Generation and application of immortalized sheep fetal fibroblast cell line.

BACKGROUND: Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure. RESULTS: In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility. CONCLUSIONS: Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.

Internalization of rabies virus glycoprotein differs between pathogenic and attenuated virus strains.

The zoonotic rabies virus (RABV) is a non-segmented negative-sense RNA virus classified within the family Rhabdoviridae, and is the most common aetiological agent responsible for fatal rabies disease. The RABV glycoprotein (G) forms trimeric spikes that protrude from RABV virions and mediate virus attachment, entry and spread, and is a major determinant of RABV pathogenesis. A range of RABV strains exist that are highly pathogenic in part due to their ability to evade host immune detection. However, some strains are disease-attenuated and can be cleared by host defences. A detailed molecular understanding of how strain variation relates to pathogenesis is currently lacking. Here, we reveal key differences in the trafficking profiles of RABV-G proteins from the challenge virus standard strain (CVS-11) and a highly attenuated vaccine strain SAD-B19 (SAD). We show that CVS-G traffics to the cell surface and undergoes rapid internalization through both clathrin- and cholesterol-dependent endocytic pathways. In contrast, SAD-G remains resident at the plasma membrane and internalizes at a significantly slower rate. Through engineering hybrids of CVS-G and SAD-G, we show that the cytoplasmic tail of CVS-G is the key determinant of these different internalization profiles. Alanine scanning further revealed that mutation of Y497 in CVS-G (H497 in SAD-G) could reduce the rate of internalization to SAD-G levels. Together, these data reveal new phenotypic differences between CVS-G and SAD-G proteins that may contribute to altered in vivo pathogenicity.

Rotavirus VP4 Epitope of a Broadly Neutralizing Human Antibody Defined by Its Structure Bound with an Attenuated-Strain Virion.

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity.

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities.

Isolation of the various serotypes of Mannheimia haemolytica and preparation of the first vaccine candidate in Iran.

BACKGROUND: Mannheimia haemolytica is one of the main agents of domestic pneumonic mannheimiosis, but a proper vaccine has not been explored in IRAN. METHODS AND RESULTS: 362 lung and nasal samples from sick domestic animal were detected by culture and PCR methods. Totally, 71 M. haemolytica isolates were identified in three main serotypes (A1, A2, and A6). Serotypes A2 (38/71; 54%) and A1 (25/71; 39%) were the most frequently detected, whereas the A6 serotype was detected with a frequency of less than 1% (1/71; 1%) and 7 isolates remained unknown (7/71; 10%). Subsequently, M. haemolytica vaccinal strain was developed and then formalin-killed vaccine was prepared. It provided the best protection against mannheimiosis in sheep which was proved by indirect ELISA. CONCLUSIONS: Our results suggest that the efficacy and safety of vaccine strain are remarkable and may serve as a new therapeutic target in mannheimiosis.

Antigenic characterization of influenza and SARS-CoV-2 viruses.

Antigenic characterization of emerging and re-emerging viruses is necessary for the prevention of and response to outbreaks, evaluation of infection mechanisms, understanding of virus evolution, and selection of strains for vaccine development. Primary analytic methods, including enzyme-linked immunosorbent/lectin assays, hemagglutination inhibition, neuraminidase inhibition, micro-neutralization assays, and antigenic cartography, have been widely used in the field of influenza research. These techniques have been improved upon over time for increased analytical capacity, and some have been mobilized for the rapid characterization of the SARS-CoV-2 virus as well as its variants, facilitating the development of highly effective vaccines within 1 year of the initially reported outbreak. While great strides have been made for evaluating the antigenic properties of these viruses, multiple challenges prevent efficient vaccine strain selection and accurate assessment. For influenza, these barriers include the requirement for a large virus quantity to perform the assays, more than what can typically be provided by the clinical samples alone, cell- or egg-adapted mutations that can cause antigenic mismatch between the vaccine strain and circulating viruses, and up to a 6-month duration of vaccine development after vaccine strain selection, which allows viruses to continue evolving with potential for antigenic drift and, thus, antigenic mismatch between the vaccine strain and the emerging epidemic strain. SARS-CoV-2 characterization has faced similar challenges with the additional barrier of the need for facilities with high biosafety levels due to its infectious nature. In this study, we review the primary analytic methods used for antigenic characterization of influenza and SARS-CoV-2 and discuss the barriers of these methods and current developments for addressing these challenges.

Survival of Brucella abortus RB51 and S19 Vaccine Strains in Fresh and Ripened Cheeses.

Brucellosis is a zoonotic infection caused by the consumption of contaminated raw milk and dairy products. This study aims to compare survival rates of Brucella abortus RB51 and S19 vaccine strains to that of virulent B. abortus 2308 strain during the manufacture of fresh and ripened cheeses. To do this, we inoculated fresh pasteurized milk with B. abortus RB51, S19, or 2308 at a 6 × 108 colony-forming unit per milliliter concentration during the cheese making process. Cheese was manufactured at room temperature, then, fresh cheeses were conserved at either 4°C or 25°C for 7 days, while ripened cheeses were conserved for 31 days at the same temperatures. We measured B. abortus survival and pH values during different stages of the process. Our results confirm that all three strains can maintain viable cells in both types of cheeses throughout the process. Survival of B. abortus RB51 was 10 times lower than was the survival of the B. abortus S19 and B. abortus 2308 strains in both fresh and ripened cheeses. Our results also suggest that both temperature and pH can condition Brucella survival. In conclusion, B. abortus RB51 and S19 vaccine strains can survive throughout the manufacture and conservation processes of both fresh and ripened cheeses. In turn, this implies a potential health risk if cheeses contaminated with these strains were to be consumed.

In ovo application of a live infectious bursal disease vaccine to commercial broilers confers proper immunity.

Infectious bursal disease (IBD) is an economically important disease of young chickens caused by the Avibirnavirus infectious bursal disease virus (IBDV). Besides biosecurity, vaccination is the most important measure for IBDV control. Sufficient levels of maternally derived antibodies (MDA) protect against early challenge and also interfere with the take of live conventional vaccines. Recently, the field surveys conducted in four countries, published by Ashash, U., Noach, C., Perelman, B., Costello, C., Sansalone, P., Brazil, T. & Raviv, Z. [(2019). In ovo and day of hatch application of a live infectious bursal disease virus vaccine to commercial broilers. Avian Diseases, 63, 713-720] using the MB-1 vaccine strain by in ovo application or sub-cutaneous route at the day of hatch seem to conflict with the rule that very early application of a conventional live vaccine in birds with significant levels of MDA has very little chance of a successful immune response. An in ovo vaccination-challenge controlled experiment with MB-1 vaccine was performed using commercial broilers with high levels of MDA against IBDV and a vvIBDV challenge at 22 or 36 days of age. Clinical signs, bursa-bodyweight ratios, histology, serology, RT-PCR, Sanger- and deep sequencing were used to study the efficacy and safety of the in ovo-applied MB1 vaccine in comparison to an established immuno-complex vaccine. The study findings confirmed that the in ovo application of the live MB-1 vaccine in commercial broilers was successful and induced full protection against a vvIBDV challenge at 22 and 36 days of age, demonstrated by the bursa lesion score and qPCR and IBDV genotyping. Comparable to the field studies, a delayed viral replication of 2-3 weeks, following the in ovo administration of the MB1 vaccine, was observed.

Live attenuated vaccines under immunosuppressive agents or biological agents: survey and clinical data from Japan.

Live attenuated vaccines are contraindicated for patients on immunosuppressive agents or biological agent, except for live attenuated varicella vaccine, although previous reports showed their effectiveness and safety. This study is the nationwide cross-sectional research about the current utilization of live attenuated vaccines for patients on immunosuppressive agents or biological agents in Japan. We sent questionnaires to pediatric centers and examined whether each institution offered live attenuated vaccines to patients with immunosuppressive agents or biological agents (institutional research). We also examined adverse events associated with live attenuated vaccines between 2013 and 2017 (patient research). In the institutional research, 46 out of 334 institutions (13.8%) administered live attenuated vaccines to patients receiving immunosuppressive agents. In contrast, only six out of 270 institutions (2.2%) administered live attenuated vaccines to patients receiving biological agents. However, 66.3% of physicians answered that patients receiving immunosuppressive agents should be immunized with live attenuated vaccines, and only 7.0% disagreed with them. In the patient research, data for 781 patients were collected. Vaccine-associated infections were observed in only two patients (0.3%), both of whom had varicella, although they recovered promptly. No life-threatening adverse events were noted. CONCLUSION: In pediatric centers, the demand for live attenuated vaccines in patients receiving immunosuppressive agents was high and most physicians think they should be immunized. Immunization with live attenuated vaccines appeared safe in patients receiving immunosuppressive agents, although further studies are needed for patients receiving biological agents What is known: • Live attenuated vaccines (LAV) are generally contraindicated for patients on immunosuppressive agents (IS) or biological agents (BA), except for live attenuated varicella vaccine, as immunocompromised patients are at greater risk for serious viral infection from the vaccine strains. • Viral infections, such as measles and varicella, cause serious complications in children receiving IS. • Several previous reports showed that LAV is relatively effective and safe for patients receiving IS. What is new: • In Japan, the demand for LAV in patients receiving IS was high, and most physicians hoped they should be immunized. • Vaccine-associated infection is rarely observed in patients with IS after LAV administration. • Immunization with LAV appeared safe in patients receiving IS. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN). TRIAL REGISTRATION NUMBER: UMIN000029176.Date of registration: 2017/09/19.

Comparative genomic analysis between newly sequenced Brucella abortus vaccine strain A19 and another Brucella abortus vaccine S19.

BACKGROUND: Brucellosis is a bacterial disease caused by Brucella infection. Brucella abortus strain A19 is a spontaneously attenuated vaccine strain that has been used in vaccination of cattle against brucellosis. Until now, the physiological and molecular mechanisms of A19 are still unknown. RESULTS: In this paper, the whole-genome sequence of B. abortus A19 was performed using Illumina Hiseq 4000 and PacBio sequencing technology and comparative genomics analysis were carried out with the whole genome sequences of B. abortus strains S19. This analysis indicated that the two vaccine strains have a high degree of similarity in genomic structure. We further analysis of the difference in genomic structure between A19 and S19. And found some differential genes such as eryC, eryD and eryF. Of the other different proteins between A19 and S19, such as outer membrane protein, 2-isopropylmalate synthase, citramalate synthase, GntR family transcriptional regulator and ABC transporters, no clear effects related to bacterial virulence were found, pending further investigation. CONCLUSION: The data presented here provide a reasonable basis for designing Brucella vaccines that can be used in other strains.

A Strategy for the Rapid Development of a Safe Vibrio cholerae Candidate Vaccine Strain.

Approximately 1/6 of humanity is at high risk of experiencing cholera epidemics. The development of effective and safe vaccines against Vibrio cholerae, the primary cause of cholera, is part of the public health measures to prevent cholera epidemics. Natural nontoxigenic V. cholerae isolates represent a source of new genetically improved and relatively safe vaccine strains. However, the genomic engineering of wild-type V. cholerae strains is difficult, and these strains are genetically unstable due to their high homologous recombination activity. We comprehensively characterized two V. cholerae isolates using genome sequencing, bioinformatic analysis, and microscopic, physiological, and biochemical tests. Genetic constructs were Gibson assembled and electrotransformed into V. cholerae. Bacterial colonies were assessed using standard microbiological and immunological techniques. As a result, we created a synthetic chromoprotein-expressing reporter operon. This operon was used to improve the V. cholerae genome engineering approach and monitor the stability of the genetic constructs. Finally, we created a stable candidate V. cholerae vaccine strain bearing a recA deletion and expressing the ß-subunit of cholera toxin. Thus, we developed a strategy for the rapid creation of genetically stable and relatively safe candidate vaccine strains. This strategy can be applied not only to V. cholerae but also to other important human bacterial pathogens.

Multi-task learning sparse group lasso: a method for quantifying antigenicity of influenza A(H1N1) virus using mutations and variations in glycosylation of Hemagglutinin.

BACKGROUND: In addition to causing the pandemic influenza outbreaks of 1918 and 2009, subtype H1N1 influenza A viruses (IAVs) have caused seasonal epidemics since 1977. Antigenic property of influenza viruses are determined by both protein sequence and N-linked glycosylation of influenza glycoproteins, especially hemagglutinin (HA). The currently available computational methods are only considered features in protein sequence but not N-linked glycosylation. RESULTS: A multi-task learning sparse group least absolute shrinkage and selection operator (LASSO) (MTL-SGL) regression method was developed and applied to derive two types of predominant features including protein sequence and N-linked glycosylation in hemagglutinin (HA) affecting variations in serologic data for human and swine H1N1 IAVs. Results suggested that mutations and changes in N-linked glycosylation sites are associated with the rise of antigenic variants of H1N1 IAVs. Furthermore, the implicated mutations are predominantly located at five reported antibody-binding sites, and within or close to the HA receptor binding site. All of the three N-linked glycosylation sites (i.e. sequons NCSV at HA 54, NHTV at HA 125, and NLSK at HA 160) identified by MTL-SGL to determine antigenic changes were experimentally validated in the H1N1 antigenic variants using mass spectrometry analyses. Compared with conventional sparse learning methods, MTL-SGL achieved a lower prediction error and higher accuracy, indicating that grouped features and MTL in the MTL-SGL method are not only able to handle serologic data generated from multiple reagents, supplies, and protocols, but also perform better in genetic sequence-based antigenic quantification. CONCLUSIONS: In summary, the results of this study suggest that mutations and variations in N-glycosylation in HA caused antigenic variations in H1N1 IAVs and that the sequence-based antigenicity predictive model will be useful in understanding antigenic evolution of IAVs.

Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo.

BACKGROUND: Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo. RESULTS: Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type. CONCLUSIONS: These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.

Development of mismatch amplification mutation assay for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates.

Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 102 and 103 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTS The full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Genome analysis of Mycoplasma synoviae strain MS-H, the most common M. synoviae strain with a worldwide distribution.

BACKGROUND: The bacterial pathogen Mycoplasma synoviae can cause subclinical respiratory disease, synovitis, airsacculitis and reproductive tract disease in poultry and is a major cause of economic loss worldwide. The M. synoviae strain MS-H was developed by chemical mutagenesis of an Australian isolate and has been used as a live attenuated vaccine in many countries over the past two decades. As a result it may now be the most prevalent strain of M. synoviae globally. Differentiation of the MS-H vaccine from local field strains is important for epidemiological investigations and is often required for registration of the vaccine. RESULTS: The complete genomic sequence of the MS-H strain was determined using a combination of Illumina and Nanopore methods and compared to WVU-1853, the M. synoviae type strain isolated in the USA 30 years before the parent strain of MS-H, and MS53, a more recent isolate from Brazil. The vaccine strain genome had a slightly larger number of pseudogenes than the two other strains and contained a unique 55 kb chromosomal inversion partially affecting a putative genomic island. Variations in gene content were also noted, including a deoxyribose-phosphate aldolase (deoC) fragment and an ATP-dependent DNA helicase gene found only in MS-H. Some of these sequences may have been acquired horizontally from other avian mycoplasma species. CONCLUSIONS: MS-H was somewhat more similar to WVU-1853 than to MS53. The genome sequence of MS-H will enable identification of vaccine-specific genetic markers for use as diagnostic and epidemiological tools to better control M. synoviae.

Bimodal Seasonality and Alternating Predominance of Norovirus GII.4 and Non-GII.4, Hong Kong, China, 2014-20171.

We report emerging subtropical bimodal seasonality and alternating predominance of norovirus GII.4 and non-GII.4 genotypes in Hong Kong. GII.4 predominated in summer and autumn months and affected young children, whereas emergent non-GII.4 genotypes predominated in winter months and affected all age groups. This highly dynamic epidemiology should inform vaccination strategies.

A real time high-resolution melting PCR assay for detection and differentiation among sheep pox virus, goat pox virus, field and vaccine strains of lumpy skin disease virus.

In this paper, we report on the development of a real time high-resolution melting (HRM) PCR assay for detection and differentiation among sheep pox virus (SPPV), goat pox virus (GTPV), field isolates and vaccine strains of lumpy skin disease virus (LSDV) based on high-resolution melting curve analysis of their target PCR amplicons. A 111 bp region of LSDV010 ORF, which harbors unique genetic differences for each of these viral species, was selected as the PCR target in this study. During the validation of this assay using DNA from clinical isolates originated from naturally infected animals from the different geographic locations and reference strains, the obtained PCR amplicons demonstrated that the melting temperature picks were specific for each tested viral species, i.e., 74.56 ±â€¯0.04 °C for field LSDV, 74.95 ±â€¯0.08 °C for vaccine LSDV, 74.24 ±â€¯0.06 °C for SPPV and 73.61 ±â€¯0.04 °C for GTPV. The assessment of the assay sensitivity utilizing a LSDV field strain as a PCR template revealed the assay detection limit as low as 0.1 TCD50 lg/ml. Overall, this assay based on Rotor-Gene Q (QIAGEN) platform was shown to be reproducible across replicates and operators and can be recommended as an additional diagnostic tool to the currently available molecular assays for detection and differentiation of the genus Capripoxvirus species, including the differentiation of vaccine strains of LSDV from field isolates. The assay can be used for detection of these viruses in animal- and insect-derived field specimens.

Varicella-zoster virus infection: natural history, clinical manifestations, immunity and current and future vaccination strategies.

Varicella-zoster virus (VZV) is the etiologic agent of varicella (chicken pox), a childhood exanthematic disease that develops as a result of primary infection, and zoster (shingles), caused by reactivation of the virus persisting in a latent form in the dorsal sensory ganglia. Although varicella is generally a mild self-limiting illness, in immunocompromised subjects and adults it can have a serious clinical course that can lead to permanent damage of the central nervous system. In these and in most zoster cases, treatment with anti-herpetic drugs and/or immunotherapy is necessary. Because it is highly contagious, varicella is one of the most common exanthematic diseases. It is preventable by vaccination with an attenuated vaccine administered around the first year of age, and with a boost vaccination in school age. This article briefly describes the natural history and pathophysiology of VZV infection and its current epidemiology and provides an overview of current and future vaccine options to protect against varicella and/or zoster.

[Oncolytic Paramyxoviruses: Mechanism of Action, Preclinical and Clinical Studies].

Preclinical studies demonstrate that a broad spectrum of human and animal malignant cells can be killed by oncolytic paramyxoviruses, which includes cells of ecto-, endo- and mesodermal origin. In clinical trials, significant reduction or even complete elimination of primary tumors and established metastases has been reported. Different routes of virus administration (intratumoral, intravenous, intradermal, intraperito-neal, or intrapleural) and single- vs. multiple-dose administration schemes have been explored. The reported side effects were grades 1 and 2, with the most common among them being mild fever. There are certain advantages in using paramyxoviruses as oncolytic agents compared to members of other virus families exist. Thanks to cytoplasmic replication, paramyxoviruses do not integrate the host genome or engage in recombination, which makes them safer and more attractive candidates for widely used therapeutic oncolysis than ret-roviruses or some DNA viruses. The list of oncolytic Paramyxoviridae members includes the attenuated measles virus, mumps virus, low pathogenic Newcastle disease, and Sendai viruses. Metastatic cancer cells frequently overexpress certain surface molecules that can serve as receptors for oncolytic paramyxoviruses. This promotes specific viral attachment to these malignant cells. Paramyxoviruses are capable of inducing efficient syncytium-mediated lysis of cancer cells and elicit strong immune stimulation, which dramatically enforces anticancer immune surveillance. In general, preclinical studies and phases I-III of clinical trials yield very encouraging results and warrant continued research of oncolytic paramyxoviruses as a particularly valuable addition to the existing panel of cancer-fighting approaches.