Prevalence of cobalamin deficiency is high especially in older patients and an immediate therapy start is necessary to prevent irreversible neurological damages. Unfortunately, the diagnosis of cobalamin deficiency is difficult and at present, there is no consensus for diagnosis of this deficiency. Therefore, we aim to elucidate a meaningful diagnostic pathway by a case report with an initially misleading medical history.A 57 year-old Caucasian man suffering from dramatic myelosis of the cervical posterior columns. Apart from associated neurological symptoms  there were no further complaints; especially no gastrointestinal, haematological or psychiatric disorders were provable. Cobalamin (vitamin B12) serum level was normal. The diagnosis of subacute combined degeneration of spinal cord was confirmed by an elevated methylmalonic acid, and hyperhomocysteinemia. Cobalamin deficiency was caused by asymptomatic chronic atrophic inflammation of the stomach with a lack of intrinsic factor producing gland cells. This was revealed by increased gastrin and parietal cell antibodies and finally confirmed by gastroscopy. Parenteral substitution of cobalamin rapidly initiated regeneration.This case demonstrates that normal cobalamin serum levels do not rule out a cobalamin deficiency. In contrast, path-breaking results can be achieved by determining homocysteine, holotranscobalamin, and methylmalonic acid. Prevalence of cobalamin deficiency in general population is about 4% . In oldeHumans are not able to synthesize cobalamin. Food of animal source is the only natural source of cobalamin in human diet. In the stomach, ingested cobalamin is detached from its protein-binding by pepsin and hydrochloric acid. Then, it is bound to the glycoproteins haptocorrin and intrinsic factor (IF) secreted by gastric mucosa. The majority of the required cobalamin uptake takes place in the terminal ileum by binding of the cobalamin-IF-complex to receptors of the mucosa cells . Inside Methylcobalamin and folates are co-factors in the methionine-synthase mediated conversion of homocysteine to methionine, which is essential for nucleotide synthesis and genomic and non-genomic methylation . TherefoClinically, cobalamin deficiency is manifested mainly by haematological and neuropsychiatric symptoms. Frequently, these symptoms arise before the lower limit value of cobalamin is reached . In contCommon reasons for cobalamin deficiencies can be divided into four groups Table : diminisFinally, cobalamin deficiency with inconspicuous blood values for cobalamin and holoTC is common in patients suffering from kidney diseases . ProbablClinical improvement and full recovery from myelopathy can occur when substitution of cobalamin and folic acid is started in the early stages of the disease .We would like to present a case, in which medical history and imaging initially pointed to a traumatic or malignant cause of solely neurological complaints. This case illustrates the need of a targeted laboratory diagnostic when clinical examination raises reasonable suspicion of SACD.Seven months before presentation, a 57 year-old Caucasian man fell off a two meter high roof, suffering from a left-hand serial rip fracture and a fracture of the processus transversi of the thoracic vertebral bodies 6 and 7. Three months later, he recognized for the first time a sustainable tactile hypaesthesia and paraesthesia beginning in both hands and extending to both shoulders and to the thorax double-sided within the following months. He also described a narrowed sensation within the thorax. Three and a half months after the start of sensibility loss a magnetic resonance tomography of the cervical spine was performed outward. There were no other diseases or allergies and no sustained medication intake. Nutritional status was normal with no restrictive dietary habits.The patient suffered from a symmetric hypaesthesia of both arms reaching from the fingers up to the middle of the upper arms and double-sided at the thorax from Th2 to Th10. He had a pathological two-point-discrimination at both arms and at the thorax (he only recognized distances >7 cm) and a disturbed stereognosis: the patient was not able to distinguish a pen from a rolled-up bandage. Additionally, he offered a reduced pallaesthesia: vibration sensibility was reduced to 3–4/8 on both sides of the distal radius, and to 0/8 on both malleoli mediales and to 6/8 on both tibiae. Perception of temperature and pain and sense of position were not affected. Further examination did not reveal any abnormalities. Gait was unremarkable, reflexes were normal: There were no pyramidal tract symptoms, no pareses and no mental abnormalities. The patient was of good general condition with a normal weight. He had no glossitis.The outward performed 3 Tesla MRI revealed a hyperintense T2w signal alteration in the dorsal cervical spine, corresponding to an intramedullar lesion reaching from the first to the fifth cervical vertebral body  detected a marginal central-motoric latency to the left arm, indicating a potential pyramidal tract lesion. Somatosensory evoked potentials (SSEP) uncovered a medianus SSEP with marginal values on the left side, normal results on the right side and pathological values of the tibialis SSEP on both sides. Visual evoked potentials (VEP) showed prolonged latencies. In gastroscopy, the mucosa was atrophic. Histology of gastric biopsy revealed a chronic-atrophic inflammation of corpus ventriculi with a lack of intrinsic factor-producing gland cells and with a micronodular hyperplasia of neuroendocrine cells, indicating an autoimmune gastritis.The laboratory diagnostic Table  revealedOur patient was treated with i.m. injections of 1000 µg cobalamin for 5 days. We advised a substitution of 1000 µg cobalamin i.m. once a week for the following 3 months, and afterward of 1000 µg/month i.m. Cobalamin substitution was accompanied by an intake of folic acid . Bimalleolar vibration sense had improved from 0/8 to 4–5/8 as assessed by the scale of the vibration tune .Gastrin level was still elevated , folic acid , cobalamin , homocysteine  and MMA  were of normal range . SACD was diagnosed by clinical signs and laboratory tests. When clinical signs suggest a lack of cobalamin, values of cobalamin might still be in the normal range. Therefore, it is important to determine more sensitive parameters: HoloTC is the earliest and MMA the most specific marker of a cobalamin deficiency. Measurement of homocysteine is inexpensive and therefore suitable for follow-up and therapy monitoring. Early and appropriate treatment reversed pathological changes in the spinal cord and dissolved associated clinical symptoms.


Viruses make headline news on an almost daily basis. Sometimes the news is positive, a report on the development of new anti-viral drugs or a reduction in transmission, perhaps. However, often the story will relate to a gloomier theme, for example, the appearance of new viral epidemics, the evolution of drug resistance, or falling vaccine coverage. The appearance of Ebola virus in West Africa since 2014 represents just the latest of a long series of devastating viruses that have emerged or expanded in humans in recent years, including Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) coronaviruses, Chikungunya virus, highly pathogenic avian influenza viruses, West Nile virus, and various human enteroviruses, and bunyaviruses. This list is both selective and anthropocentric and excludes numerous new epidemics in livestock , crop , and wild animal populations . The impacts of viral epidemics may extend beyond death and illness to cause substantial economic losses and social instability. Such effects are not limited to new or exotic viruses, as established and well-characterized viral diseases persist despite tremendous efforts to control and eradicate them. Important pathogens in this category include HIV/AIDS, human influenza viruses, dengue viruses, hepatitis viruses, human papillomaviruses, and rabies virus.One reason that viruses are such potent adversaries is their great potential for genetic diversity and evolvability, a characteristic that they owe to a combination of short generation times, very large population sizes, and  error-prone replication mechanisms. Strains that escape host immune responses, are resistant to antiviral drugs, or, in the case of plant viruses, that break transgenic or natural genetic resistance, may arise in a viral population soon after it is challenged by the corresponding antiviral measure. Together, the outlook is mixed: the perspectives for future eradication or control are balanced by the emergence or re-emergence of new foes.Although genetic diversity is an essential part of virus biology, classical approaches to virus control often ignore evolutionary processes and focus on understanding in great detail the molecular bases of pathogenesis, virus–host interaction, and drug–virus interference. This is despite the fact that evolutionary concepts are key to the correct interpretation of molecular variation among virus strains. Some experimental biologists, on the other hand, have taken advantage of the rapid evolution of many viruses and chosen to use them as model organisms for the investigation of fundamental questions in evolutionary biology e.g., . MoreoveThe study of virus evolution in its own right has flourished in the last 25 years and the subject undoubtedly gained greater recognition within the biological sciences after the discovery that evolution is an essential component of HIV infection. Since then, the number of published articles that use the term ‘virus evolution’ in the article title or summary has grown exponentially and doubled every 6–7 years a. An equWhere do all these manuscripts get published? Despite an explosion in virus evolution research activity, publications on the topic are scattered among a large number of journals that belong to a variety of categories from the Institute of Scientific Information (ISI). Although many studies appear in evolutionary biology journals, particularly those on viral experimental evolution, mathematical modeling, molecular evolution, and phylogenetics, a large proportion are submitted to journals that focus on virology and pathogenesis. In these disciplines, some editors express a preference for ‘mechanistic’ studies within a clear hypothetico-deductive framework and may not appreciate the importance of inferential and observational work within population and evolutionary biology. Further, several virology journals focus on either animal and plant viruses, so that relevant articles may not come to the attention of researchers from the other field. Viruses of bacteria, archaea, fungi, and protists are served comparatively poorly by the current literature, yet these groups are very likely to comprise the majority of viral genetic diversity on Earth. To add further fragmentation, some important theoretical work on virus variability and evolution is published in specialized mathematical journals that will not be well known to laboratory and field researchers.Virus Evolution with this aim in mind, and we hope that it will grow into a successful and dynamic inter-disciplinary community of researchers interested in understanding why and how viruses have and continue to evolve. We aim to cover all aspects of virus evolution, ecology, and diversity with no restriction on host range or research methodology. To achieve this goal, we have assembled an Editorial Board whose members have made many important contributions to the field. The Board has expertise in animal, plant, and bacterial viruses and in a wide range of techniques, including experimental evolutionary biology, molecular epidemiology, metagenomics, structural biology, population genetics, ecology, and molecular virology. One benefit of a focused journal, such as Virus Evolution, is that the Editorial Board shares with the authorship a passion for the subject.We believe that the study of virus evolution would benefit from a common forum in which findings and ideas can be shared. We have established the journal Virus Evolution exists first and foremost to serve its authors and readers. To make publication in the journal a more pleasant experience, we impose no specific formatting requirements at submission: the manuscript can be provided in any style so long as it is readable by reviewers. Standard formatting will be requested only ‘after’ a paper has been accepted, at which point it should seem less of a chore. We will operate a traditional peer-review process but one that will emphasize the quality of reviews as well as their speed. Published papers will be available to read by all under an Open Access model that is compliant with all major funding bodies including the USA National Institutes of Health and UK Wellcome Trust. Lastly, accepted manuscripts will be visible online in the shortest possible time after acceptance.Our editorial philosophy is that Virus Evolution a vibrant and successful home for your research.We very much look forward to receiving your submissions and to begin working with the community to make


Arthropod-borne viruses (arboviruses) are the largest biologic group of vertebrate viruses and constitute important emerging infectious disease agents globally. Arthropod transmission provides a way for viruses to cross species barriers since the same arthropod may bite animals that rarely or never come into close contact in nature. In Nigeria, arboviruses have, over several decades, caused severe diseases in livestock resulting in great economic losses and, sometimes, infection of humans leading to morbidity and mortality. In the present review, a computerized search of existing literature was conducted using the Google search engine and PubMed electronic database to identify and review relevant publications on arboviral diseases of livestock in Nigeria. The keywords used were ‘arbovirus’, ‘arthropod-borne viral diseases’ or ‘livestock diseases’ and ‘Nigeria’ while the Boolean operator ‘OR’ was used to combine and narrow the searches. Additional information was obtained by searching the veterinary libraries for journals not listed in the database. The available publications were thereafter reviewed and findings qualitatively described. Our findings revealed that although there were several studies on arboviruses and the livestock diseases they cause in Nigeria, most of such reports were made four to six decades ago, with only a few reported recently. Consequently, the true economic and public health impact of these diseases are likely to be underestimated, mainly due to under-reporting or lack of awareness of them. Thus, it is essential to update information on arboviral diseases in Nigeria in order to increase awareness of the diseases and facilitate their prompt identification and reporting. The importance of routine surveillance for arbovirus livestock diseases and sentinel herd monitoring as basis for development of an early warning and alert system to prevent future outbreaks is discussed. Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae and Asfarviridae (Table - ticks) or members of the class Insecta: mosquitoes (family Culicidae), biting midges (family Ceratopogonidae), sand flies (subfamily Phlebotominae) and cimicid bugs (family Cimicidae) . This rhabdovirus is also pathogenic for several laboratory hosts such as mice and cell culture [Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus which is classified as the type species of the genus ph nodes . A uniquph nodes , it is pph nodes , 155. Thph nodes . BEF alsph nodes . BEFV ha culture . The fir culture . This wa culture , who dem culture , the disWesselsbron virus (WSLV), a mosquito-borne flavivirus, is the aetiological agent of a severe disease of sheep characterized by high fever, severe leucopenia in adult sheep, abortion in pregnant ewes and high mortality in lambs. The virus was originally isolated during a severe epizootic in sheep in South Africa  but was An emerging viral disease is one that is newly recognized or newly evolved, or that has occurred previously but shows an increase in incidence or expansion in geographical, host, or vector range . In receBunyaviridae, genus Orthobunyavirus and is an enveloped, negative-sense, segmented, single-stranded RNA virus that is closely related to Shamonda virus, which belongs to the Simbu serogroup of viruses that includes Sango, Sabo and Shuni viruses [in utero [Schmallenberg virus (SBV) was first detected in November 2011 in a pool of blood samples from clinically affected dairy cows following reports of hyperthermia and drop in milk production in adult dairy cows in North-west Germany and The Netherlands . The vir viruses , 170. Si viruses –173. SBV viruses  and has in utero , 176. Thin utero , 178.Culicoides which has been identified as the main vector [Culicoides species identified as vectors for BTV also act as vectors for the transmission of SBV [Several studies have reported the presence of SBV in different species of n vector –182. Then of SBV , 184. Inn of SBV , 185–187n of SBV , SBV antAedes vexans and Culex tritaeniorhynchus) in 1959 [Orthobunyavirus of the family Bunyaviridae, in the Simbu serogroup of viruses [Culicoides-borne virus that replicates in many kinds of natural host species and in several experimental animals [Akabane virus (AKAV) is named after the Japanese village where the virus was first isolated from mosquitoes ( in 1959 . The vir viruses . Akabane animals . Based o animals , 192. AK animals .In Africa, limited information based on virus isolation and/or serology revealed the presence of AKAV in Kenya , 194, SuArthropod-borne viruses cause severe diseases of livestock resulting in great economic losses. Further, such infections often extend to the human population leading to morbidity and mortality with significant public health impact. In Nigeria, rapid urbanization coupled with the encroachment of individuals and their livestock into arthropod habitats has heightened the risks of animal to human transmission of viruses. However, despite these attendant risks and consequences of arboviral infections, the true economic and public health impact of these diseases are most likely underestimated, mainly due to under-reporting of disease events. Therefore, there is a need for increased awareness on the existence of these arboviral agents as well as continuous surveillance for them in order to facilitate their prompt identification and reporting to relevant government agencies. Routine serological, virological, entomological and molecular surveillance for these arboviral agents among livestock herds should be encouraged as their early detection would enable the deployment of pragmatic preventive measures to forestall their ‘escape’ into the human population. In addition, since the incidence of these diseases is generally higher during the rainy season due to increased vector activity, sentinel herd monitoring will yield useful data that can serve as the basis for an early warning and alert system to prevent future outbreaks.


We explored whether analogous mutations in the IBV polymerase subunits would result in a stable virus with an att phenotype. The PB1 subunit of the influenza B/Brisbane/60/2008 strain was used to incorporate ts mutations and a C-terminal HA tag. Such modifications resulted in a B/Bris att strain with ts characteristics in vitro and an att phenotype in vivo. Vaccination studies in mice showed that a single dose of the B/Bris att candidate stimulated sterilizing immunity against lethal homologous challenge and complete protection against heterologous challenge. These studies show the potential of an alternative LAIV platform for the development of IBV vaccines.Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages cocirculate worldwide with little cross-reactivity. Live attenuated influenza virus (LAIV) vaccines have been shown to provide better cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity and systemic B cell- and T cell-mediated memory responses. We have shown previously that incorporation of temperature-sensitive (IMPORTANCE A number of issues with regard to the effectiveness of the LAIV vaccine licensed in the United States (FluMist) have arisen over the past three seasons . While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial, this problem highlights the critical importance of continued investment in LAIV development and creates an opportunity to improve current strategies so as to develop more efficacious vaccines. Our laboratory has developed an alternative strategy, the incorporation of 2 amino acid mutations and a modified HA tag at the C terminus of PB1, which is sufficient to attenuate the IBV. As a LAIV, this novel vaccine provides complete protection against IBV strains. The availability of attenuated IAV and IBV backbones based on contemporary strains offers alternative platforms for the development of LAIVs that may overcome current limitations. Orthomyxoviridae family (––––Influenza B virus (IBV) is an enveloped virus with a negative-sense, segmented, single-stranded RNA genome in the e family . Eight ve family –4. IBV i family ––8. In thfamily ––. For thefamily ––, 10. Phyfamily –––13 that amily ––––14. AlthVaccines against seasonal influenza viruses are manufactured to confer protection against IAVs and IBVs. For protection, current vaccines rely primarily on the antibody response to the viral surface protein hemagglutinin (HA). The antigenic drift of HA requires influenza vaccines to be updated regularly so that they antigenically match the currently circulating strains , 16. Sea11–ca), temperature sensitive (ts), in vivo attenuated (att) viruses that grew well at 25°C . The ca, ts, and att mutations in MDV-B were mapped to PB2 (S630R), PA (V341M), NP , and M1 (H159Q and M183V), whereas those in MDV-A lie within PB2 (N265S), PB1 , and NP (D34G) , recombinant influenza vaccines (rIV), or live attenuated influenza virus (LAIV) vaccines. LAIV vaccines are produced using master donor viruses (MDVs), which carry a series of mutations that restrict virus replication to the upper respiratory tract  . In the  at 25°C –23. AlthP (D34G) –29. Thests phenotype in vitro. However, an adequate att phenotype in vivo was achieved only after the incorporation into PB1 of a C-terminal epitope tag consisting of a 9-amino-acid sequence derived from the H3 HA gene segment (HA tag) in the context of the ts mutations (–att viruses modified as described above (ts mutations plus HA tag) are safe and efficacious as LAIVs for mice, chickens, and swine and are amenable to intranasal (i.n.) administration. For chickens, the IAV att vaccines are also suitable for immunization in ovo  and the three PB1 mutations  from the MDV-A strain  results in viruses with a utations –32. Furtn in ovo –34.ts mutations plus the HA tag in the IAV att strains would result in an att phenotype in the context of a prototypic IBV strain. Mutations analogous to those found in our IAV att alternative live virus vaccine were introduced into a prototypic B/Vic lineage strain, B/Brisbane/60/2008 (B/Bris). Specifically, mutations were engineered in PB2 (K267S) and PB1 . In addition, the PB1 segment was modified with the C-terminal HA tag in the presence or absence of temperature-sensitive mutations. Attempts to maintain the PB2 K267S and PB1 K391E mutations stably in B/Bris failed. However, stability studies showed that the E580G and S660A mutations and the C-terminal HA tag were stably maintained over 15 passages in specific-pathogen-free (SPF) eggs and 20 passages in tissue culture cells. Safety and vaccination studies showed that the B/Bris strain with the PB1 segment carrying the E580G and S660A mutations and HA tag (referred to here as B/Bris att) was stable, attenuated in vivo, and immunogenic. In mouse studies, intranasal immunization with B/Bris att resulted in sterilizing immunity against homologous challenge (with B/Bris), and complete protection was achieved following heterologous challenge with a B/Yam lineage strain, B/Wisconsin/01/2010 (B/Wis). These studies show the potential of the alternative LAIV platform for the development of IBV vaccines.Despite some conserved features in virus structure, genome organization, and the regulation of virus replication and transcription , 35–37, 35–ts phenotype of the MDV-A strain have been mapped to the viral RNP (vRNP) complex and have been shown to be transferable to other IAV backbones (in vivo (defined as either no replication in the lower respiratory tract or a significant reduction in replication). The additional in-frame incorporation of a C-terminal tag in the PB1 segment of IAVs (of either avian or swine origin) was required in order to achieve adequate attenuation in vivo . Attempts to rescue an IBV carrying the PB2 K267S mutation resulted in instability at this position; therefore, our efforts were redirected to producing a virus with alternative PB2 mutations. IBVs carrying either the F406Y or the W359F mutation in PB2 were produced, since either one of those mutations affects the activities of the cap-binding protein and the viral polymerase (att) was stable over several passages in either tissue culture cells or SPF eggs, it was further characterized in order to establish whether it showed a ts phenotype in vitro and whether it was attenuated in vivo.In this study, we sought to evaluate whether analogous modifications and the resulting o an IBV . Specifilymerase . Therefolymerase . Indepenlymerase , 38, theIn vitro characterization of the B/Bris att vaccine candidate began by evaluating polymerase activity (measured by levels of Gaussia luciferase [GLuc] expression) in vRNP minigenome assays (att and wild-type (WT) RG-B/Bris vRNP complexes displayed similar levels of GLuc reporter activity at either 24 hpt  or 48 hpt  . A temperature increase to 37°C further accentuated the statistically significant differences  in levels of luciferase activity between the B/Bris att and WT RG-B/Bris polymerase complexes at both time points analyzed, suggesting that modifications in PB1 confer a ts phenotype on B/Bris att. As expected, at 39°C, both the B/Bris att and WT RG-B/Bris polymerase complexes displayed only residual levels of luciferase activity.e assays  and C. Tts phenotype, the growth kinetics of the B/Bris att and WT RG-B/Bris viruses were evaluated. Confluent monolayers of MDCK cells were infected at a multiplicity of infection (MOI) of 0.01, and virus growth kinetics were monitored at 33, 35, 37, 37.5, and 39°C. Overall, the growth kinetics results showed a trend similar to that observed with the vRNP reconstitution assays. The B/Bris att and WT RG-B/Bris viruses grew to similar titers at 33°C . In contrast to the results of the virus polymerase complex assay, the B/Bris att and WT RG-B/Bris viruses also grew to similar titers at 35°C . At 37°C, the WT RG-B/Bris virus grew to levels similar to those observed at 33°C and 35°C (att virus showed impaired growth at 37°C at 24 (P < 0.01), 48 (P < 0.0001), and 72 (P < 0.001) hpi relative to that of the WT RG-B/Bris virus  relative to that of the WT RG-B/Bris virus  (mock inoculation), 106 50% egg infective doses (EID50) of the B/Bris att virus, or 106 EID50 of the WT RG-B/Bris virus. Clinical signs of disease, body weight changes, and mortality were recorded daily. Body weight monitoring revealed no weight loss following inoculation of the B/Bris att vaccine candidate  were euthanized at 3 and 5 days postinoculation (dpi) in order to determine the levels of virus replication in nasal turbinates (NT) and lungs . Both the B/Bris att and the WT RG-B/Bris virus replicated well in the upper respiratory tract, as evidenced by virus detection in nasal turbinates  (P < 0.05) and 5 (P = 0.001) dpi. These results are further supported by the histopathology findings for lung samples collected at 5 dpi. Lungs were scored on a scale from 0 to 4, where a score of 4 indicates severe lung pathology. Mice that were either mock inoculated or inoculated with the B/Bris att virus had an average whole-lung score of 0 at 5 dpi  . These r   at 3  had HI antibody titers of ≥40 against the homologous virus  were challenged at 21 dpi with 107 EID50 per mouse of the B/Bris PB2-F406Y virus via the i.n. route. The B/Bris PB2-F406Y virus was chosen over the WT B/Bris virus because of its increased virulence (n = 16). B/Bris att-vaccinated mice had no apparent signs of disease following the B/Bris PB2-F406Y virus challenge. The changes in body weight in the vaccinated and challenged mouse group were indistinguishable from those seen in the unchallenged control group  (att-vaccinated mice at either 3 or 5 dpc (att-vaccinated mice showed an increase in HI antibody titers against the homologous B/Bris virus at 21 dpc (att vaccine candidate confers sterilizing immunity against the homologous virus on mice.To assess the efficacy of the B/Bris irulence . An addiol group . All B/B 0.0001) . In cont 0.0001) . Lungs aor 5 dpc  and D. Aor 5 dpc  and D. It 21 dpc . Overallatt-vaccinated mice were challenged at 21 dpi with 107 EID50 per mouse of the B/Wis PB2-F406Y virus. All B/Bris att-vaccinated mice (n = 8) survived the challenge with the heterologous B/Wis PB2-F406Y virus, whereas ∼40% of challenged mock-vaccinated mice (P < 0.05) either succumbed to virus infection or were euthanized due to severe clinical disease and/or weight loss  and nasal turbinate  tissues collected showed lower virus loads in B/Bris att-vaccinated mice than in challenged mock-vaccinated mice  cleared the challenge virus infection by 5 dpc (Live attenuated influenza virus (LAIV) vaccines have been shown to provide more–cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity in addition to systemic B and T cell-mediated responses. In order to evaluate the extent of cross-protection, mock- or B/Bris ght loss  and B. Ihallenge . At 3 anted mice  and D. At 21 dpc . B/Bris by 5 dpc  and D, sby 5 dpc .att-vaccinated mice challenged with the B/Bris PB2-F406Y virus received an overall histopathology score of 0, like lungs from mock-vaccinated, mock-challenged mice  were milder than those for the challenged mock-vaccinated group  or in MDCK cells . Following serial passage, isolated RNA from both allantoic fluid and the tissue culture supernatant was subjected to whole-IBV-genome amplification and next-generation sequencing (NGS). Sequencing analysis of the PB1 segment confirmed that all modifications introduced  were stably maintained following successive passages in SPF eggs and/or MDCK cells . More importantly, no additional mutations were found within the sequenced PB1 segment from nasal turbinate samples. These results emphasize the safety and stability of the B/Bris att virus in vivo.Ascertaining genetic stability remains an essential step in the development of LAIV candidates. Thus, the B/Bris CK cells . In PB1,CK cells . The vRNatt. The selection of mutations in PB2 and PB1 and the incorporation of the HA epitope tag into PB1 were based on our previous experience with IAV att virus vaccines and the assumption that the analogous amino acids in IBV overlap regions with similar functions. The original goal was to incorporate all possible analogous mutations; however, a limited set of those were tolerated, were stable, and provided the desired attenuated phenotype. The PB2 K267S mutation (N265S in the IAV PB2) was deleterious for virus rescue but not for polymerase activity, which was reduced in the context of PB1 att modifications, in minigenome assays (not shown). Surprisingly, alternative mutations in PB2, previously known to impair cap-binding activity (F406Y or W359F), did not attenuate B/Bris virulence in mice but instead increased it over that of the WT strain. It is well established that IBV strains are usually not lethal in mice unless the virus is forcibly adapted in this species. We took advantage of these new findings and used the B/Bris PB2-F406Y virus for lethal challenge in subsequent vaccine efficacy studies. Similarly, the B/Wis PB2-F406Y virus was created for the heterologous challenge, which was more lethal to mice than the WT B/Wis virus but less lethal than the B/Bris PB2-F406Y virus.This study describes the development of an IBV vaccine candidate with a modified PB1 segment, B/Bris att virus within and beyond the engineered mutations and HA tag addition. Whole-genome sequences of the early passages of the B/Bris att virus obtained from SPF eggs or tissue culture cells were compared to those obtained by passaging the virus multiple times in either SPF eggs or MDCK cells. These sequences were further compared to IBV PB1 sequences obtained from the Influenza Research Database (IRD) (www.fludb.org). More than 4,000 complete IBV PB1 sequences are available in the IRD, but only 793 differ from each other in at least one amino acid, demonstrating remarkable stability for this particular segment.Sanger-based sequencing and next-generation sequencing (NGS) were utilized to better understand the stability of the B/Bris ts (no HA tag) that also maintained the K391E mutation after 9 serial passages in eggs (sE9) (data not shown). The PB1 E48K mutation is unique to the recombinant virus prepared in this study; no other IBV PB1 segment appears to favor such a mutation in nature. How the PB1 E48K mutation could affect the virus phenotype in vitro and in vivo requires further studies beyond the scope of the present report.The PB1 K391E and E580G mutations (the same as in IAV PB1) were tolerated and resulted in adequate polymerase activity; however, the K391E mutation was not always stably maintained in SPF eggs. This mutation was present in viruses grown in MDCK cells (P1) but was lost upon passage in SPF eggs (E2). The reason for the instability of the K391E mutation in SPF eggs is unknown. Further studies will be required, particularly considering the fact that the mutation was stable in virus serially passaged in MDCK cells (sP1 through sP20). The emergence of the PB1 E48K mutation may have played a compensatory role and contributed to maintaining the K391E mutation in MDCK cells. It must be noted that the PB1 E48K mutation also emerged in an alternative virus rescue iteration of B/Bris att virus, although it does not appear to be highly favored in nature: only 4 of the 793 unique IBV PB1 sequences analyzed contained G580 . The PB1 G580 mutant strains were not found to be associated with any particular season or location; they represent isolates cocirculating with the more-favored PB1 E580 (wild-type) strains. It would be of interest to determine whether naturally occurring strains containing the PB1 G580 mutation are less fit for growth in vitro or in vivo than the more-common PB1 E580 strains. If this is the case, it will support the notion that the PB1 G580 mutation contributes to the safety of the B/Bris att backbone.The PB1 E580G mutation was stably maintained in MDCK cells, in SPF eggs, and after replication for at least 5 days in mice. This mutation is not unique to the B/Bris att virus, was stable over multiple serial passages in either SPF eggs or MDCK cells and contributed to the ts phenotype of B/Bris.The design of the PB1 S660A mutation (A661T in IAV PB1) was counterintuitive. In IAV, the hydrophobic-to-polar amino acid change in the PB1 A661T mutation contributes to its attenuation. We simply assumed that the opposite change in IBV PB1, the polar-to-hydrophobic amino acid mutation S660A, would be stable and would contribute to the virus's attenuation. In fact, the S660A mutation, unique to the B/Bris in vivo. The combination of the E580G and S660A mutations with the C-terminal HA tag in PB1 yielded the B/Bris att virus with a ts phenotype in vitro and attenuation in vivo. The ts phenotype was evident by reduced polymerase complex activity and decreased virus growth kinetics at elevated temperatures (≥37°C). The defective replication of the B/Bris att virus in the lungs of mice suggests that the virus is attenuated in vivo. The PB1 E580G, S660A, and HA tag modifications were stably maintained not only after multiple passages in either SPF eggs or MDCK cells but also for at least 5 days in viruses isolated from the nasal turbinates of infected mice.The incorporation of the C-terminal HA tag in B/Bris PB1 was also well tolerated and remarkably stable over multiple serial passages in either SPF eggs or MDCK cells. Just as in the modified IAVs, the HA tag does not severely affect the polymerase activity of IBV and is not sufficient to attenuate the virus att virus showed one additional amino acid mutation in PB1, V474I. The presence of PB1 I474 is fairly common among IBV field isolates. Neither V474 nor I474 is fixed in the IBV PB1 population. PB1 I474 is present in 100% of strains isolated from 1940 until 2000, including the cold-adapted B/Ann Arbor/1/66 strain. Therefore, it is safe to speculate that the V474I mutation is unlikely to affect the in vitro or in vivo phenotype of B/Bris att. It remains to be determined whether the V474I mutation reflects egg-grown adaptation. In addition, in the sE15 virus, single amino acid mutations were found in PA (K489Q) and NP (D377N). The PA K489Q mutation appears to be unique to the sE15 B/Bris att virus, whereas the NP D377N mutation is found in at least one field isolate. The effects of these mutations on virus attenuation are unknown. Other segments remained unchanged; neither amino acid nor nucleotide changes from the predicted wild-type sequence were observed in the sE15 virus. Likewise, no amino acid changes from predicted sequences were observed in the PB2, PA, HA, NP, NB, M, BM, and NEP open reading frames (ORFs) of the sP20 virus. The sP20 virus did show two amino acid changes from the sP1 virus, one in NA (K371E) and one in NS1 (M106T). Neither of these mutations is unique to the sP20 virus, and the NA K371E mutation is fairly common among IBV strains. Next-generation sequencing allows for an unprecedented level of detail in examining the stability of live virus vaccines. Although amino acid changes were observed upon serial passage, the B/Bris att virus showed no changes in HA and great stability at the engineered sites in PB1. Other changes do not appear to change the virus's attenuated phenotype.The sE15 B/Bris att virus are ideal for a safe LAIV vaccine  cells in the lungs after influenza virus infection -mediated immune responses, instead of simply the humoral (IgG) response associated with inactivated vaccines and some universal vaccines  backbones, highlight the critical importance of continued investment in LAIV development and create an opportunity to improve the current strategy so as to develop more-efficacious vaccines. A variety of strategies have been employed to develop alternative experimental LAIV vaccines. Such strategies include, but are not limited to, whole and partial gene knockouts, the insertion of foreign sequences, and manipulation of the HA cleavage site. Extensive work has been done on NS, M, NA, and PB2 partial and full knockout vaccines, and while such vaccines have proven effective, there are drawbacks (–in vitro (A number of issues with regard to the effectiveness of the licensed LAIV vaccine in the United States (FluMist) have arisen over the past three seasons . While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial and are a subject of ongoing investigation, possible culprits include the suboptimal performance of the H1N1 component, the inclusion of a second IBV strain, and reduced immunogenicity due to sequential exposure to the same LAIV vaccine backbones over the years , 56. Therawbacks –59. For in vitro , 61. Finin vitro , 60, 62,in vitro .in ovo as well as in mouse, chicken, and pig models. Furthermore, we have shown that viruses generated using this strategy grow to high titers in established cell lines and SPF eggs  are analogous to a subset of the mutations present in the A/Ann Arbor ca backbone. The availability of contemporary attenuated IAV and IBV (this study) backbones offers an alternative platform for the development of LAIVs that may overcome current limitations.Our laboratory has developed an alternative IAV strategy, which incorporates the PB2 and PB1 mutations found in the A/Ann Arbor cold-adapted backbone , 28. Add30– eggs 30–. The cur2 atmosphere. The B/Brisbane/60/2008 (WT B/Bris) and B/Wisconsin/01/2010 (WT B/Wis) influenza B virus strains were a gift from Ruben Donis, Centers for Disease Control and Prevention, Atlanta, GA. Virus stocks were amplified in specific-pathogen-free (SPF) embryonated chicken eggs  and were stored at −80°C. The viruses used in this report are summarized in Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution . Cells were propagated at 37°C in a humidified incubator under a 5% COFive- to 6-week-old female DBA/2J mice  were used in all mouse experiments. All animal studies were performed under animal biosafety level 2 (ABSL-2) containment conditions according to protocols approved by the respective Institutional Animal Use and Care Committees (IACUC) at the University of Georgia and the University of Maryland, College Park. Mice that experienced significant weight loss (21 to 25%) or scored 3 or higher on a 4-point scale of disease severity were humanely euthanized.att), a modified HA tag was first cloned in-frame at the C terminus of PB1 by inverse PCR. The cloning strategy was based on the HA tag cloning procedure described previously  corresponding to the modified HA tag. Subsequently, the K391E, E580G, and S660A mutations were introduced into PB1 via site-directed mutagenesis using PCR. The K267S, F406Y, or W359F mutation was introduced into the PB2 segment of the B/Brisbane/60/2008 genome by site-directed mutagenesis with the QuikChange II XL kit . PCRs were performed with either Pfu Ultra DNA polymerase AD  or Phusion High Fidelity DNA polymerase . All plasmids constructed were sequenced, and no unwanted mutations were identified. A schematic overview of all the modifications introduced into the IBV genome and their respective outcomes is given in The reverse genetics system for the WT B/Bris strain has been described elsewhere . The HA eviously , with miatt, B/Bris PB2-F406Y, and B/Bris PB2-W359F viruses, the respective mutant plasmid was paired with the 7 remaining WT B/Bris plasmids. The B/Wis PB2-F406Y virus is a 2:6 reassortant carrying the surface genes of WT B/Wis and the internal gene constellation of the B/Bris PB2-F406Y virus. Following transfection, transfected cells were incubated at 35°C. After 24 h of incubation, the medium was replaced with Opti-MEM I  containing 1 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin  and antibiotic/antimycotic solution. Following virus rescue (passage 0 [P0]), the tissue culture supernatant was used to inoculate fresh MDCK cells to amplify the virus once (P1). Viruses in P1 were used for further amplification in MDCK cells (P2) and then in SPF eggs (E1). The viruses obtained in E1 were used to grow virus stocks in SPF eggs (E2). P1, P2, E1, and E2 virus amplifications were carried out at 33°C. Virus stocks were titrated both by 50% tissue culture infectious doses (TCID50) and by 50% egg infectious doses (EID50). Virus titers were determined by the Reed-Muench method (Viruses were rescued using a coculture of 293T and MDCK cells as described previously . To geneh method .att virus (E2) was serially passaged either as many as 20 times in MDCK cells or as many as 15 times in SPF eggs. RNA from tissue culture supernatants and allantoic fluid, respectively, was purified using the RNeasy minikit . Isolated RNA served as the template for the amplification of the whole IBV genome by one-step reverse transcription-PCR (RT-PCR) (att virus following immunization.All the virus stocks were sequenced by either Sanger sequencing or next-generation sequencing (NGS), or both, to assess the presence of the inserted mutations in rescued viruses. To ascertain the stability of attenuation markers, the B/Bris (RT-PCR)  and NGS (RT-PCR) . Additio(RT-PCR)  and was Gaussia luciferase (GLuc) reporter plasmid (pBNPGLuc) was constructed. The open reading frame (ORF) of GLuc was amplified by PCR from pGLuc-Basic  and was subcloned into pDP2002. The plasmid was engineered such that the GLuc ORF was flanked by the 5′ and 3′ untranslated regions (UTRs) of the IBV NP segment. This construct was further modified by removing the pCMV promoter immediately upstream of the 5′ UTR. The resulting plasmid was verified by sequencing, which revealed no unwanted modifications. The vRNP minigenome assay was reconstituted in 293T cells cotransfected with pCMV-SEAP, pBNPGLuc, and B/Bris PB2, PB1, PA, and NP plasmids using the TransIT-LT1 transfection reagent  according to the manufacturer's instructions. The pCMV-SEAP plasmid expresses the secreted alkaline phosphatase and was used to normalize for transfection efficiency. At 24 and 48 hpt, tissue culture supernatants from transfected cells were collected to measure reporter activity using a Victor X3 multilabel plate reader . GLuc activity was assessed using the Biolux Gaussia luciferase assay kit , while phosphatase activity was measured using the Phospha-Light SEAP reporter gene assay system . Experiments were carried out independently at least twice, and all transfection conditions were tested in triplicate per experiment.To assess viral polymerase activity, a ts, B/Bris att, or 7attWF10:1malH7) at an MOI of 1 for 1 h at 37°C. The 7attWF10:1malH7 virus is a reassortant IAV att control virus that has been described previously . Each mouse was anesthetized with isoflurane and was subsequently inoculated via the intranasal (i.n.) route. Group 1 received 50 μl of PBS and served as a control group. Each mouse in group 2, 3, or 4 was inoculated with 105 EID50 of the B/Bris PB2-F406Y, B/Bris PB2-W359F, or WT RG-B/Bris virus, respectively, in 50 μl of PBS. Clinical signs of disease, body weight changes, and mortality were monitored daily.To evaluate the impact of the PB2 F406Y and W359F mutations on increased IBV virulence, DBA/2J mice  were randomly distributed into 4 groups  were randomly distributed into 5 groups . Each mouse was anesthetized with isoflurane and was subsequently inoculated i.n. Groups 1, 2, and 3 received 50 μl of PBS alone as mock-vaccinated control groups. Groups 4 and 5 were inoculated with 106 EID50 per mouse of the B/Bris att virus in 50 μl of PBS. An additional group (n = 8) received 106 EID50 per mouse of the WT RG-B/Bris virus and served as a control for in vivo evaluation of the attenuated phenotype of B/Bris att. At 3 and 5 days postinoculation (dpi), 3 or 4 animals from each group were humanely euthanized for tissue collection. Nasal turbinates and lungs were collected for virus titer quantification and histopathological examination. The day before challenge (20 dpi), mice were bled from the submandibular vein to measure neutralizing antibody responses. At 21 dpi, group 1 remained unchallenged, while groups 2, 3, 4, and 5  were challenged i.n. with 107 EID50 per mouse of either the B/Bris PB2-F406Y or the B/Wis PB2-F406Y virus in 50 μl of PBS. Natural isolates of IBV are usually not lethal to mice unless the virus is serially adapted in this species or a high virus dose is used for inoculation. The increased virulence observed in mice inoculated with the B/Bris PB2-F406Y or B/Bris PB2-W359F virus  were bled from the submandibular vein for serology. Clinical signs of disease, body weight changes, and mortality were monitored daily throughout the study.To evaluate the immune response and protective efficacy of B/Bris 9F virus  and B prSerum samples collected at 20 dpi and at 21 dpc were assayed for the presence of neutralizing antibodies by an HI assay as described elsewhere  using thAt 5 dpc, lungs from mice euthanized postvaccination and postchallenge were collected and preserved in 10% formalin for histopathological examination by hematoxylin-and-eosin (H&E) staining. Tissue sections from bronchi/bronchioles, pulmonary vasculature, and alveoli were examined, and the overall extent of pulmonary lesions was scored on a scale from 0 to 4, based on increasing levels of tissue damage and inflammation. Representative images of lung histopathology were taken at ×20 magnification.in vitro assays were performed at least twice in triplicate. For multiple comparisons, two-way analysis of variance (ANOVA) was performed, followed by a post hoc Bonferroni test. Differences in survival curves were analyzed using the log rank test. A P value below 0.05 was considered significant.All data analyses were performed using GraphPad Prism software, version 7 . All


In December 2016, the Wisconsin Department of Health Services (WDHS) notified CDC of a patient hospitalized with fever, leukopenia, elevated transaminases, and proteinuria. The patient owned and operated an in-home rattery, or rat-breeding facility, with approximately 100 Norway rats, primarily bred as pets. A family member developed similar symptoms 4 weeks later, but was not hospitalized. Because both patients were known to have rodent contact, they were tested for hantavirus infections. In January 2017, CDC confirmed recent, acute Seoul virus infection in both patients. An investigation was conducted to identify additional human and rat infections and prevent further transmission. Ultimately, the investigation identified 31 facilities in 11 states with human and/or rat Seoul virus infections; six facilities also reported exchanging rats with Canadian ratteries. Testing of serum samples from 183 persons in the United States and Canada identified 24 (13.1%) with Seoul virus antibodies; three (12.5%) were hospitalized and no deaths occurred. This investigation, including cases described in a previously published report from Tennessee . Rats infected with Seoul virus are asymptomatic, but can transmit the virus to humans through infectious saliva, urine, droppings, or aerosolization from contaminated bedding. Human signs and symptoms range from mild influenza-like illness to hemorrhagic fever with renal syndrome (HFRS). HFRS causes acute renal failure and can result in death; however, asymptomatic Seoul virus infections also occur. Wild Norway rats in the United States have been known to harbor Seoul virus infection  and from (trace-forward) the rattery to identify suspected and confirmed facilities. Trace-back investigations initially extended back 2 months prior to onset of clinical disease, based on the known maximum incubation period for Seoul virus in humans. As additional confirmed facilities were identified, tracing focused instead on interactions with known infected facilities, sometimes as much as 1 year prior. Suspected facilities included ratteries, homes, or pet stores that sold rats to a confirmed facility (a facility where at least one human or rat tested positive for Seoul virus infection) or housed rats that lived at or comingled with rats from a confirmed facility. Once a suspected facility was identified, local or state health officials interviewed persons with a history of rodent contact associated with the facility about their rat exposure and health history. Additionally, the primary rodent caretaker was interviewed using a standardized questionnaire to identify movement of rats into and out of the facility, including dates and locations where the rats were obtained. Local or state health officials offered laboratory testing for Seoul virus infection to all persons with rodent contact. Officials recommended testing for persons with a history of febrile illness and exposure to rats from a confirmed facility and for rats at suspected and confirmed facilities. Trace-forward and trace-back investigations of rat shipments at confirmed facilities identified additional suspected facilities, which were similarly assessed.A suspected human case of Seoul virus infection was defined as a febrile illness (recorded temperature >101°F [38.3°C] or subjective history of fever) or an illness clinically compatible with Seoul virus infection  without laboratory confirmation in a person reporting contact with rats from a confirmed or suspected facility. Human Seoul virus infections were laboratory-confirmed by detection of Seoul virus-specific immunoglobulin M (IgM) and/or immunoglobulin G (IgG) (By March 16, 2017, trace-forward and trace-back investigations identified approximately 100 suspected facilities in 21 states. Among these, 31 facilities in 11 statesOn January 24, CDC issued a Health Alert Notice to notify health departments and health care providers of the Seoul virus investigations.Health departments notified suspected and confirmed facilities and placed those facilities under quarantine, allowing no rats to enter or leave. Rat contact was limited to as few persons as possible to reduce transmission. In suspected facilities, CDC recommended rat testing be performed under the supervision of a public health official or licensed veterinarian. The quarantine was lifted when at least 4 weeks had elapsed since the newest animal was introduced, and all rats subsequently tested negative. Rats belonging to owners who refused to test their animals could remain quarantined for life or be euthanized. CDC recommended euthanasia of all rats in confirmed facilities as the most effective method to prevent transmission, although control recommendations differed by state and country according to local policies and response capacities. If euthanasia was not possible, then owners could either quarantine all rats for life or pursue quarantine with testing and culling. The testing and culling strategy entailed testing all rats and euthanizing only infected rats. Testing and euthanasia were repeated at 4 week intervals until all rats tested negative and the quarantine was lifted. In Canada, public health officials opted for education and a voluntary testing and culling approach to control Seoul virus transmission.This outbreak report, in parallel to the previously described investigation in Tennessee (,Pet rat owners should be aware of the potential for Seoul virus infection. To keep themselves and their pets healthy, all persons with rodent contact should avoid bites or scratches and practice good hand hygiene, especially children and persons with compromised immune systems (Rattery owners are encouraged to quarantine any newly acquired rats for 4 weeks and to test these rats for Seoul virus antibodies before allowing them to comingle with other rats. Commercial laboratories can perform Seoul virus testing of rodent blood samples, and comparisons of results from shared samples have been concordant with CDC’s ELISA and RT-PCR assays. To prevent transmission to humans, CDC recommends euthanasia of all rats in facilities with human or rat Seoul virus infections. Further guidance on methods to eradicate Seoul virus from infected ratteries should be obtained from local or state health departments.Seoul virus, a type of hantavirus, is carried by Norway rats. Humans become infected through contact with virus shed in rat urine or droppings, or inhalation of virus particles in dust from contaminated bedding. Infected rats do not develop disease, but humans can experience symptoms ranging from mild influenza-like illness to severe disease with kidney failure and death. Although infections have been previously reported in humans after contact with wild rats, Seoul virus infections had not been reported in pet rats in the United States or Canada.This report describes the first known outbreak of Seoul virus infections in humans from contact with pet rats in the United States and Canada. This investigation identified 31 U.S. facilities with human and/or rat Seoul virus infections in 11 states, including six that exchanged rats with Canadian ratteries. Seventeen persons had recent infection with Seoul virus, eight became ill, and three were hospitalized and recovered.Human hantavirus infections are reportable to state or local health departments in the United States. Clinicians should consider Seoul virus infection in patients with a history of rat contact and compatible symptoms. Pet rat owners and breeders should also be aware of Seoul virus and should practice good hand hygiene and safe rodent handling to prevent infection.


Recent studies indicated that the Zika virus genome could be detected in the amniotic fluid and the fetal brain, which confirms that the virus can cross the placental barrier. Secretory autophagy or exosome pathways may participate in this virus transfer. Autophagy modulators regulate autophagosome formation or membrane fusion with lysosomal vesicles and therefore inhibit viral nucleocapsid releasing or virus transfer to the fetus hypothetically. However, some autophagy modulators may enhance virus replication. Autophagy inhibitors may arrest placental development; while exaggeration of autophagy in human placenta may be associated with the fetal growth restriction. Therefore, autophagy modulators should be used carefully due to their complex clinical effects. Alternatively, exosome-specific inhibitors might be also considered, although their safety of both maternal and fetal conditions must be carefully assessed before any advancement to human clinical trials. A widespread epidemic of Zika virus (an emerging mosquito-borne flavivirus) infections was found in Central and South America recently. The main concern is the significant increasing of microcephaly in the fetus born to the mother of Zika virus infection  pathways, may enhance antigen presentation and processing and improve maintenance of memory CD8 cytotoxic T lymphocytes after influenza virus infections Fedson, . CarbamaAmong the common autophagy stimulators, only rapamycin (a mTORC1 inhibitor) has been shown to inhibit exosome release evidently  may be only one of the complex mechanisms of Zika virus penetration through the placental barrier. Zika virus infection triggers apoptosis and vascular damages in the placenta, which may increase the permeability of the placenta (Miner et al., When Zika virus has penetrated the placenta, it may damage the fetal brain directly or indirectly, because of its neurotropic property (Mlakar et al., ZZ contributed to the discussion of ideas and helped with the writing. ZL contributed to the discussion of ideas and the writing. SY conducted the literature search and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IEC and handling Editor declared their shared affiliation and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Aedes aegypti mosquitoes. To determine the extent to which Ae. albopictus mosquitoes from the United States are capable of transmitting Zika virus and the influence of virus dose, virus strain, and mosquito species on vector competence, we evaluated multiple doses of representative Zika virus strains in Ae. aegypti and Ae. albopictus mosquitoes. Virus preparation (fresh vs. frozen) significantly affected virus infectivity in mosquitoes. We calculated 50% infectious doses to be 6.1–7.5 log10 PFU/mL; minimum infective dose was 4.2 log10 PFU/mL. Ae. albopictus mosquitoes were more susceptible to infection than Ae. aegypti mosquitoes, but transmission efficiency was higher for Ae. aegypti mosquitoes, indicating a transmission barrier in Ae. albopictus mosquitoes. Results suggest that, although Zika virus transmission is relatively inefficient overall and dependent on virus strain and mosquito species, Ae. albopictus mosquitoes could become major vectors in the Americas.In the Western Hemisphere, Zika virus is thought to be transmitted primarily by  Flaviviridae, genus Flavivirus) is the latest in a series of arboviruses to successfully invade the Americas; cases locally acquired in the Western Hemisphere were first identified in Brazil in May 2015 (http://www.paho.org). Zika virus was first isolated in Uganda in 1947 .The New York State Department of Health (NYSDOH) Arbovirus Laboratory isolated Zika virus HND  from serum from a patient who had traveled to Honduras in early 2016. Amplification was obtained by inoculating 100 μL of serum into shell vials  confluent with Vero cells , followed by centrifugation at 700 × t-test. Growth kinetics were compared by using repeated measured analysis of variance (ANOVA) and Tukey post hoc tests .We inoculated confluent monolayers of Vero and C6/36 cells with Zika virus strains in duplicate at a multiplicity of infection of 0.01 PFU/cell. After a 1-hour absorption period at 37°C (Vero) or 28°C (C6/36), the inoculum was removed and cells were washed twice with appropriate maintenance media. Cultures were maintained in 6-well plates with 3 mL of maintenance media  and incubated at 37°C (Vero) or 28°C (C6/36). Samples of 100 μL supernatant were harvested at days 1–4 (Vero) or 1–7 (C6/36) after infection, diluted 1:10 in media containing 20% fetal bovine serum, and stored at −80°C. Titrations were performed in duplicate, by plaque assay on Vero cells  were originally collected in Suffolk County in 2014 and subsequently colonized in the NYSDOH Arbovirus Laboratory. F5–F7 female mosquitoes from New York were used for experimental feedings. Ae. aegypti mosquitoes used for preliminary experiments were collected by C. Mangudo in Salta, Argentina, in 2014 and initially colonized by V. Micieli and L.D. Kramer at the Centro de Estudios de Parasitología y Vectores  before being shipped to the NYSDOH Arbovirus Laboratory for maintenance. F4–F5 females from Argentina were used for experimental feedings. Ae. aegypti mosquitoes  were originally collected in Poza Rica, Mexico. F7–F8 females from Mexico were used for experimental feedings. For preliminary blood feeding experiments, Ae. aegypti mosquitoes from Argentina were fed Zika virus PR stock virus diluted 1:1, 1:5, or 1:20 in defibrinated sheep blood  with 2.5% sucrose. For feedings with freshly propagated virus, supernatant from infected C6/36 cultures was harvested at 96 h after infection (multiplicity of infection ≈1.0) and diluted 1:1 with blood-sucrose mixture without freezing. Female mosquitoes, 4–7 days of age, were deprived of sucrose for 18–24 h and offered blood meal mixtures by use of a Hemotek membrane feeding system  with a porcine sausage casing membrane. For all subsequent experiments assessing dose-dependent vector competence, similarly prepared fresh C6/36 cultures of Zika virus HND and Zika virus CAM were used to feed Ae. aegypti mosquitoes from Mexico and Ae.albopictus mosquitoes from New York. In addition to undiluted supernatant, 1:20, 1:400, and 1:8,000 dilutions were made in C6/36 maintenance media before being mixed with blood. 2 after 1 h of feeding, and fully engorged mosquitoes were transferred to 0.6-L cartons and maintained at 27°C for experimental testing. Infection, dissemination, and transmission rates were determined as previously described . Zika virus HND was also modestly attenuated in mammalian cell culture, replicating to titers 2–3.3-fold lower than the titers achieved by Zika virus PR and Zika virus CAM, respectively . Peak titer for Zika virus CAM was statistically higher than that for Zika virus HND (p = 0.04 by t-test), yet Zika virus PR replicated to an intermediate value and was statistically equivalent to both Zika virus HND and CAM.Sequencing analysis of these strains revealed 1.7% divergence and 16 aa differences distributed throughout the genome . These dAe. aegypti mosquitoes. No infection was identified at 14 days after feeding for mosquitoes fed 6.0 log10 PFU/mL, and only 3 (10%) of 30 mosquitoes were Zika virus positive when the dose was increased to 7.4 log10 PFU/mL  of 25 mosquitoes were Zika virus positive. Of the 24 positive mosquitoes, 22 (91.6%) had disseminated infections and 13 (54.2%) had Zika virus–positive saliva. To clarify the extent to which differences in infectivity were the result of virus titer or preparation (freshly propagated vs. frozen virus stocks), we fed a subset of mosquitoes the same blood meal (titer 9.1 log10 PFU/mL) after freezing at −80°C for 2 weeks. Although feeding rates were poor and survival was low for this cohort (n = 12), only 2 of the mosquitoes surviving to day 14 after feeding were Zika virus positive, which equated to a significantly lower infection rate than that obtained with freshly propagated virus  and 8.6 (Zika virus CAM) log10 PFU/mL were used for Ae. albopictus feedings . Although differences were also measured at the lower doses, deviation is higher and statistical power is constrained by the smaller sample sizes. Viral loads among mosquitoes of each species were similar for Zika virus CAM, yet significantly higher than Zika virus HND in Ae. albopictus mosquitoes (t-test), indicating an influence of mosquito species and of virus strain on Zika virus replication.After isolation, Zika virus HND was used as the representative Western Hemisphere strain and compared with Zika virus CAM for all experiments assessing the relationships between dose, vector competence, virus strain, and mosquito species. To maximize transmission potential, we waited until day 21 after mosquito feeding to conduct these studies. For feedings . For ZikAe. albopictus mosquitoes. This difference was highly significant for the Zika virus HND 1:20 dilution (≈7.5 log10 PFU/mL), for which 93.3% of Ae. albopictus mosquitoes were infected compared with 46.7% of Ae. aegypti mosquitoes (p<0.001 by Fisher exact test). Although the higher infection rate measured in Ae. albopictus mosquitoes was not significant at the 1:400 dilution , it is notable that the input titer was ≈5-fold lower for the Ae. albopictus mosquito feeding (6.6 vs. 5.9 log10 PFU/mL), consistent with the increased infectiousness of Zika virus HND in this species.Infection rates for high-dose feedings were 80%–100% . Suscept50, DD50, TD50, respectively; Ae. albopictus relative to Ae. aegypti mosquitoes for both strains. Infectiousness was determined both by statistical comparison of Y-intercepts by linear regression analyses (p<0.05) and comparisons of calculated ID50s. The ID50 of Zika virus HND was found to be greater than a log lower in Ae. albopictus compared with Ae. aegypti mosquitoes (6.1 vs. 7.5 log10 PFU/mL) and ≈5-fold lower for Zika virus CAM  was with Zika virus CAM in Ae. aegypti mosquitoes. Despite increased infection rates for Ae. albopictus mosquitoes, dose dependence for Zika virus HND transmission was significantly lower than that for Ae. aegypti mosquitoes . Previous studies have noted differences in arbovirus infectivity and vector competence when use of artificial feeding was compared with feeding on experimentally infected hosts  adjacent to New York City, which is among the largest centers for the movement of Zika virus–exposed travelers. Indeed, the highest number of confirmed travel-associated cases of Zika virus infection in the United States are reported from New York state (http://www.cdc.gov/zika/geo/united-states.html). The combination of the recent success of Ae. albopictus mosquitoes in the region (http://diseasemaps.usgs.gov). Despite this potential vulnerability, transmission intensity is dependent on more than host and vector competence and it is the frequent and highly anthropophilic feeding of Ae. aegypti mosquitoes that often results in their relative success as vectors, even in the absence of high competence (Ae. albopictus mosquitoes does not translate to increased overall competence; transmission efficiency was significantly higher for Ae. aegypti mosquitoes. These data are consistent with a midgut escape barrier and, more significantly, with a salivary gland infection or transmission barrier. Although it is well documented that these barriers are capable of preventing transmission (Ae. albopictus mosquitoes is notable because the increased infectivity we measured in Ae. albopictus mosquitoes translates to more opportunity for adaptive events that could increase fitness or competence over time. The potential epidemiologic consequences for adaptation to Ae. albopictus mosquitoes are well documented for chikungunya virus, for which primary and secondary epistatic mutations increased competence in mosquitoes of this species and facilitated the explosive outbreaks in the islands of the Indian Ocean and beyond (Ae. albopictus mosquito populations, together with continued genetic and phenotypic monitoring of circulating strains, will help elucidate the potential for similar adaptive events enabling increased transmission efficiency of Zika virus in the Americas.We have demonstrated that US populations of


Ixodes scapularis, and the availability of tick-cell lines as convenient models to investigate interactions. One of the by-products of these investigations has been the observation of familiar proteins in new situations. One such protein family is Toll and Toll-like receptors that in vertebrates play a key role in detection of microorganisms, including viruses. But does Toll signaling play a similar role in detection of virus infection in ticks, and if it does, how does this affect the maintenance of viruses within the tick?Ticks are important vectors of viruses that infect and cause disease in man, livestock, and companion animals. The major focus of investigation of tick-borne viruses has been the interaction with the mammalian host, particularly the mechanisms underlying disease and the development of vaccines to prevent infection. Only recently has research begun to investigate the interaction of the virus with the tick host. This is striking when considering that the virus spends far more time infecting the tick vector relative to the vertebrate host. The assumption has been that the tick host and virus have evolved to reach an equilibrium whereby virus infection does not impede the tick life cycle and conversely, the tick does not restrict virus replication and through blood-feeding on vertebrates, disseminates the virus. The development and application of new technologies to tick-pathogen interactions has been fuelled by a number of developments in recent years. This includes the release of the first draft of a tick genome, that of  The phylum Arthopoda emerged during the “Cambrian explosion” (540–485 million years ago) creating numerous groups, many that have survived to the present day. One of these, the Chelicerata, contains the order Acari, which in turn contains species that obtain nutrition through blood feeding on vertebrates, collectively termed ticks. Fossil records indicate that ticks have been present from at least the Cretaceous period (146–65 million years ago) where they could feed on mammals  and LGTV  and TBEV. One striking observation from this study was the up-regulation of a single Toll-like protein transcript (ISCW022740) following infection with the viruses but not the bacterium. Three other Toll transcripts showed very little change to infection with the pathogens. This suggests that transcript ISCW022740 may play a role in the antiviral response. This strongly mirrors the mammalian response to infection where Toll-like receptors such as TLR3 are upregulated in response to infection (McKimmie et al., An alternative approach is to measure Toll activity in ticks in response to microbial infection. Mansfield et al.  have recin vitro infection of tick cells with tick-borne viruses shows no apparent cellular changes in stark contrast to the lytic cytopathic effect observed in many mammalian cells infected with the same virus. However, an alternative interpretation could be that infection stresses the cell. Cell-lines used in such studies are often derived from embryonic tissue and stress could lead to induction of transcripts associated with a developmental response. Ticks appear to tolerate virus infection but further investigation is required to understand what mechanisms tick cells use to control virus infection and why this does not lead to elimination of the virus analogous to the response in vertebrate hosts.The association between ticks and viruses is a fascinating one and a growing field of investigation. Ticks harbor a vast array of endogenous viruses (Bell-Sakyi and Attoui, The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dear Editor,Flavivirus) epidemic in the Western Hemisphere has been declared a public health emergency. The virus can cause transplacental infections in pregnant women, resulting in microcephaly of the fetus, intrauterine growth restriction and abortions.1 In addition, regions with the heaviest ZIKV burden are endemic for dengue virus (DENV). It is possible that ZIKV infection or vaccination may enhance DENV infections, and render mild dengue fever into a life-threatening disease.2 Animal models to study ZIKV infection and ZIKV co-infections with other flaviviruses are crucial for further fundamental studies and development of effective interventions. To date, significant efforts have been made to develop only mouse and non-human primate models3 for ZIKV infection. The identification of another mammalian species able to at least partially model ZIKV infections in humans has significant value. It has been previously reported that flaviviruses, including DENV,4 West Nile virus5 and Japanese encephalitis virus,6 can infect domestic pigs. Therefore, we designed this preliminary study to determine whether neonatal pigs are susceptible to ZIKV infection.The current Zika virus /mL of ZIKV strain PRVABC59 (GenBank: KU501215), which was isolated from human serum collected in Puerto Rico in December 2015 and was provided by the Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Fort Collins, Colorado, USA. Virus stocks were prepared by inoculating a confluent monolayer of Vero E6 cells. We selected a viral challenge dose within the previously reported range for West Nile virus: 102–107 infectious viral particles during a mosquito bite.7 A prior study also indicated a ZIKV load of up to 108 TCID50 in the salivary gland of the Aedes aegypti mosquito.8 Eleven piglets were inoculated intracerebrally (IC), six piglets were inoculated intradermally (ID) in four sites on the neck skin and six piglets were inoculated intraperitoneally (IP). Three control mock-inoculated (IC-inoculated) and non-manipulated piglets were also included in the study. Animals were monitored daily for clinical signs. Blood was collected at three, five and seven days post virus inoculation (dpi). At each sampling time point, two piglets in the ID and IP groups and three to four piglets in the IC group were euthanized to collect urine (aspirated from the bladder), brain and spleen. Tissues were snap-frozen for virological analyses. A previously published ZIKV-specific real-time reverse transcription polymerase chain reaction (RT-PCR) SYBR Green assay was used to identify and quantify ZIKV RNA. Zika virus titers were identified by inoculation on Vero E6 cells and subsequent staining with ZIKV-specific antibodies . The infectious virus in brain tissues was also isolated on mosquito C6/36 cells. For quantification of ZIKV-specific IgM, IgG Ab and neutralizing Ab (NAb), modified immunoperoxidase monolayer assay and neutralizing assay were used, respectively. A detailed description of experimental methods is provided in First, we determined the susceptibility of one-day-old piglets to ZIKV infection. Two pregnant Landrace-cross sows were housed at the VIDO-InterVac. After farrowing, newborn piglets were challenged with 1  mL of inoculum containing 5.8 log9 and a human adult who died because of encephalitis associated with ZIKV infection.10 In the IC-inoculated piglets, ZIKV RNA in sera was detected at 3, 5 and 7 dpi, whereas ID- and IP-inoculated piglets were positive at 3 and 5 dpi (P=0.02). ZIKV RNA and live virus were also identified in urine (IC and ID groups), brain (only IC group) and spleen  at 7 dpi. In addition, weak NAb activity was observed in sera  at 7 dpi.Mock-inoculated and non-manipulated animals did not exhibit any clinical signs, ZIKV RNA, live virus titers or virus-specific Ab. At 5 dpi, two ZIKV IC-inoculated piglets of eleven exhibited leg weakness, ataxia and tremor . Similarnd 5 dpi . In the nd 5 dpi . Intradepensions  resultedpensions . Serum s10 TCID50 of ZIKV  had viremia at 4–6 dpi  co-infections and ADE of infection in infants.Most importantly, pigs inoculated with ZIKV via different routes developed IgM, IgG, and neutralizing Abs, which may provide opportunities to study the antibody-dependent enhancement (ADE) of other flavivirus infections. For example, the major risk factor for developing severe dengue fever is ADE of infection caused by the presence of DENV-reactive Ab from a previous infection with a different DENV serotype. Antibodies induced by previous infections with other related flaviviruses may cause the same detrimental effects. In support, polyclonal mouse Ab against ZIKV enhanced DENV infection Beyond modeling ZIKV infection, our data may have epidemiological applications. Given that ZIKV is an emerging pathogen in the Americas, it is difficult to predict potential reservoirs of the virus as it spreads in the new environment. Japanese encephalitis virus, another member of the family Flaviviridae, uses pigs as amplifying hosts with subsequent mosquito-mediated transmission to humans. Therefore, additional studies are necessary to clarify if ZIKV has the potential to establish endemic infections in swine herds and a sylvatic cycle involving pigs.


The Journal of Applied Clinical Medical Physics (JACMP) is here because of a revolution in publishing technology. That publishing revolution has greatly lowered some of the costs of publishing scholarly articles. However, this is not true of all costs across the board, as some do not change. For example, it costs as much for copy‐editing, layout production and quality assurance services for the JACMP as for any print journal. Still, take a look as some of the costs associated with a print and electronic journal:A library will typically pay $1.00 per print article per year to obtain, own and archive that articleA library will typically pay far less than $0.10 per article per year to provide access to all the open source scholarly articles on the internetA scholar will typically pay $0.50 per print article per year to have a journal subscription containing the articles of interestA scholar will typically pay less than $0.05 to access and print an open source article from the internet.These are examples of “transaction costs”, the cost of exchanging or transferring information from one point to another. The internet only / open source model lowers transaction costs across the board for stakeholders by factors of between 5 and 20. The total cost to publish an article online in the JACMP is approximately $300 per article. Presently, that cost is borne by our advertisers, and that is how we provide the JACMP to a worldwide audience, to anyone with web access.Our advertisers also experience the benefit of lowered transaction costs. Generally, it costs less to buy a pair of eyes to look at a vendor web site in the electronic media than in the print media. I sincerely hope you will support our vendors and use the vendor links in the JACMP. We track the number of click‐throughs in order to justify to our vendors the cost of a banner ad. If you just make it a habit to visit vendor web sites using JACMP banner ads, it helps us keep the journal available to you without cost.Let me express my very best wishes and thanks to Walter Huda and John Horton for their dedicated support as Associate Editors over the past several years. Both are leaving the JACMP, and we appreciate all the volunteer work they have done.Michael D. Mills, PhD


In this study, nanoindentation was conducted to extract the load-displacement behaviour and the nanomechanical properties of asphalt concrete across the mastic, matrix, and aggregate phases. Further, the performance of hydrated lime as an additive was assessed across the three phases. The hydrated lime containing samples have greater resistance to deformation in the mastic and matrix phases, in particular, the mastic. There is strong evidence suggesting that hydrated lime has the most potent effect on the mastic phase, with significant increase in hardness and stiffness. Asphalt concrete (AC) is widely used around the world due to its primary function in road and pavement construction. AC is a composite material composed of a heterogeneous mix of asphalt binder and mineral aggregates. It is generally agreed that AC consists of three distinct phases: mastic, matrix, and aggregate phases . The masThe capacity to study the mechanical behaviour of materials is expanding with the introduction of new technologies that can probe locally at nanoscale resolutions and with great control over experimental variables. Using nanoindentation, a multi-structured, multi-phased material like AC can be examined under a variety of loading conditions. Whilst nanoindentation has been used as a means of testing material properties for decades ,3,4, stuIn order to modify the properties of asphalt binder and asphalt concrete mixes various fillers (aggregate particles fine than 75 μm) are added. Some of the commonly used fillers include Portland cement, hydrated lime, fly ash, limestone dust and clay particles . It is wCalcium hydroxide, known more commonly as hydrated lime, is one of the fillers frequently used in AC to improve the asphalt-aggregate bond and the binder’s resistance to water-induced damage ,17,18,19Two hot mix AC were selected for testing in this study (one with hydrated lime and one without). Each mix contained a heterogeneous blend of crushed granite aggregates (10 mm) and asphalt binder. A 10 mm size was chosen as this is a common fine aggregate size used on roads, pathways and car parks. The aggregates were sourced from Boral and BGC and dried in the Geomechanics Lab of Curtin University Australia. The standard addition of hydrated lime in AC mixes in Australia is 1.5% of the total weight of the dry mix . TherefoThe samples were mixed and compacted as per AS 2150–2005 [Nanoindentation tests were carried out using Agilent Nano Indenter G200 . Poisson’s ratio for both AC and indenter tip were assumed based on average values from previous literature . In ordeSet 1: Two indents on a randomly selected area of mastic and two indents on a randomly selected area of aggregate . This waSet 2: A 10 × 10 grid (100 indents) on a manually selected area of each sample containing mastic, matrix, and aggregate . The griThe relative difference of each of the phases in AC without hydrated lime is more apparent when displayed on the same scale as shown in Overall, there is reasonable confirmation that the hydrated lime containing samples have greater resistance to deformation in the mastic and matrix phases, in particular, the mastic. Again, there appears to be a negative differential in the aggregate phase, although 16 nm could be accounted for as natural variance within the material. There is certainly no indication from the results that hydrated lime acts as a stiffener in the aggregate phase.The nanomechanical properties (Young’s modulus and hardness) in sample without hydrated lime are shown in Using nanoindentation with a Berkovich indenter, it was possible to successfully capture the heterogeneity of asphalt concrete, with distinctly different properties for mastic, matrix, and aggregate phases. A dwell time of 20 s was sufficient in limiting creep for the majority of indents, except those on very soft mastic. The effects of creep were present on the unloading curve for indents with a dwell time under 20 s. The Young’s modulus and hardness for samples with hydrated lime are higher than the samples without them. In the mastic phase, there is an increase of 31% in the Young’s modulus and 153% in the hardness due to the addition of hydrated lime. In the matrix phase, there is an increase of 6% in the Young’s modulus and 114% in the hardness due to the addition of hydrated lime. Future study on correlating the nanoscale testing with the macroscale testing presents exciting possibilities to improve current understanding of asphalt concrete.