epidemiologia , Animais , Chlorocebus aethiops , Comorbidade , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morbidade , Células VeroRESUMO
Usutu virus (USUV; Flavivirus) has caused massive die-offs in birds across Europe since the 1950s. Although rare, severe neurologic disease in humans has been reported. USUV is genetically related to West Nile virus (WNV) and shares an ecological niche, suggesting it could spread from Europe to the Americas. USUV's risk of transmission within the United States is currently unknown. To this end, we exposed field-caught Aedes japonicus, Culex pipiens pipiens, and Culex restuans-competent vectors for WNV-to a recent European isolate of USUV. While infection rates for each species varied from 7%-21%, no dissemination or transmission was observed. These results differed from a 2018 report by Cook and colleagues, who found high dissemination rates and evidence of transmission potential using a different USUV strain, U.S. mosquito populations, temperature, and extrinsic incubation period. Future studies should evaluate the impact of these experimental conditions on USUV transmission by North American mosquitoes.
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Aedes/virologia , Culex/virologia , Infecções por Flavivirus , Flavivirus/patogenicidade , Mosquitos Vetores/virologia , Animais , Aves/virologia , Infecções por Flavivirus/transmissão , Infecções por Flavivirus/virologia , HumanosRESUMO
Usutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. Here, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in mammalian and insect cells. Additionally, we observed similar levels of replication and disease in two mouse models. These clones will aid the study of USUV infection, transmission, diagnostics, and vaccines.
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Infecções por Flavivirus/virologia , Flavivirus/genética , Flavivirus/fisiologia , Animais , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Flavivirus/classificação , Flavivirus/patogenicidade , Humanos , Masculino , Camundongos , Mutação , Filogenia , Viremia , Replicação ViralRESUMO
Rhizoctonia solani is one of the most devastating pathogens. R. solani AG-1 IA causes sheath blight in rice, maize, and other Gramineous plants. Accurate identification is essential for the effective management of this pathogen. In the present study, a set of four primers were designed viz. RSPG1, RSPG2, RSPG4, and RSPG5 for polygalacturonase (PG) gene, an important virulence factor in phytopathogenic fungi. All four primer sets showed specific amplification of 300 bp (RSPG1F/R), 375 bp (RSPG2F/R), 500 bp (RSPG4F/R) and 336 bp (RSPG5F/R) amplicons. q-PCR detection using each primer sets could detect up to 10 pg of DNA. We also designed six primers (RS_pg_F3_1/RS_pg_B3_1, RS_pg_FIP_1.1/RS-pg_BIP_1.1, and RS_pg_LF_1/RS_pg_LB_1) for PG gene. Further, a colorimetric LAMP assay developed yielded visual confirmation of the pathogen within 45 min of sample collection when coupled with rapid high throughput template preparation method (rHTTP) from infected samples. The sensitivity of the LAMP assay was as low as 1.65 fg/µl of template DNA and could effectively detect R. solani AG-1 IA from diseased plant tissues and soil samples. The LAMP assay was highly specific for R. solani as it did not show any amplification with other AG groups of R. solani and closely related fungal and bacterial outgroups. This study will help in designing an effective point of care diagnostic method for early monitoring of R. solani and thereby planning timely preventive measures against the pathogen.
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Colorimetria , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Oryza/microbiologia , Doenças das Plantas/microbiologia , Rhizoctonia/isolamento & purificação , Biomarcadores/metabolismo , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase , Padrões de Referência , Reprodutibilidade dos Testes , SoloRESUMO
Simple and rapid stability-indicating HPLC methods were developed for the individual analysis of aconitine (ACN) and piperine (PIN) in Mahamrutynjaya rasa, an herbal dosage form containing Aconitum ferox, Piper nigrum, and Piper longum in combination. Separation of the ACN from its major and minor degradation products was successfully achieved on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size), with isocratic elution using a mixture of acetonitrile-KH2PO4 buffer (10 mM, pH 8 +/- 0.1; 50 + 50, v/v) at flow rate of 0.7 mL/min with UV detection at 227 nm. PIN separation was performed on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size), with isocratic elution in acetonitrile-KH2PO4 buffer (10 mM, pH 7 +/- 0.1; 35 + 65, v/v) at a flow rate of 1 mL/min with UV detection at 343 nm. The methods were validated with respect to linearity, precision, accuracy, specificity, system suitability, and robustness. The responses were linear in the drug concentration range of 10-100 microg/mL for both ACN and PIN. The percent recoveries of both the markers from a mixture of degradation products were in the range between 98-101%. The utility of the procedures was verified by their application to marketed formulations that were subjected to accelerated degradation studies. The methods could distinctly separate the drug and degradation products. The products formed in the marketed tablets were similar to those formed in the laboratory during stress studies.
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Aconitina/análise , Alcaloides/análise , Benzodioxóis/análise , Piperidinas/análise , Preparações de Plantas/análise , Alcamidas Poli-Insaturadas/análise , Calibragem , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Combinação de Medicamentos , Estabilidade de Medicamentos , Temperatura Alta , Umidade , Concentração de Íons de Hidrogênio , Luz , Modelos Lineares , Oxirredução , Padrões de Referência , Reprodutibilidade dos Testes , Soluções , Comprimidos , Água/químicaRESUMO
Background: Mahamrutyunjaya rasa is an ayurvedic formulation used in the treatment of cardiac disorders. It contains the purified roots of Visa (Aconitum ferox), Brihati (Solanum indicum), fruits of Pippali Kana (Piper longum), Marica (Piper nigrum), Gandhaka (Sulfur), Hingula (Cinnabar) and Tankana (Sodium metaborate) as per Bhaishajya Ratnavali. The purification (shodhana) process changes the physiochemical properties of the raw materials which need to be studied and understood. Objective: The present work aims to perform a comprehensive physicochemical characterization of raw materials, intermediates and the final product obtained during purification, using modern analytical techniques. Materials and methods: The standard methods as per traditional text were followed and the physicochemical changes were also investigated by collecting samples at different steps of purification. The samples were analysed using various techniques, viz. Fourier transform infra-red spectroscopic (FTIR), X-ray diffraction (XRD), Differential Scanning Calorimeter (DSC) and High Performance thin Layer chromatography (HPTLC). Results: The FTIR and HPTLC analysis of the alkaloidal extracts of Visa showed loss of an ester group with shift in the peaks from 1720 cm-1 (C[bond, double bond]O stretching of esters) to 1676 cm-1 (C[bond, double bond]O stretching of Ketone) which signifies the conversion of alkaloid Aconitine (LD50 - 0.08 mg/kg) to Benzoylaconine (LD50 - 24 mg/kg) improving its safety. The analysis of gandhaka by XRD and DSC showed that purification brought about transformation of orthorhombic sulphur into monoclinic sulphur and it reverted back to original form with higher purity. The treatments given to gandhaka and hingula with organic compounds made them homologous to the body tissues. Analysis of purified tankana showed that the processing led to loss of water and slight change in the crystal structure with the shift in the endothermic peak from 110.6 °C to 104.2 °C. Conclusion: Thus, the present study provides a scientific backing to the methodologies used by Ayurvedic practitioners. The study also provides physicochemical fingerprints for the standardization as well as characterization of raw materials and forms a technical platform for manufacturers to develop quality control standards.
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The study describes development and subsequent validation of a stability indicating reverse-phase high-performance liquid chromatography method for the simultaneous estimation of clidinium bromide (CLI) and chlordiazepoxide (CHLOR) from their combination drug product. Chromatographic separations are performed at ambient temperature on a Phenomenex Luna C(18) (250 mm x 4.6 mm, i.d., 5 microm) column using a mobile phase consisting of potassium dihydrogen phosphate buffer (0.05 M, pH 4.0 adjusted with 0.5% orthophosphoric acid)-methanol- acetonitrile (40:40:20, v/v/v). The flow rate is 1.0 mL/min, and the detection wavelength is 220 nm. The method is validated with respect to linearity, precision, accuracy, system suitability, and robustness. The utility of the procedure is verified by its application to marketed formulations that were subjected to accelerated degradation studies. The method distinctly separated the drug and degradation products even in actual samples. The products formed in marketed tablet dosage forms are similar to those formed during stress studies.