Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic “GII.4 2012” variants in UK coastal waters during 2013

Pandemic norovirus in coastal blue mussels during summer in UK This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic “GII.4 Sydney variants” whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks

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

Drug resistance mutations in HSV-1 UL5 selected using a helicase–primase inhibitor: frequency and effects on virus growth and pathogenicity

Helicase–primase inhibitors (HPIs), e.g. BAY 57-1293, are extremely active against HSV in cell culture and animal infection models. They target the helicase–primase (HP) complex which is involved in virus DNA replication. Using BAY 57-1293 at inhibitory concentrations (e.g. 10–100 times the IC50) it was possible to detect HPI-resistant viruses in two different laboratory working stocks of HSV-1 following a single passage with the inhibitor in Vero cells. Furthermore, resistance selection occurred when the inhibitor was continuously present from prior to virus inoculation suggesting that certain resistance mutations may pre-exist in virus populations at relatively high frequency. PCR data will be presented to confirm these observations. It was shown subsequently that 2 out of 10 recent clinical isolates of HSV-1 also contained BAY 57-1293-resistant variants at 10−4 to 10−5 p.f.u. This is similar to the laboratory isolates and 10–100 times the previously reported spontaneous rate for HPI-resistance mutations (10−6) in plaque-purified HSV-1 strains. The most common resistance mutations involved three amino acid residues just down-stream from the predicted helicase motif IV in HSV-1 UL5 and one residue near the C-terminus of the primase (UL52). We also showed that certain HPI-resistance mutations in UL5 are associated with increased or decreased virus growth in tissue culture with concomitant effects on pathogenicity

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

Occult hepatitis B virus infections (often with human herpesvirus 7 co-infection) detected in Pityriasis rosea patients: A pilot study

Background: The etiopathogenesis of Pityriasis rosea (PR), a papulo-squamous skin disease, remains elusive and hypothesized to be caused primarily by human herpesvirus (HHV) 6 or 7 or immune dysfunction. Aims: The recent increasing incidences of hepatitis B virus (HBV) infections, including asymptomatic occult HBV infections (OBIs), in a densely populated city in India, prompted us to investigate whether PR patients (from varied socioeconomic and immune status) harbor the underlying HBV infections. These cases were also investigated for HHV 6 and 7 infections. Materials and Methods: DNA from ethylenediaminetetraacetic acid blood samples from PR-diagnosed individuals (n = 13; mostly young adults) and healthy controls (n = 11) were subjected to virus gene-specific polymerase chain reactions (PCRs) for HBV and HHV 6 and 7. PCR products of expected length, when observed, were sequenced (bidirectional) using overlapping primers. Sequences were identified by NCBI BLAST and analyzed by multiple sequence alignment and phylogenetic studies. The blood samples were tested for HBsAg by EIA. Results: In 5/13 PR samples, only HBV DNA (4/5 being HBsAg negative) was detected, providing first-time evidence that PR may be manifested in asymptomatic HBV carriers. 6/13 cases were HHV 7 (not HHV 6) DNA positive, providing confirmatory molecular genetic evidence for the first time of PR association with HHV 7 from India. Surprisingly, 5/6 HHV 7-positive PR cases were also HBV positive. Overall, 10/13 PR samples showed evidence of HBV infection. 8/13 were OBI, harboring at least one OBI-signature S protein mutation. All healthy controls were HBsAg EIA and PCR negative. Conclusions: 77% of PR patients presented the evidence of underlying HBV infection (genotype D2), suggestive of horizontal HBV transmission. This warrants for mass HBV vaccination. PR patients should be tested for underlying virus infections for appropriate therapy and management

Helicase-primase inhibitors for herpes simplex virus: looking to the future of non-nucleoside inhibitors for treating herpes virus infections

Helicase-primase inhibitors (HPIs) are the first new family of potent herpes virus (herpes simplex and varicella-zoster virus) inhibitors to go beyond the preliminary stages of investigation since the emergence of the original nucleoside analog inhibitors. To consider the clinical future of HPIs, this review puts the exciting new findings with two HPIs, amenamevir and pritelivir, into the historical context of antiviral development for the prevention and treatment of herpes simplex virus over the last century and, on this basis, the authors speculate on the potential evolution of these and other non-nucleoside inhibitors in the future

Effect of butyrophilin gene polymorphism on milk quality traits in crossbred cattle

A genetic polymorphism study on butyrophilin gene was carried out to explore variability of this gene and to estimate effects of such variability on milk quality traits in crossbred cattle. Polymorphism was unraveled by conducting Hae III PCR-RFLP of this gene. Three genotypes such as AA, BB and AB and two alleles namely A and B were observed in crossbred population. The frequencies of genotypes and alleles were 0.78, 0.17 and 0.04 for AA, AB and BB genotypes, respectively, and 0.87 and 0.13 for A and B alleles, respectively. The nucleotides, which have been substituted from allele A to B, were observed as C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitutions at 71st, 86th and 377th position of the fragment were found as silent mutations whereas nucleotide changes at 217th, 258th and 371st positions were detected as substitution of amino acid lysine with arginine, valine with isoleucine, and leucine with proline from allele A to B. The genotypes had significant effects (p??.05) on total milk solid%, fat%, SNF%, while showing nonsignificant impact on total protein%. AA genotype produced highest average yield for all the traits

Effects of therapy using a helicase-primase inhibitor (HPI) in mice infected with deliberate mixtures of wild-type HSV-1 and an HPI-resistant UL5 mutant

Point mutations in the HSV-1 UL5 (helicase) gene confer resistance to helicase-primase inhibitors (HPIs), e.g. BAY 57-1293. Such mutations normally occur at a frequency of < or =10(-6)PFU. However, individual HSV-1 laboratory strains and some clinical isolates contained resistance mutations (e.g. UL5: Lys356Asn) at 10(-4)PFU. To address the possibility that pre-existing mutants at high frequency might have an impact on therapy using HPIs, deliberate mixtures were prepared to contain the SC16 UL5: Lys356Asn mutant in SC16 wild-type in the proportion of 1/500 or 1/50PFU. Mice were infected in the neck-skin with 5x10(4)PFU/mouse of wt alone, mutant alone, or the respective mixture. The mutant could not be detected in infectious virus yields from mice inoculated with the 1/500 mixture. However, resistant mutant was recovered from some treated mice inoculated with the 1/50 mixture. All mice inoculated with mixtures remained responsive to BAY 57-1293-therapy with no increase in clinical signs compared to treatment of wt-infected mice

A mutation in helicase motif IV of herpes simplex virus type 1 UL5 that results in reduced growth in vitro and lower virulence in a murine infection model is related to the predicted helicase structure

A variant was selected from a clinical isolate of herpes simplex virus type 1 (HSV-1) during a single passage in the presence of a helicase-primase inhibitor (HPI) at eight times the IC(50). The variant was approximately 40-fold resistant to the HPI BAY 57-1293 and it showed significantly reduced growth in tissue culture with a concomitant reduction in virulence in a murine infection model. The variant contained a single mutation (Asn342Lys) in the UL5 predicted functional helicase motif IV. The Asn342Lys mutation was transferred to a laboratory strain, PDK cl-1, and the recombinant acquired the expected resistance and reduced growth characteristics. Comparative modelling and docking studies predicted the Asn342 position to be physically distant from the HPI interaction pocket formed by UL5 and UL52 (primase). We suggest that this mutation results in steric/allosteric modification of the HPI-binding pocket, conferring an indirect resistance to the HPI. Slower growth and moderately reduced virulence suggest that this mutation might also interfere with the helicase-primase activity