IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans.

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.

Evidences support that dengue virus can impart broad-spectrum immunity against betacoronaviruses in dengue endemic regions.

COVID-19 tended to be less aggressive in dengue endemic regions. Conversely, dengue cases plummeted in dengue endemic zones during the active years of the pandemic (2020-2021). We and others have demonstrated serological cross-reactivity between these two viruses of different families. We further demonstrated that COVID-19 serum samples that were cross-reactive in dengue virus (DV) serological tests, "cross-neutralized" all DV serotypes in Huh7 cells. Here we showed by co-immunoprecipitation (Co-IP) and atomic force microscopy (AFM) imaging that severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 (SARS-CoV-2) spike (S) protein subunit S1 and S2 monoclonal antibodies can indeed, bind to DV particles. Likewise, DV envelope antibodies (DV E Abs) showed high docking frequency with other human pathogenic beta-CoVs and murine hepatitis virus-1 (MHV-1). SARS-CoV-2 Ab didn't show docking or Co-IP with MHV-1 supporting poor cross-protection among CoVs. DV E Abs showed binding to MHV-1 (AFM, Co-IP, and immunofluorescence) and prepandemic dengue patients' serum samples even "cross-neutralized" MHV-1 plaques in cell culture. Furthermore, dengue serum samples showed marked inhibition potential in a surrogate virus-based competitive enzyme-linked immunosorbent assay, used for determining neutralizing Abs against SARS-CoV-2 S protein receptor-binding domain in COVID-19 serum samples. We therefore, provide multiple evidence as to why CoVs are epidemiologically less prevalent in highly dengue endemic regions globally.

Cross-reactivity of SARS-CoV-2 with other pathogens, especially dengue virus: A historical perspective.

Dengue is a vector-borne viral disease caused by a Flavivirus whereas the COVID-19 pandemic was caused by a highly contagious virus, SARS-CoV-2 belonging to the family Coronaviridae. However, COVID-19 severity was observably less in dengue-endemic countries and vice versa especially during the active years of the pandemic (2019-2021). We observed that dengue virus (DENV) antibodies (Abs) could cross-react with SARS-CoV-2 spike antigen. This resulted in SARS-CoV-2 false positivity by rapid Ab test kits. DENV Abs binding to SARS-CoV-2 receptor-binding domain (and the reverse scenario), as revealed by docking studies further validated DENV and SARS-CoV-2 cross-reactivity. Finally, SARS-CoV-2 Abs were found to cross-neutralize DENV1 and DENV2 in virus neutralization test (VNT). Abs to other pathogens like Plasmodium were also cross-reactive but non-neutralizing for SARS-CoV-2. Here, we analyze the existing data on SARS-CoV-2 cross-reactivity with other pathogens, especially dengue to assess its impact on health (cross-protection?) and differential sero-diagnosis/surveillance.

Non-structural protein 1 (NS1) variants from dengue virus clinical samples revealed mutations that influence NS1 production and secretion.

Dengue diagnosis primarily relies on NS1 ELISA and serological (IgG/IgM) tests. There are reports of low and variable sensitivity of the widely used NS1 ELISA tests. Poor sensitivity has been attributed to patient's infection status, prevalent serotypes, and the geographical origin of the samples. We investigated whether NS1 mutations directly have any impact on NS1 ELISA-based dengue virus (DENV) detection in clinical samples. Fifty-eight serum samples were collected from dengue-endemic area during 2015-2017 and tested with three commonly used NS1 ELISA kits. The samples were subjected to diagnostic RT-PCR and sequencing of structural gene(s). Sequencing of NS1 gene revealed amino acid changes which were transferred to respective wild type NS1 backbone to determine their effects on NS1 production and secretion in Huh-7, Vero, and A549 cells. Eighty-seven percent samples were virus RNA-positive but 65% of these were NS1 ELISA-positive. NS1-gene mutations like Val236➔Ala (DENV2) or Trp68➔stop codon in DENV3 were associated with decreased NS1 production and secretion. These mutations were originally identified in NS1 ELISA-negative clinical isolates. All DENV1 and > 80% DENV2 were NS1 ELISA-positive. The three NS1 ELISA could not detect recently circulating DENV3 single infections despite being RNA-positive. Among serotypes 1-3, wild-type NS1 production was highest for DENV1 and lowest for DENV3 in all cell lines tested. Mutations in circulating DENV directly correlated with NS1 production and secretion and, hence, ELISA-based NS1 detection. Further studies to define more NS1 mutations in clinical samples are needed to optimize ELISA kits for more sensitive dengue diagnosis.

Glucogallin Attenuates the LPS-Induced Signaling in Macrophages and Protects Mice against Sepsis.

The anti-oxidant and anti-inflammatory effect of beta-glucogallin (BGG), a plant-derived natural product, was evaluated in both in vitro and in vivo studies. For the in vitro study, the ability of BGG pre-treatment to quench LPS-induced effects compared to LPS alone in macrophages was investigated. It was found that BGG pre-treatment showed a significant decrease in ROS, NO, superoxide, and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-17, IL-1ß, and IL-6) and increased reduced glutathione coupled with the restoration of mitochondrial membrane potential. Gene profiling and further validation by qPCR showed that BGG pre-treatment downregulated the LPS-induced expression of c-Fos, Fas, MMP-9, iNOS, COX-2, MyD88, TRIF, TRAF6, TRAM, c-JUN, and NF-κB. We observed that BGG pre-treatment reduced nuclear translocation of LPS-activated NF-κB and thus reduced the subsequent expressions of NLRP3 and IL-1ß, indicating the ability of BGG to inhibit inflammasome formation. Molecular docking studies showed that BGG could bind at the active site of TLR4. Finally, in the LPS-driven sepsis mouse model, we showed that pre-treatment with BGG sustained toxic shock, as evident from their 100% survival. Our study clearly showed the therapeutic potential of BGG in toxic shock syndrome.

In Vitro Anticancer Activity of Methanolic Extract of Justicia adhatoda Leaves with Special Emphasis on Human Breast Cancer Cell Line.

Natural products are being targeted as alternative anticancer agents due to their non-toxic and safe nature. The present study was conducted to explore the in vitro anticancer potential of Justicia adhatoda (J. adhatoda) leaf extract. The methanolic leaf extract was prepared, and the phytochemicals and antioxidant potential were determined by LCMS analysis and DPPH radical scavenging assay, respectively. A docking study performed with five major alkaloidal phytoconstituents showed that they had a good binding affinity towards the active site of NF-κB. Cell viability assay was carried out in five different cell lines, and the extract exhibited the highest cytotoxicity in MCF-7, a breast cancer cell line. Extract-treated cells showed a significant increase in nitric oxide and reactive oxygen species production. Cell cycle analysis showed an arrest in cell growth at the Sub-G0 phase. The extract successfully inhibited cell migration and colony formation and altered mitochondrial membrane potential. The activities of superoxide dismutase and glutathione were also found to decrease in a dose-dependent manner. The percentage of apoptotic cells was found to increase in a dose-dependent manner in MCF-7 cells. The expressions of caspase-3, Bax, and cleaved-PARP were increased in extract-treated cells. An increase in the expression of NF-κB was found in the cytoplasm in extract-treated cells. J. adhatoda leaf extract showed a potential anticancer effect in MCF-7 cells.

Leptomonas seymouri narna-like virus 1 and not leishmaniaviruses detected in kala-azar samples from India.

The great majority of kala-azar/visceral leishmaniasis (VL) cases, which are caused by Leishmania donovani (LD), are reported in Asia. We investigated whether leishmaniaviruses (LRVs) are present in LD isolates. These dsRNA viruses contribute to hyperpathogenicity, as observed in the case of other members of the genus Leishmania. However, LRVs could not be detected in 22 Indian LD isolates tested in the present study, while 70% of these original LD isolates harboured a virus that was not of LD but instead of Leptomonas seymouri (LS) origin. LS is another protozoon that parasitizes the sandfly vector of LD. Historically, LD clinical isolates from India often showed high incidence of LS coinfection. LS was detected in 20 out of the 22 (91%) above-mentioned LD isolates. Leptomonas seymouri narna-like virus 1 (Lepsey NLV1) was identified by whole-genome sequencing in an LD-LS coinfected sample, and its presence was confirmed by PCR and sequencing in 15 (75%) of the 20 LD-LS coinfected samples. The LS-negative LD samples were also virus negative by PCR. That the human host is exposed to an RNA virus in LS, another coinfecting parasite with LD, i.e., the "LD-LS-Lepsey NLV1 triple pathogen" phenomenon, unveils a new paradigm of research towards revisiting the mysteries of Indian leishmaniasis pathogenesis and management.

Pharmacokinetics-pharmacodynamics of the helicase-primase inhibitor pritelivir following treatment of wild-type or pritelivir-resistant virus infection in a murine herpes simplex virus 1 infection model.

Herpes simplex virus (HSV) infections can cause considerable morbidity. Transmission of HSV-2 has become a major health concern, since it has been shown to promote transmission of other sexually transmitted diseases. Pritelivir (AIC316, BAY 57-1293) belongs to a new class of HSV antiviral compounds, the helicase-primase inhibitors, which have a mode of action that is distinct from that of antiviral nucleoside analogues currently in clinical use. Analysis of pharmacokinetic-pharmacodynamic parameters is a useful tool for the selection of appropriate doses in clinical trials, especially for compounds belonging to new classes for which no or only limited data on therapeutic profiles are available. For this purpose, the effective dose of pritelivir was determined in a comprehensive mouse model of HSV infection. Corresponding plasma concentrations were measured, and exposures were compared with efficacious concentrations derived from cell cultures. The administration of pritelivir at 10 mg/kg of body weight once daily for 4 days completely suppressed any signs of HSV infection in the animals. Associated plasma concentrations adjusted for protein binding stayed above the cell culture 90% effective concentration (EC90) for HSV-1 for almost the entire dosing interval. Interestingly, by increasing the dose 6-fold and prolonging the treatment duration to 8 days, it was possible to treat mice infected with an approximately 30-fold pritelivir-resistant but fully pathogenic HSV-1 virus. Corresponding plasma concentrations exceeded the EC90 of this mutant for <8 h, indicating that even suboptimal exposure to pritelivir is sufficient to achieve antiviral efficacy, possibly augmented by other factors such as the immune system.

Murid herpesvirus-4 exploits dendritic cells to infect B cells.

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells.

Identification and validation of novel non-nucleoside class of molecules inhibiting the dengue virus replication.

There is currently no drug available to treat mosquito-borne dengue. The C-terminal RNA-dependent RNA polymerase (RdRp) domain in the non-structural type 5 (NS5) protein of the dengue virus (DENV) is essential for viral RNA synthesis and replication, and therefore, it is an attractive target for the anti-dengue drug development. We report herein the discovery and validation of two novel non-nucleoside classes of small molecules as DENV RdRp inhibitors. Firstly, using the refined X-ray structure of the DENV NS5 RdRp domain (PDB-ID: 4V0R), we conducted docking, binding free-energy studies, and short-scale molecular dynamics simulation to investigate the binding sites of known small molecules that led to the optimized protein-ligand complex. Subsequently, protein structure-based screening of a commercial database (∼500,000 synthetic compounds), pre-filtered for the drug-likeness, led to the top-ranked 171 molecules, which was then subjected to structural diversity analysis and clustering. This process led to six structurally distinct and best-scored compounds that were procured from the commercial vendor, and then subjected to the in vitro testing in the MTT and dengue infection assays. It revealed two unique and structurally unique compounds, KKR-D-02 and KKR-D-03, exhibiting 84 and 81% reductions, respectively, in DENV copy number in repeated assays in comparison to the virus-infected cell controls. These active compounds represent novel scaffolds for further structure-based discovery of novel candidate molecules for the intervention of dengue.Communicated by Ramaswamy H. Sarma.

Non-cytopathic herpes simplex virus type-1 isolated from acyclovir-treated patients with recurrent infections.

Herpes simplex virus (HSV) usually produces cytopathic effect (CPE) within 24-72 h post-infection (P.I.). Clinical isolates from recurrent HSV infections in patients on Acyclovir therapy were collected between 2016 and 2019 and tested in cell cultures for cytopathic effects and further in-depth characterization. Fourteen such isolates did not show any CPE in A549 or Vero cell lines even at 120 h P.I. However, these cultures remained positive for HSV-DNA after several passages. Sequence analysis revealed that the non-CPE isolates were all HSV-1. Analysis of the thymidine kinase gene from the isolates revealed several previously reported and two novel ACV-resistant mutations. Immunofluorescence and Western blot data revealed a low-level expression of the immediate early protein, ICP4. Late proteins like ICP5 or capsid protein, VP16 were almost undetectable in these isolates. AFM imaging revealed that the non-CPE viruses had structural deformities compared to wild-type HSV-1. Our findings suggest that these strains are manifesting an unusual phenomenon of being non-CPE herpesviruses with low level of virus protein expressions over several passages. Probably these HSV-1 isolates are evolving towards a more "cryptic" form to establish chronic infection in the host thereby unraveling yet another strategy of herpesviruses to evade the host immune system.

Monoclonal antibodies: a remedial approach to prevent SARS-CoV-2 infection.

SARS-CoV-2, the newly emerged virus of the Coronaviridae family is causing havoc worldwide. The novel coronavirus 2019 was first reported in Wuhan, China marked as the third highly infectious pathogenic virus of the twenty-first century. The typical manifestations of COVID-19 include cough, sore throat, fever, fatigue, loss of sense of taste and difficulties in breathing. Large numbers of SARS-CoV-2 infected patients have mild to moderate symptoms, however severe and life-threatening cases occur in about 5-10% of infections with an approximately 2% mortality rate. For the treatment of SARS-CoV-2, the use of neutralizing monoclonal antibodies (mAbs) could be one approach. The receptor binding domain (RBD) and N-terminal domain (NTD) situated on the peak of the spike protein (S-Protein) of SARS-CoV-2 are immunogenic in nature, therefore, can be targeted by neutralizing monoclonal antibodies. Several bioinformatics approaches highlight the identification of novel SARS-CoV-2 epitopes which can be targeted for the development of COVID-19 therapeutics. Here we present a summary of neutralizing mAbs isolated from COVID-19 infected patients which are anticipated to be a better therapeutic alternative against SARS-CoV-2. However, provided the vast escalation of the disease worldwide affecting people from all strata, affording expensive mAb therapy will not be feasible. Hence other strategies are also being employed to find suitable vaccine candidates and antivirals against SARS-CoV-2 that can be made easily available to the population.

COVID-19 serum can be cross-reactive and neutralizing against the dengue virus, as observed by the dengue virus neutralization test.

OBJECTIVES: Observing the serological cross-reactivity between SARS-CoV-2 and dengue virus (DV), we aimed to elucidate its effect on dengue serodiagnosis and infectivity in a highly dengue-endemic city in India. METHODS: A total of 52 COVID-19 (reverse transcription-polymerase chain reaction [RT-PCR] positive) serum samples were tested in rapid lateral flow immunoassays and DV immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) to detect DV or SARS-CoV-2 IgG/immunoglobulin M. The COVID-19 antibody (Ab) positive samples were subjected to a virus neutralization test (Huh7 cells) using DV type 1 (DV1) clinical isolate. RESULTS: Most (93%) of the SARS-CoV-2 Ab-positive serum samples cross-reacted with DV in rapid or ELISA tests. All were DV RNA and nonstructural protein 1 (NS1) antigen-negative. COVID-19 serum samples that were DV cross-reactive neutralized DV1. Of these, 57% had no evidence of DV pre-exposure (DV NS1 Ab-negative). The computational study also supported potential interactions between SARS-CoV-2 Ab and DV1. CONCLUSION: DV serodiagnosis will be inconclusive in areas co-endemic for both viruses. The COVID-19 pandemic appears to impart a protective response against DV in DV-endemic populations.

Detection of Leptomonas seymouri narna-like virus in serum samples of visceral leishmaniasis patients and its possible role in disease pathogenesis.

Kala-azar/Visceral Leishmaniasis (VL) caused by Leishmania donovani (LD) is often associated with Leptomonas seymouri (LS) co-infection in India. Leptomonas seymouri narna-like virus 1 (Lepsey NLV1) has been reported in multi-passaged laboratory isolates of VL samples which showed LD-LS co-infection. A pertinent question was whether this virus of LS is detectable in direct clinical samples. DNA from the serum of twenty-eight LD diagnosed patients was subjected to LD-specific and LS-specific PCR to reconfirm the presence of LD parasites and to detect LD-LS co-infections. RNA extracted from same samples was subjected to RT-PCR, qRT-PCR and sequencing using virus-specific primers to detect/identify and quantify the virus. The presence of the virus was confirmed in thirteen of eighteen (72%) recently collected VL and PKDL samples. Cytokine profiling showed significantly elevated IL-18 in only LD infected patients compared to the virus-positive LD and control samples. IL-18 is crucial for Th1 and macrophage activation which eventually clears the parasite. The Lepsey NLV1 interaction with the immune system results in reduced IL-18 which favors LD survival and increased parasitic burden. The study emphasizes the need to revisit LD pathogenesis in the light of the association and persistence of a protozoan virus in kala-azar and PKDL patients.

Virophages: association with human diseases and their predicted role as virus killers.

The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.

A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons.

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient's serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

Computational modelling supports that dengue virus envelope antibodies can bind to SARS-CoV-2 receptor binding sites: Is pre-exposure to dengue virus protective against COVID-19 severity?

The world is going through the scourge of the COVID-19 pandemic since January 2020. However, the pandemic appears to be less severe in highly dengue endemic countries. In this connection, several studies reported that sero-diagnostic tests for dengue virus (DV) yielded considerable false-positive results for SARS-CoV-2 and vice versa in dengue endemic regions, thereby indicating towards potential cross-reactivity between these two viruses. We anticipated that SARS-CoV-2 and DV might share antigenic similarity and performed computational docking studies to test this hypothesis. Our results predicted with high confidence that human DV antibodies can indeed, bind to RBD of SARS-CoV-2 Spike protein. Some of these interactions can also potentially intercept human ACE2 receptor binding to RBM. Dengue serum samples predating the COVID-19, had been found to cross-react with SARS-CoV-2 Spike and this provides direct experimental validation of our predictions. Our analysis also showed that m396 and 80R antibodies (against SARS-CoV-1) did not dock with RBM of SARS-CoV-2, a fact already proven experimentally. This confirmed reliability and robustness of our approach. So, it is highly probable that immunological memory/antibodies to DV in endemic countries may reduce the severity and spread of COVID-19. It is not known whether SARS-CoV-2 antibodies will hinder DV infections by binding to DV particles and reduce dengue incidences in the future or, augment DV infection and severity by deploying antibody-dependent enhancement.

Archived dengue serum samples produced false-positive results in SARS-CoV-2 lateral flow-based rapid antibody tests.

Co-endemicity of SARS-CoV-2 and dengue virus (DV) infection is becoming a matter of serious concern as it has been already reported that antibodies (Ab) elicited by SARS-CoV-2 infection can produce false-positive results in dengue IgG and IgM rapid tests and vice versa. Here we communicate that five of thirteen DV antibody-positive serum samples from Kolkata, archived in 2017 (predating the COVID-19 outbreak), produced false-positive results in SARS-CoV-2 IgG/IgM lateral flow-based rapid tests. Our results emphasize the importance of implementing tests with higher specificity to conduct sero-surveillance for accurate estimation of SARS-CoV-2/DV prevalence in regions where both viruses now co-exist.

Mismatch primer-based PCR reveals that helicase-primase inhibitor resistance mutations pre-exist in herpes simplex virus type 1 clinical isolates and are not induced during incubation with the inhibitor.

OBJECTIVES: Previous studies suggested that helicase-primase inhibitor (HPI) resistance mutations can be selected at relatively high frequency from some isolates of herpes simplex virus type 1 (HSV-1). An intentional mismatch primer (IMP) PCR was developed to detect three known HPI resistance mutations well above the expected background frequency. The objective of this study was to provide proof that HPI resistance mutations pre-exist at relatively high frequency in some clinical isolates obtained from individuals naive to HPIs. METHODS: Three different IMP PCRs were standardized to detect critical HPI resistance mutations (K356N or K356T in UL5, or A899T in UL52) at 10-100 times the expected background frequency (<10(-6)). Thirty HSV-1 clinical isolates were then screened for the resistance mutations in the absence of the inhibitor using IMP PCR. RESULTS: Among 30 clinical isolates that were all susceptible to the HPI, BAY 57-1293, 5 were shown to contain UL5 mutations at 10-100 times higher than the expected frequency. No UL52 resistance mutations were encountered in this study. CONCLUSIONS: The detection of HPI-resistant mutations in some clinical isolates by means of IMP PCR proved that the mutations pre-exist and showed that they are not induced during incubation with the inhibitor.

Cloning and characterization of DGAT1 gene of Riverine buffalo.

The present study was carried out to characterize the DGAT1 gene of Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and DGAT1cDNA were synthesized by RT-PCR, then cloned using pDRIVE cloning vector and sequenced. The sequencing revealed that the size of DGAT1 gene was 1470 bp with GC content of 62.30%. The gene encoded for 489 amino acid precursors and that it possessed 32 amino acids signal peptide. The similarity of buffalo DGAT1 mRNA sequence with that of cattle, pig, monkey, human, mice and rat were determined as 98.4, 90.7, 85.4, 85.0, 77.4 and 77.1%, respectively. Phylogenetic tree constructed from the derived DGAT1 protein sequences of 15 different species illustrated a unique branches for mammals, fly, nematode and plants. Among mammals, cattle and buffalo grouped together, whereas swine formed another group in the same branch. Four motifs were predicted in buffalo DGAT1 peptide sequence, one N-linked glycosylation site (246th position), two putative tyrosine phosphorylation site (316 and 261), one putative diacylglycerol binding site (382-392 amino acid position) and a conserved domain MBOAT (membrane bound acyl transferase from 150 to 474 amino acids) with a histidine as an active residue.