Evaluation of SDS and GRAS liquid disinfectants for mitigation of hepatitis A virus contamination of berries.

AIM: To evaluate generally recognized as safe (GRAS) liquid wash formulations against hepatitis A virus-contaminated strawberries and blackberries in order to identify a formulation suitable for reducing virus contamination. METHODS AND RESULTS: Formulations included the surfactant sodium dodecyl sulfate (SDS; 0·5% w/v) by itself, and in combination, with lactic acid (LA; 0·5% v/v), levulinic acid (LVA; 0·5% v/v) and 3 ppm aqueous chlorine dioxide (ClO2 ). After contamination and drying overnight, the average total extracted contamination for both untreated strawberries and blackberries was 4·4 log PFU. Three successive distilled H2 O only treatments reduced total contamination by up to 1·8 log PFU for both strawberries and blackberries, while wash formulations showed significant (P ≤ 0·05) total reductions ranging from 2·1 to 2·9 log PFU. CONCLUSIONS: Considering results for both berry types, the combination of ClO2 and SDS was the most effective. Overall results indicate that adding surfactant and several types of sanitizers to berry wash can enhance HAV reduction on berries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that industry could enhance the virologic safety of ready-to-eat berries by the combined use of surfactant and sanitizer.

Evaluation of 405-nm monochromatic light for inactivation of Tulane virus on blueberry surfaces.

AIM: The study aim was to evaluate the potential of 405-nm light as a virus intervention for blueberries. METHODS AND RESULTS: Tulane virus (TV)-inoculated blueberries were treated with 4·2 mW cm-2 of 405-nm light for 5-30 min. To mitigate thermal heating due to the intense light, a dry ice-chilled, nitrogen-based cooling system was utilized. Blueberries were rotated to ensure exposure of all surfaces to 405-nm light. Five-, 15- and 30-min treatments resulted in little or no inactivation of TV on blueberries (average log reductions of -0·18; -0·02; and +0·06 respectively). Since 405-nm light's inactivation mechanism may involve singlet oxygen, two singlet oxygen enhancers, riboflavin and rose bengal, were used to coat the blueberries prior to 405-nm light treatment. When 0·1% riboflavin or rose bengal was added, resulting in an average PFU reduction of -0·51 and -1·01 logs respectively. However, it was noted that the addition of riboflavin and rose bengal in the absence of 405-nm light treatment produced some inactivation. Average untreated log reductions for riboflavin and rose bengal were -0·13 and -0·66 respectively. Also, 60-30-s 405-nm light pulses with 2-min ambient cooling periods without the dry ice nitrogen cooling system did not inactivate TV, suggesting that oxygen limitation by the nitrogen CO2 mixture was not the cause of limited inactivation. CONCLUSIONS: Overall results indicate that 405-nm light has some potential to inactivate viruses if singlet oxygen enhancers are present. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of visible monochromatic violet/blue light (405 nm) as a nonthermal intervention for viruses on foods, such as berries that are prone to norovirus contamination, had not been previously evaluated. Use of food-grade singlet oxygen enhancer compounds in combination with visible spectra light may offer a means to inactivate foodborne viruses.

Outbreak of Hepatitis A in Italy Associated with Frozen Redcurrants Imported from Poland: A Case Study.

Hepatitis A virus (HAV) was detected in a batch of imported non-packaged frozen redcurrants purchased in a Bari grocery. Sequence and phylogenetic analysis showed the HAV strain clustered tightly with the HAV strain from the 2013 Italian epidemic, providing additional evidence that frozen redcurrants were the main vehicle of the HAV outbreak.

High hydrostatic pressure processing of murine norovirus 1-contaminated oysters inhibits oral infection in STAT-1(-/-)-deficient female mice.

We have previously demonstrated that high pressure processing (HPP) is effective in preventing in vitro replication of murine norovirus strain 1 (MNV-1), a human norovirus surrogate, in a monocyte cell line following extraction from MNV-1-contaminated oysters. In the present study, the efficacy of HPP to prevent in vivo replication within mice fed HPP-treated MNV-1-seeded oyster extracts was evaluated. Oyster homogenate extracts seeded with MNV-1 were given 5-min, 400-MPa (58,016-psi) treatments and orally gavaged into immunodeficient (STAT-1(-/-)) female mice. Mice orally gavaged with HPP-treated MNV-1 showed significant (P ≤ 0.05) weight loss leading to enhanced morbidity, whereas those given 100 and 200 PFU of HPP-treated MNV-1 were comparable to uninfected controls. MNV-1 was detected, via real-time PCR, within the liver, spleen, and brain of all mice fed non-HPP-treated homogenate but was not detected in the tissues of mice fed HPP-treated homogenates or in uninfected control mice. Hepatocellular necrosis and lymphoid depletion in the spleen were observed in non-HPP-treated MNV-1 mice only. These results clearly show that HPP prevents MNV-1 infection in vivo and validates that viral inactivation by HPP in vitro is essentially equivalent to that in vivo. Further, the data suggest that HPP may be an effective food processing intervention for norovirus-contaminated shellfish and thus may decrease risk to both immunocompromised and immunocompetent individuals who consume shellfish.

Rapid and efficient extraction method for reverse transcription-PCR detection of hepatitis A and Norwalk-like viruses in shellfish.

As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine-PEG-Tri-reagent-poly(dT) method can be performed in less than 8 h on hard-shell clams (Mercenaria mercenaria) and Eastern oysters (Crassostrea virginica) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR50 units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as Ruditapes philippinarum) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.

A discrete stage of baculovirus GP64-mediated membrane fusion.

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

Infectious laryngotracheitis virus, an alpha herpesvirus that does not interact with cell surface heparan sulfate.

Among the alpha herpesviruses studied to date, the initial stage of wild-type virus attachment involves an interaction between virally encoded structural envelope glycoproteins (predominantly glycoprotein C) and cell surface heparan sulfate proteoglycans. An analysis of the infectious laryngotracheitis virus (ILTV) glycoprotein C and glycoprotein B sequences suggested that these proteins lacked consensus heparin-binding domains. This indicated that ILTV might attach to its host cell in a heparan-independent manner, distinct from other alpha herpesviruses. The infectivity of two ILTV strains, a tissue-culture-adapted vaccine strain and a virulent field challenge strain, were found to be insensitive to the presence of exogenous heparin or chondroitin. Furthermore, infectivity was retained in chicken embryonic liver cells treated with heparinase. However, 4 degrees C attachment studies and penetration studies in the presence of citrate buffer clearly demonstrated that ILTV attaches stably to and effectively penetrates chicken embryonic liver cells. Consequently, ILTV represents an alpha herpesvirus whose initial attachment step does not involve interactions with heparan or chondroitin sulfate containing proteoglycans.

Identification and characterization of the infectious laryngotracheitis virus glycoprotein C gene.

The infectious laryngotracheitis virus (ILTV) gene encoding a homologue to the glycoprotein C gene of herpes simplex virus has been sequenced and identified based on its genomic location, comparative analysis to other gC proteins, and the identification of a glycosylated protein product. Located near the small subunit ribonucleotide reductase gene, the ILTV gC gene is 1242 bp in length and is predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, five potential N-linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains. Antibodies raised in rabbits against a Cro-ILTV-beta-galactosidase fusion protein expressed in Escherichia coli recognize a 60-kDa ILTV-specific glycoprotein from infected cell extracts. Transcriptional analysis, using a portion of the open reading frame as a probe, identified a 1.55-kb transcript expressed with late gene kinetics. Comparison to other herpesvirus gC proteins revealed limited amino acid sequence homology and the absence of a charged extracellular region, which would normally interact with cell surface proteoglycans.

Identification of the thymidine kinase gene of infectious laryngotracheitis virus.

An an initial step in the development of a recombinant poultry infectious laryngotracheitis virus (ILTV) vaccine, we report on the identification, cloning, and sequencing of a thymidine kinase (tk) gene from a virulent U.S. field isolate of ILTV. Degenerate oligonucleotide primers for the consensus nucleotide (ATP) binding site and the nucleoside (thymidine) binding site of other herpesvirus tk genes were used in the polymerase chain reaction (PCR) to amplify a fragment of ILTV DNA. The 344-base-pair (bp) amplified fragment was cloned into plasmid pKSII and used in Southern hybridizations to locate the ILTV tk gene on a 2.4-kb HindIII fragment. Upon cloning and sequencing this fragment, a 1089-bp open reading frame was identified, which is predicted to encode a protein demonstrating 27.9% amino acid homology to the herpes simplex virus type 1 (HSV-1) thymidine kinase protein. Analysis of the sequence revealed one region of difference from that reported for the Thorne strain of ILTV. In addition, the portion of the TK protein corresponding to the nucleotide binding domain is highly conserved among the avian herpesviruses.

The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12.

The nucleotide sequences of rbsD, rbsA, and rbsC have been determined. These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M., Mahoney, W.C., Hope, J.N., Furlong, C.E., Robb, F.T., Zalkin, H., and Hermodson, M.A. (1983) J. Biol. Chem. 258, 12952-12956) and rbsK (Hope, J.N., Bell, A.W., Hermodson, M.A., and Groarke, J.M. (1986) J. Biol. Chem. 261, 7663-7668), they complete the nucleotide sequence of the first five genes of the rbs operon. Nuclease S1 mapping places the transcriptional start site for the operon 29 base pairs upstream from the most likely translational start site for rbsD. The open reading frames of rbsD, rbsA, and rbsC encode proteins of 139, 501, and 321 amino acid residues, respectively. The character of the proteins varies widely, from very hydrophilic for the rbsA product to exceedingly hydrophobic for the rbsC product. The intercistronic spaces between the three genes are very short, with the stop codons of the upstream genes overlapping the ribosome-binding sites of the downstream genes. This may imply translational control of expression of these genes, the products of which presumably form a membrane-bound transport complex.