Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage.

The Nairoviridae family of the Bunyavirales order comprises tick-borne, trisegmented, negative-strand RNA viruses, with several members being associated with serious or fatal diseases in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely distributed tick-borne pathogen and is associated with devastating human disease, with case fatality rates averaging 30%. Hazara virus (HAZV) is closely related to CCHFV, sharing the same serogroup and many structural, biochemical, and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed, for the first time, a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetic analysis of nairoviruses without the need for high-level biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an uncleavable DQVE sequence was rescued and exhibited growth parameters equivalent to those of wild-type virus in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly uncleavable AQVA sequence could not be rescued despite repeated efforts. Together, these results highlight the importance of this caspase cleavage site in the HAZV life cycle but reveal the DQVD sequence performs a critical role aside from caspase cleavage.IMPORTANCE HAZV is classified within the Nairoviridae family with CCHFV, which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL2 protocols. Here, we report a system that is able to rescue HAZV from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection and this sequence performs a critical role in the virus life cycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL2 protocols.

Hazara nairovirus elicits differential induction of apoptosis and nucleocapsid protein cleavage in mammalian and tick cells.

The Nairoviridae family within the Bunyavirales order comprise tick-borne segmented negative-sense RNA viruses that cause serious disease in a broad range of mammals, yet cause a latent and lifelong infection in tick hosts. An important member of this family is Crimean-Congo haemorrhagic fever virus (CCHFV), which is responsible for serious human disease that results in case fatality rates of up to 30 %, and which exhibits the most geographically broad distribution of any tick-borne virus. Here, we explored differences in the cellular response of both mammalian and tick cells to nairovirus infection using Hazara virus (HAZV), which is a close relative of CCHFV within the CCHFV serogroup. We show that HAZV infection of human-derived SW13 cells led to induction of apoptosis, evidenced by activation of cellular caspases 3, 7 and 9. This was followed by cleavage of the classical apoptosis marker poly ADP-ribose polymerase, as well as cellular genome fragmentation. In addition, we show that the HAZV nucleocapsid (N) protein was abundantly cleaved by caspase 3 in these mammalian cells at a conserved DQVD motif exposed at the tip of its arm domain, and that cleaved HAZV-N was subsequently packaged into nascent virions. However, in stark contrast, we show for the first time that nairovirus infection of cells of the tick vector failed to induce apoptosis, as evidenced by undetectable levels of cleaved caspases and lack of cleaved HAZV-N. Our findings reveal that nairoviruses elicit diametrically opposed cellular responses in mammalian and tick cells, which may influence the infection outcome in the respective hosts.

Observation as the initial management strategy in patients with mantle cell lymphoma.

BACKGROUND: Patients with mantle cell lymphoma (MCL) follow a heterogeneous clinical course. While they generally require treatment initiation shortly after diagnosis, it is unclear whether deferring treatment in selected patients with an indolent clinical behavior affects their overall outcome. PATIENTS AND METHODS: In this population-based study, all patients diagnosed with MCL during 1998-2014 were identified in the British Columbia Cancer Agency Lymphoid Cancer Database. The associations between clinico-pathologic characteristics, including the expression of Ki67, SOX11, and TP53, and time to treatment (TtT) and OS were analyzed. RESULTS: A total of 440 patients with MCL were evaluated: 365 (83%) received early treatment and 75 (17%) were observed ≥3 months. In the observation group, 54 (72%) patients had a nodal presentation, 16 (21%) a non-nodal presentation, and 5 (7%) had only gastrointestinal involvement. Characteristics associated with deferred treatment included good performance status, no B symptoms, low LDH, non-bulky disease, non-blastoid morphology, and lower Ki67 values. The median TtT in the observation group was 35 months (range 5-79), and 60 (80%) patients were observed beyond 12 months. The median OS was significantly longer in the observation group than in the early treatment group (72 versus 52.5 months, respectively, P = 0.041). In multivariable analysis, treatment decision was not associated with OS [HR 0.804 (95% CI 0.529-1.221), P = 0.306]. CONCLUSIONS: A subgroup of patients with MCL may be safely observed from diagnosis without negatively impacting their outcomes, including patients with non-nodal presentation as well as asymptomatic patients with low burden nodal presentation and a low proliferative rate.

BCL2 mutations in diffuse large B-cell lymphoma.

BCL2 is deregulated in diffuse large B-cell lymphoma (DLBCL) by the t(14;18) translocation, gene amplification and/or nuclear factor-κB signaling. RNA-seq data have recently shown that BCL2 is the most highly mutated gene in germinal center B-cell (GCB) DLBCL. We have sequenced BCL2 in 298 primary DLBCL biopsies, 131 additional non-Hodgkin lymphoma biopsies, 24 DLBCL cell lines and 51 germline DNAs. We found frequent BCL2 mutations in follicular lymphoma (FL) and GCB DLBCL, but low levels of BCL2 mutations in activated B-cell DLBCL, mantle cell lymphoma, small lymphocytic leukemia and peripheral T-cell lymphoma. We found no BCL2 mutations in GC centroblasts. Many mutations were non-synonymous; they were preferentially located in the flexible loop domain, with few in BCL2-homology domains. An elevated transition/transversions ratio supports that the mutations result from somatic hypermutation. BCL2 translocations correlate with, and are likely important in acquisition of, additional BCL2 mutations in GCB DLBCL and FL. DLBCL mutations were not independently associated with survival. Although previous studies of BCL2 mutations in FL have reported mutations to result in pseudo-negative BCL2 protein expression, we find this rare in de-novo DLBCL.

Is Tayside becoming a Scottish hotspot for Lyme borreliosis?

The epidemiology of Lyme borreliosis (LB) in Tayside was studied and compared with Highland (an area of high endemicity) and the rest of Scotland. From April 2001 to March 2008 the incidence of LB in Tayside rose from an estimated 2.57 to 5.84 per 100,000 population. In 2008/09 the incidence of LB in Tayside increased further to an estimated 13.85 per 100,000 population. This rise was significant and, although numerically less than that in Highland (37.24 to 49.69 per 100,000 population), it was proportionally much larger (137% vs 33%) and confirmed that LB in Tayside has diverged from that in non-endemic Scottish regions. The dramatic rise of LB in Tayside cannot be accounted for by changes in laboratory protocol or changes in the number or demographics of patients tested. However, changes in climatic conditions and alterations in clinical presentations may have contributed to this significant rise.

Food safety begins on the farm: the viewpoint of the producer.

Consumers expect the food they purchase to be safe. Governments seek to provide them with assurances of food safety through regulation, but additional steps are needed to more fully address the issue. Producers are increasingly aware of their responsibility in this area and are working in concert with other segments of the agri-food industry. Hazard analysis critical control point-based (HACCP) quality assurance programmes are being developed and implemented at the farm level for most species, in many countries. These approaches will enhance food safety for consumers everywhere. Producers continue to demonstrate that they respond positively to programmes based on science and good management practices. The authors conclude that the use of HACCP programmes will continue to increase.

Routine double treatments of superficial basal cell carcinomas using aminolaevulinic acid-based photodynamic therapy.

BACKGROUND: Superficial basal cell carcinomas of the skin (sBCC) often respond poorly to single-treatment aminolaevulinic acid-based photodynamic therapy (ALA-PDT), with a number of reports indicating a relapse rate of 50% or more. OBJECTIVES: To determine whether a second treatment at seven days can improve the response. METHODS: Twenty-six lesions were treated twice with ALA-PDT, with an interval of 7 days between the two treatment sessions. RESULTS: We observed a complete response rate of 100% 1 month after treatment. Only one lesion relapsed (16 months post-PDT), a relapse rate of 4% (median follow up 27 months; range 15-45 months). Cosmetic results were excellent. CONCLUSIONS: We consider routine double treatments with ALA-PDT to be an effective approach to the management of sBCC, particularly those located in anatomically difficult, or cosmetically sensitive, sites.

Site-selective electronic Raman excitation spectroscopy of the lowest 4f ? 5d transitions in Ce(3+):Y(2)O(3).

The intensities of electronic Raman-scattering transitions between the ground state and the first excited state of the (2)F(7/2) manifold of Ce(3+) in both the C(2) and C(3i) sites of Y(2)O(3) are measured as a function of excitation frequency in the region of the lowest 4f ? 5d intermediate-state resonance with a wavelength-tunable pulsed dye laser. As the dye laser is tuned through the center of the resonance, we observe a 2-order-of-magnitude enhancement in electronic Raman-transition intensity over that for nonresonant excitation. The electronic Raman excitation profiles (resonance enhancement as a function of frequency) in the 15 949- to 23 810-cm(-1) (627-420-nm) range provide spectrally resolved information about the lowest 4f ? 5d transitions of both the C(2) and C(3i) centers that could not be obtained from the strongly overlapping absorption spectra.

A longitudinal epidemiological study on dental plaque and the development of dental caries--interim results after two years.

1. During the two-year period, caries developed at 20% of the target premolar sites. The attack rate for these surfaces was similar in the plaque panel and the other subjects in the study. 2. The microbial composition of plaque samples from caries-free sites and from carious sites before and after radiographic detection of lesions was broadly similar. 3. Numerical domination of particular sites by S mutans before detection of caries can occur, but has only been observed so far in 2 of 15 sites. 4. Pooled date from sites which have developed lesions indicate a rise in the isolation frequency and mean numbers of S. mutans after detection of caries. This trend was particularly obvious in the one subject who developed bilateral lesions by the second examination and in three of four sites where caries was detected at the fourth examination. Similar observations have been made with lactobacilli. 5. In two of 15 instances no isolations of S mutans were made from sites which developed caries. 6. To date, no single species appears to be uniquely associated with the onset of dental caries.