Serological tests for Johne s disease

Serological tests for Johne\’s disease have low sensitivity but reasonable specificity. Testing of individual milk specimens yielded a sensitivity of 28% (Collins et al., 2005), slightly higher than serum, and sensitivity increased with age of animal tested (Nielsen et al., 2013). PCR can also be used to test for the presence of MAP DNA in milk (Buergelt and Williams, 2004), as can the peptide-mediated magnetic separation-phage (PMS-phage) assay (Foddai et al., 2011). However, advances in PCR testing for MAP in faeces could negate the need to use antibody based tests.
Antibody based tests (ELISA) are available to measure myeloperoxidase in bulk tank milk to the abomasal parasite O. ostertagi (Forbes et al., 2008). Only an association between ELISA values and milk yield can be made using these test results, rather than confirming true positive nematode infections in the herds, so additional diagnostic testing is required to establish the parasite status of the herd.
In sheep, Q-fever (Coxiella burnetii; Klaasen et al., 2014), Brucella melintensis (Hamidi et al., 2015) and Mycoplasma agalactiae (Poumarat et al., 2012) can be tested for using milk; in goats, milk specimens can be used to test for caprine arthritis and encephalitis (Nagel-Alne et al., 2015). Q-fever outbreaks in humans are associated with C. burnetii infection in small milking ruminants in Africa (Klaasen et al., 2014). Shedding of the organism is intermittent, thus infection was not always detected by PCR and serological tests might also be required. In contrast, PCR testing of milk for B. melintensis detected was more sensitive than serology in one study (Hamidi et al., 2015). Accurate serological classification of the M. agalactiae status of sheep is difficult and PCR testing myeloperoxidase of milk specimens with two PCRs should be used to confirm the presence of the organism. The resultant PCR results also require cross checking with a dot-immunobinding technique (Poumarat et al., 1991).
Milk testing can be utilised for detection of non-infectious conditions. For example, lateral flow devices to test for progesterone concentrations in milk present opportunities to define the oestrus cycle and pregnancy status of cows (Waldmann and Raud, 2016) and technological modifications may allow for testing to occur during milking (Dobson, 2016).

Colostrum is another medium that can be used for animal disease testing instead of milk. Its availability is restricted to a shorter time period, but provides other testing and diagnostic advantages. Testing colostrum for antibodies (as an alternative or add-on to milk testing) is potentially useful because of the higher concentration of immunoglobulins in colostrum compared to milk. The concentration of IgG is estimated to be up to 100 times higher than milk in the first few days after parturition (Korhonen et al., 2000). Recent work in cattle suggests that testing colostrum increases analytical and diagnostic sensitivity compared to milk. This could be most useful for on-going surveillance of animal diseases requiring less frequent checks, as colostrum is only available during the perinatal period (Jenvey et al., 2012, 2015; Cockcroft et al., 2014).
The drawback of colostrum testing is the small window of availability and the difficulty of collection in some species. In dairy cattle, sampling of colostrum might be most usefully applied for diseases where diagnostic tests are hampered by low analytical and/or diagnostic sensitivity (such as is the case with Johne\’s disease; Reichel et al., 1999). Colostrum has also been used successfully for testing for rotavirus and mycoplasma infections (Corthier and Franz, 1981; Zimmermann et al., 1986; Rautiainen, 1998).

Hair and ear notch skin specimens
Hair and ear notch specimens have been used successfully to detect BVDV persistently infected animals (Hill et al., 2007; Lanyon et al., 2014c) and formed the basis of the recent successful Swiss BVDV eradication campaign (Presi and Heim, 2010). In this campaign, detection of BVDV antigen in skin was the specimen of choice to identify persistently infected calves, and was preferred over serum. After the ingestion of colostrum, maternal anti-BVDV antibodies can bind to BVDV antigen and prevent its successful detection in the routinely used antigen-capture ELISA (Fux and Wolf, 2012). Using ear notch skin specimens reduces this complication, as there are fewer antibodies in ear notch tissue. Heating of serum specimens under specific conditions to break up antibody-antigen complexes can overcome the interference of maternal antibody and allow successful serum testing. This adds extra steps to the procedure, but has been used effectively in BVDV testing (Lanyon and Reichel, 2016) and in heartworm serology (Little et al., 2014a, 2014b; Velasquez et al., 2014).

The interplay of host agent and environmental factors is often

The interplay of host, agent and environmental factors is often discussed in the context of infectious disease in animal populations, yet this interaction is less frequently considered in relation to the occurrence of antimicrobial resistance in such groups. Instead, when discussing antimicrobial resistance, attention is usually focussed on the relationship between antimicrobial use and resistance. Whilst antimicrobial use is undoubtedly the primary selective pressure for the emergence and persistence of antimicrobial resistance, and might be considered as a ‘host’ factor, the role played by other host, agent and environmental factors in the occurrence of resistance is often overlooked. The influence of such factors is demonstrated by Dr. Karla Cameron-Veas and colleagues, of the Centre de Recerca en Sanitat Animal (CReSA), Barcelona, Spain, in their paper on cholesterol absorption inhibitor of cephalosporin resistant in pigs treated with antimicrobial agents, published recently in ().
In this study, the selective effect of use of antimicrobial agents was demonstrated by an increase in the numbers of cephalosporin resistant (CR-) in faeces following the use of a single injection of ceftiofur at a therapeutic dose in piglets already colonised with CR-. However, treatment with ceftiofur was not a significant predictor of the occurrence of resistance in the study group as a whole. Instead, farm of origin (an environmental factor) had the greatest influence on the occurrence of cephalosporin resistance, with the selective pressure of antimicrobial use only exerting an effect in those herds where CR- was already present. Furthermore, the study showed that the proportion of animals shedding CR- and the numbers of CR- shed decreased with animal age (a host factor).
Resistance to third generation cephalosporins in is mediated by a number of β-lactamases, the extended spectrum β-lactamase (ESBL) and acquired AmpC-type β-lactamases being of particular current concern. These resistance phenotypes have their origins in other Gram-negative bacteria and their emergence and spread is due to horizontal transfer of the associated resistance genes on mobile genetic elements (). Therefore, in contrast to other resistance mechanisms, these are not likely to arise as a result of mutation of existing bacterial genes during antimicrobial treatment. This may explain why CR- did not emerge during treatment on all farms in this study.
Farm related factors that may influence the occurrence of antimicrobial resistance in of porcine origin include production type, housing type, hygiene status and stress (). In the study by , the authors suggest that the origin and housing of sows may be part of the farm effect observed. These findings offer some hope that farms and animals can be kept free of certain resistant organisms through strict biosecurity and management practices, such as the careful sourcing of breeding stock. However, the efficacy of such interventions needs to be evaluated fully before they can be recommended.
The decrease in the proportion of animals shedding CR- and in the numbers of CR- shed may be explained, in part, by the time elapsed after the removal of the selective pressure of antimicrobial use. However, similar results have been reported in other studies from pigs, independent of antimicrobial use, which suggest that increasing host age is associated with decreased resistance (). It has been proposed that these changes over time reflect changes in the cholesterol absorption inhibitor microbiota of the porcine intestine, but it has not been determined whether these bacteria enjoy a primary selective advantage in the juvenile intestine as a consequence of their resistance status or if this results from co-selection with other advantageous traits. In either case, colonised sows might be considered as ‘reservoirs’ of resistant organisms (with shedding at lower levels due to increased age) for the colonisation of young pigs, which in turn act as ‘amplifiers’ to allow for the persistence of resistance within the herd and environment.

br Experimental methods br Results

Experimental methods

Results and discussion
A typical TEM image of the K12 sample is presented in Fig. 1a, showing that this sample is composed mainly by nanoribbons with narrow size distribution. The ribbons have some micrometers in length and diameters of 50nm approximately. They are arranged in form of bundles, as can be seen in Fig. 1b. The sample morphology is similar to that reported in literature [14,17]. All samples that have been studied in this work presented the same morphology, once that the p coumaric acid washing process does not affect the nanoribbons form.
The powder XRD pattern of K12 sample is presented in Fig. 2a–I is similar to those reported by other authors [14–18]. The real structure of the potassium titanates synthesized by the hydrothermal method is still under discussion. The most acceptable candidates are K2Ti8O17[14–16] and K2Ti6O13[17,18], both belonging to a monoclinic structure of the (C2/m) space group. However only the crystallographic data are not able to define the correct composition. Raman measurement of the K12 sample was done in order to elucidate such point, as can be seen in Fig. 2b, where the positions of the observed peaks are indicated. Comparing with bulk potassium hexa and octatitanates Raman spectra reported by Bamberger et al. [23], it can be noticed that the spectrum of K2Ti8O17 is in best agreement with that presented by the K12 sample. Table 1 presents the positions of the depicted K12 nanoribbon Raman modes, compared to those of K2Ti8O17 bulk sample. These modes have been obtained by adjusting the experimental spectra with Lorentzian lines. An example of a fitted spectrum is given in detail in Fig. S1 in the Supplementary Part. Once the preliminary analyses by XRD and Raman spectroscopy have indicated that K2Ti8O17 is the most probable composition for our K12 sample, XRD profiles for a bulk and nanoscale K2Ti8O17 crystals were simulated using the GSAS software [24,25] and the results are shown in Fig. 2a–II and a–III. The simulated XRD patterns was based on the monoclinic structure of K2Ti8O17 (ICSD-69878 [26]) crystal using the Le Bail method. As it can be observed, the peak positions are basically the same of our sample with a reasonable agreement. The lattice parameters obtained were a=19.005(5)Å, b=3.771(2)Å, c=11.938(1)Å and β=94.5(1)°. The relatively broad peaks may be associated with size effects, microdeformations and defects. Some peaks overlapping with each other are due to nanometer size of nanowires.
As presented in Fig. 2c, the potassium octatitanate has a tunnel structure with four distinct positions for Ti atoms, each one surrounded by six oxygen ions, yielding in distorted TiO6 octahedra, as reported by Sasaki et al. [24]. These octahedra form ribbons composed by four edge-shared octahedra in the (0 1 0) plane. All oxygens are coordinated by titanium atoms which keep the lamellas connected, yielding the tunnel like structure along the [010] vector. The potassium atoms are located in the tunnel space and are statistically distributed over two distinct crystallographic sites, assigned as P1 and P2 in Fig. 2c. Site occupations are 0.68 and 0.32, respectively.
Potassium and sodium content of the samples were investigated by AAS measurements. The results are shown in Table 2. The K12 sample has 16.63wt% of potassium, which is higher than the expected content of 10.66wt% for K2Ti8O17. Such discrepancy can be explained by the fact that the amount of potassium atoms in the alkaline solution used in the synthesis was sixty times higher than necessary to completely convert 2.00g of TiO2 into 3.83g of K2Ti8O17. Furthermore, even with the washing process applied, the pH of the supernatant was at about 12, which revealed a good amount of KOH in the solution. Therefore, the potassium content obtained from AAS measurements represent not only the percentage of potassium in the sample, but also a certain of amount of remaining potassium. The results for the others samples showed that the acid and base washing processes led to a K-H and H-Na ion exchange, respectively. This kind of replacement has already been shown for some lamellar titanates [8,27], although Papp et al. [6] showed that the sodium hexatitanate lost only 1% of their initial sodium content during the treatment with HCl. These authors concluded that only the sodium atoms in the surface and in a few open channels can be changed. Despite the tunnel like structure of the potassium titanate nanoribbons, their good ion exchange capacity may be related to their small dimension, which results in a big surface area. This property may be responsible for a bigger amount of exchangeable sites and, consequently, a better ion exchange capacity than the tunnel like structured bulk materials. Table 2 shows that the potassium content in K1 and KNa12 samples are almost the same. It can be inferred that only the hydrogen atoms were replaced by the sodium atoms. The K samples may be expressed by general chemical formula KHTi8O17 and the corresponding values of x and y are listed in Table 2. The AAS results presented in this work could not determine the x values for the KOH in the K12 and K8 samples due to their undesired K contents. The KNa12 sample can be expressed by KNaHTi8O17, where the x and y depends on HCl and NaOH washing processes. The x and y values found for the KNa12 sample are 0.8 and 1.4, respectively. These values could be considered as exceed ones, however, considering the error, the KNa12 sample could be expressed as K0.7Na1.3Ti8O17. This result suggests that there is almost no hydrogen in this sample.

Strategic outcomes of normalization of relations

Strategic outcomes of normalization of relations from the point of views of Turkey are distinct from others. The renormalization of Turkey–Armenia relations is considered a kind of diplomacy to establish peace (Sanberk, 2009). From the point of view of Turkey that has taken important step for its dream membership of the European Union, relations based on peace and vegf tyrosine kinase inhibitor with neighboring and peripheral countries enjoy special importance. Under the framework of multi-dimensional foreign policies, Turkey, on the one hand follows the policy of rapprochement and resolving problems with its neighbors through dialog and on the hand, with the aim to create a peace band in its surrounding, it poses itself a mediating player in regional crises. In fact, if nothing is achieved from Turkey–Armenia ties, the essence of Turkey\’s peace efforts will be in question. With the idea of assistance to establish a ground of cooperation in the region and Turkey\’s collaboration to establish regional stability was aimed at normalizing bilateral relations. Boosting its relations, Turkey would certainly evolve itself a big regional power (Aras & Özbay, 2009: 7–8).

Analysis & findings



Communications and values in the morphology of the cultural landscape of Urals and Siberia
The increasing attention of today\’s society to the concept of “cultural landscape” attests to the relevancy and urgency of conducting comprehensive research into the inextricable links between the various aspects of human existence and their contexts. This research must take great care to avoid the extremes of atomistic, “one-dimensional” interpretations of culture – for example, by regarding a given culture simply as a collection of material artifacts or as a strictly linear unfolding of defined vegf tyrosine kinase inhibitor social and demographic processes (Birks, 1988; Nassauer, 1995; Rubinshtein, 2010; Salter, 1971; Sauer, 1925, pp. 36–48; Sauer, 1927, pp. 154–214; Wallach, 2004).
The manner in which each of these forms of communication unfolds mirrors a particular system of cultural values that is characteristic either of the regional economic structure or of a historical era. In this way, we can speak generally about the values of pre-industrial, industrial, and post-industrial development as addressed in the conceptual schema of periodic societal development proposed by Alvin Toffler and Daniel Bell (Bell, 1973; Toffler, 1980).
Frame-based approaches to spatial analysis of social and economic processes were first proposed in Russia by Nikolay Baranskij in the 1950s (Baranskij, 1980). The concept of support frame as pelagic zone was subsequently developed holds that a set of junction (urban) and linear (transportation network) elements provides the foundation for development of spatial models of individual, administrative, economic, and socio-demographic aspects of regional life activities. In particular, this approach treats a given region\’s support frame as the structural and communicative basis of its identity.

Trans-Siberian communication modalities as the linear basis of the spatial structure of Urals and Siberian cultural landscapes
The Babinov road, constructed shortly after the camping of Yermak\’s (Yermak (born between 1532 and 1542 – died 1584) was a Cossack who led the Russian conquest of Siberia) can be considered as the basis of the first linear route of Siberian colonization (Witzenrath, 2007; Yermak\’s Campaign in Siberia, 1975). During this period, the availability of reliable land routes was necessary for Russia to secure its eastern frontier beyond the Urals. Construction of the road started in 1595 and continued for two years. In 1597 the road reached Nerom-Car – the settlement of Siberian natives Voguls, located at the head waters of the Tura River. Establishment of the town of Verkhoturye occurred one year later.
Soon this road was extended to the first Russian towns founded in Siberia – Tyumen and Tobolsk. Throughout the seventeenth century and the first half of the eighteenth century, it was the only official line of communication between the European part of Russia and Siberia. This was primarily due to the opening of the Verkhoturye customs house in 1600. Without exception, everyone traveling to and from Siberia was obliged to undergo the customs process, which consisted mainly of levying the tax on furs that was of great value to the Russian state. For nearly 150 years, the Babinov Road, named after Artemiy Babinov, the peasant who founded it, served as the route for the colonization of Siberia (Lincoln, 2007).

Afghan conflict has typically seen the movement

Afghan conflict has typically seen the movement of migrants to neighbouring Iran and Pakistan. And, those who have resources and education seek refuge in Western countries. A fraternity also brings in many Afghans to India. But, Central Asia is also appearing as an important destination for many Afghans. They are comfortable there because of less violence and culturally it represents a Muslim society, where some degree of membership on that account is affordable. There is a fear that after the international forces leave Afghanistan, Central Asia could be the most probable destination for many Afghans, especially, the non-Pashtuns (, 2013). Most of the Afghans in Central Asia would wish to see a resettlement into lucrative destinations, such as, North America or Australia. Tajikistan has numerous refugees coming from cities of northern Afghanistan, like, Kunduz and Mazar-i-Sharif. The ethnic background becomes important and in this case it is largely the Tajiks from northern Afghanistan who have been seeking asylum in Tajikistan. Among the Afghan refugee in Pakistan only 7.3% were Tajiks in 2005 (, 2004). Afghans in Kyrgyzstan are coming as migrants, students, business people and refugees largely located in the urban areas, like Osh, Bishkek and Jalalabad. Many of them have settled and married Azerbaijani, Kyrgyz, Ukrainian or Russian woman. Several have also been granted citizenship. The Dostum foundation has been sending students to American University of Central Asia (AUCA), Bishkek. The refugees mention ideology (communism) as a major cause of seeking shelter. But, largely the secure environment and stable living conditions attract most of the Afghans (Kazemi, 2013).

Economic and how to determine molarity linkages
Afghanistan served as gateway to littoral Asia for Silk route trade passing across Central Asia. The reason holds perennially good as the US looks for a revival of those links that can form the basis for sustained economic development of Afghanistan and the region at large. The Heart of Asia Process is being a regional initiative that is aimed at reviving the spirit of Silk Route with special focus on trade, transit, energy and communication routes (Ministry of Foreign Affairs, Afghanistan, 2012). The initiative extends from Turkey to China. The first meeting held in Istanbul readied the roadmap, where Afghanistan was not to be seen as roundabout but a significant diver of economic growth in the region. The economic empowerment precedes the political and strategic empowerment of the Afghan State. Heart of Asia Conference brought together nearly 40 countries and international organisations that matter to the future of stable Afghanistan (The Hindu, 2013). A significant challenge in years to come is the justifiable appropriation of water resources, namely, hydropolitics. Central Asia has been in the grip of these conflicts, with lower riparian states of Uzbekistan and Kazakhstan pressuring Kyrgyzstan and Tajikistan to abandon their upstream hydel projects (Coskun, 2004). Already there is opposition to Tajik Rogun hydel project and Kyrgyz Upper-Naryn power plants. Germany has come to assistance with the launch of Central Asia Water Initiative (Berlin Process) for transboundary water management. The situation is not far when this might involve Afghanistan into the regional muddle. “According to research officer Omar Nesar, Institute of Oriental Studies, Russia, founder of Analytic Centre for Modern Afghanistan Studies, Moscow, Afghan officials believe rivers starting in Afghanistan unfairly flow into other countries and they should build dams to keep water in the country. But is it a right way out?” (Dudka, 2013). The physical connections between Central Asia and Afghanistan remained seasonal during the Cold War. It remained open when the two sides on friendly terms and remained closed otherwise. The post-Cold war period saw variegated options exercised by individual neighbours. For example, during the Taliban occupation it was only Turkmenistan who maintained steady contact with the Afghan state (Radio Liberty, 2011). The Friendship Bridge at Khairaton marks an important cross-over which was opened in 2002. The Airitom Customs Complex was opened in Termex in 2003. Uzbekistan is playing vital role in electricity and transportation to Afghanistan. The rail lines between Hairatan and Mazar-i-Sharif have been laid, and are operated since 2011 (Laruelle, Peyrouse, & Axyonova, 2013). Tajikistan is next important player with surplus electricity that it wishes to supply to South Asia; Afghanistan would be inevitable beneficiary. The CASA-1000 will connect Tajik power generating plant to cities of Afghanistan, like, Kunduz, Baghlan, Pul-i-Kumri and Kabul (Starr & Farhadi, 2012). Turkmenistan has also restored the Soviet era cross-border rail link between Kushka and Turghundi. It is allowing more people to people contact along the border. Besides, its supply of electricity and facilitating NATO supplies makes it a vital partner. Kazakhstan though does not share border, but its economic pull is good enough as it is the biggest trading partner with Afghanistan among the 5 Central Asian Countries.

br Case report A year old woman

Case report
A 56-year-old woman presented to our clinic with rapidly decreasing vision and increasing pain in her right eye over a 2-day period. The best corrected visual acuity was hand motion with her right eye and counting fingers with her left eye. Slit-lamp examination of her right eye revealed conjunctival xerosis, a 6×6mm2 central corneal epithelial defect with ring infiltration, and disciform stromal edema with Descemet membrane detachment (Fig. 1). The anterior chamber showed a severe reaction with 4+ cells. The left eye exhibited conjunctival keratinization and diffuse superficial punctate keratopathy. Both eyes showed a decrease in corneal sensation. The tentative diagnosis was xerophthalmia of both eyes and herpetic keratouveitis in the right eye, because the results of clinical imaging and the polymerase chain reaction test for herpes simplex virus-1 (HSV-1) was positive in the aqueous humor. An initial treatment consisted of 500mg of acyclovir administered intravenously every 12 hours because the patient had an oral ulcer and was thus incapable of swallowing large tablets. Tobramycin (3mg/mL) eye drops and artificial tears (Vidisic gel, Dr. Gerhard Mann Chem.-Pharm. Berlin, Germany) were administered four times a day in both eyes. On the second day of hospitalization, the anterior chamber reaction had worsened in her right eye and a 1×1mm2 corneal epithelial defect appeared in the left eye (Fig. 2). Dexamethasone 1.5mg (5mg/1mL) was therefore injected into the subconjunctival space of the right eye. The reaction of the anterior chamber was eased after this igf ir procedure.
Unfortunately, on the evening of the fourth day, the density of the ring infiltrate and hypopyon increased in the right eye. A 2×2mm2 paracentral corneal ulceration with minimal hypopyon was also noted in the left eye. Furthermore, a purulent discharge developed the next morning, and the condition of both eyes worsened (Fig. 3). A superimposed bacterial infection in both eyes was thus suspected and an hourly treatment of topical fortified gentamicin (15mg/mL), alternating with cefazolin (50mg/mL) every hour, was started. Additionally, 1g of cefazolin was administered every 8 hours and 120mg of gentamicin twice a day, both intravenously. Smear tests and bacterial cultures proved that both eyes were infected with Pseudomonas aeruginosa.
Meanwhile, we conducted a systemic survey on the patient. The biochemical analysis on her blood sample showed that the serum albumin was 3.4g/dL (normal range 3.5∼5.2g/dL), total protein was 5.9g/dL (normal range 6.2∼8.3g/dL), cholesterol was 91mg/dL (normal range 130∼200mg/dL), and triglyceride was 32mg/dL (normal range 35∼200mg/dL). No remarkable result was found on the hematologic, immunologic, and rheumatologic evaluations, except for mild anemia (hemoglobin 10.1g/dL, hematocrit 31.5%). Abdominal sonography showed that her liver and kidneys were normal except for a gallbladder polyp. On reviewing her medical history, we found that the patient had difficulty in eating solid food because she had biting pain with tooth mobility for months and had a long followed strict vegetarian diet, she also disliked carrots, sweet potatoes, pumpkins, and asparagus, and was restricted from eggs and milk. No major surgery or medical disease in previous years was mentioned, except for a history of dry eye syndrome. The patient had been informed of the diagnosis of vitamin A deficiency due to conjunctival xerosis (Fig. 4) 2 months ago. Artificial tears and vitamin A ointment had been prescribed. The patient was asked to take vitamin A pills and more vitamin A containing food such as papaya, spinach, etc. However, she did not seem to follow these suggestions. Her weight was 38kg and height was 150cm. Her skin was scaling and dry. This general appearance in conjunction with the aforementioned ocular signs strongly suggested that the patient had severe vitamin A deficiency and malnutrition. As such, she was placed on a high-protein diet as monitored by a nutritionist and asked to ingest multivitamin pills, including 5,000IU of vitamin A daily.

Pathogenesis from members within Alphabaculovirus

Pathogenesis from members within Alphabaculovirus and Betabaculovirus may differ, although fewer studies have described betabaculovirus pathogenesis. Briefly, alphabaculoviruses and betabaculoviruses infect the midgut epithelium of insect hosts. In alphabaculoviruses, virions reach tissues in the insect hemocoel producing more virions. The product of the very late gene P10 is responsible for nuclear lysis (van Oers et al., 1993), releasing ODV in the environment after the insect cadaver liquefies. In betabaculoviruses, the nucleus of infected Otamixaban enlarges, followed by nuclear membrane breakage. The genes and mechanisms affecting this early nuclear membrane rupture are not known. Nucleocapsids are then enveloped and occluded in this nucleocytoplasmic compartment. Betabaculovirus pathogenesis differs in tissue tropism, from viruses being midgut restricted, to infecting midgut epithelium and fat body tissue, to infecting several tissues of the host. In addition, dispersal of ODV may differ from dispersal in diarrheal secretions to dispersal following complete insect liquefaction (reviewed Otamixaban in Federici, 1997).
Degradative enzymes have been reported in baculoviruses, including viral-chitinase (v-chitinase) which digests chitin, the main component of the insect exoskeleton; and viral-cathepsin (v-cathepsin), which is involved in the degradation of internal larval tissues (Ohkawa et al., 1994; Slack et al., 1995). The concerted activity of these two enzymes enables host liquefaction which allows virus release from the infected cadaver and dissemination to other hosts (Hawtin et al., 1997; Kang et al., 1998a). The betabaculovirus Cydia pomonella granulovirus (CpGV) v-cathepsin was shown to be a functional protease and necessary for larval melanization and liquefaction or softening of larval cadavers, depending on the virus background in which it was tested (Hilton et al., 2008; Kang et al., 1998b). Similarly, the CpGV v-chitinase expressed from a Bombyx mori NPV (BmNPV) recombinant virus allowed larval liquefaction and interacted with BmNPV v-cathepsin (Daimon et al., 2007).
The role of another degradative enzyme, the viral MMP, found in betabaculoviruses, has not been studied extensively. To date, there is only one functional study on baculovirus MMPs, characterizing the Xestia c-nigrum granulovirus (XcGV) MMP (XcGV-MMP). XcGV-MMP is a functional MMP involved in larval melanization and thought to have a role in degradation of host basement membranes during the late stages of infection (Ko et al., 2000). Open reading 46 (ORF46) encoded in the CpGV genome predicts a protein, CpGV-MMP, which shows significant similarity to MMPs (Luque et al., 2001). However, its role in viral pathogenesis has not been described.



Materials and methods

We are grateful to Anna C. Rogers for preliminary results, cloning CpGV-ORF46 from the CpGV genome, and providing other reagents. We thank Monique van Oers, Susuma Katsuma, and Jeffrey Slack for providing Ac-ΔCCBac, anti-chitinase serum, and Cath (-), respectively.
This work was supported in part by the National Institutes of Health Award 5RO1AI091972-3 to A. L. P. This is contribution number 15-186-J from the Kansas Agricultural Experiment Station.

It is well known that mammalian influenza viruses undergo antigenic drift through acquisition of amino acid substitutions within the viral glycoproteins haemagglutinin (HA) and neuraminidase (NA), leading to the occurrence of disease epidemics. For this reason, vaccine strains to influenza viruses eventually become ineffective, unless they are updated regularly. The HA1 domain appears to be under the most selective pressure, consistent with its role in induction of neutralising antibodies (Nelson and Holmes, 2007). Substitutions in human H3 viruses have been associated with changes in charge, acquisition of glycosylation sites and also alteration of receptor binding avidity (Blackburne et al., 2008; Kobayashi and Suzuki, 2012; Lin et al., 2012). Changes in HA are often accompanied by substitutions in NA and it is believed that the activity of HA and NA should be balanced (Mitnaul et al., 2000; Kaverin et al., 1998; Baigent and McCauley, 2001). The rate of antigenic change differs between influenza viruses of different species: human H3N2 viruses appear to drift more rapidly than either swine H3N2 or equine H3N8 viruses, as assayed by haemagglutination inhibition (HI) with ferret antisera. This has been demonstrated by antigenic cartography, which indicated that human H3N2 viruses underwent multiple ‘cluster jumps’ between 1968 and 2003 (Smith et al., 2004) whereas equine and swine viruses evolved fewer antigenic clusters over a similar period of time (de Jong et al., 2007; Lewis et al., 2011).

br Materials and methods br

Materials and methods

Galectin-3 (Gal-3) is a member of the β-galactoside-binding lectin family of proteins, which bind to HMBA Linker Supplier (reviewed by Boscher and Nabi, 2013; Vasta, 2012). Specifically, Gal-3 binds to the β1,6 branched N-glycans of glycoproteins on the cell surface, which are HMBA Linker Supplier the products of the Golgi enzyme β1,6-acetylglucosaminyltransferase 5 (Mgat5) (reviewed by Boscher et al., 2011). On the cell surface, Gal-3 interacts with several ligands, including integrins and epidermal-growth factor receptor (EGFR) (reviewed by Boscher et al., 2011). Therefore, it has been proposed that Gal-3 modulates the clustering and signaling activity of receptors on the plasma membrane (Boscher et al., 2011; Goetz, 2009). Similarly, through its interactions with Mgat5 modified N-glycans of various cell surface receptors and proteins, Gal-3 modulates several cellular functions, such as signal transduction on the cell surface and cell adhesion, motility, growth, and differentiation (Dennis et al., 2009b). Since some of these functions are altered in tumorigenic cells, Gal-3 regulates many physiological and pathological cellular processes. However, a role for Gal-3 in viral infection has only recently been discovered. In a proteomic study, Gal-3 and its binding protein, Gal-3-binding protein (Gal-3-BP), were identified as cellular partners of the parvovirus minute virus of mice prototype strain (MVMp) (Garcin et al., 2013). In addition, a screen for serum proteins that interact with different adeno-associated virus (AAV; another parvovirus) types identified Gal-3-BP as a binding partner of AAV (Denard et al., 2012). It was subsequently shown that Gal-3 is required for efficient MVMp cellular uptake and infection, but not for viral binding to the plasma membrane (Garcin et al., 2013), and that Gal-3-BP reduces AAV-6 transduction (Denard et al., 2012). In addition to these parvoviruses, it has also been reported that herpes simplex virus type 1 (HSV-1) uses extracellular Gal-3 for cell entry (Woodward et al., 2013).
MVM is a small (26nm in diameter), non-enveloped virus with a single-stranded DNA genome that belongs to the Parvoviridae family (Cotmore et al., 2014). The MVM genome is protected by a capsid assembled from 60 copies of two size variants of the capsid proteins VP1 and VP2, which are identical, except for the unique N-terminal sequence (140 amino acids) of VP1 (Tattersall et al., 1977). Several strains of MVM exist, but the best characterized strains are the prototype MVMp that infects cells of fibroblast origin and the immunosuppressive strain MVMi that infects T lymphocytes (Tattersall and Bratton, 1983). This MVM tropism is controlled predominantly by the capsid protein VP2, with only two amino acid substitutions on this protein making the virus fibrotropic or lymphotropic (Ball-Goodrich and Tattersall, 1992).
Several parvoviruses attach to their target cells via binding of capsid proteins to glycan receptors (reviewed by Huang et al., 2014). For example, human parvovirus B19 uses gangliosides (Cooling et al., 1995), AAV2 uses heparan sulfate proteoglycan (Kern et al., 2003), and rat H-1 parvovirus, porcine parvovirus, and MVM use sialic acid (Allaume et al., 2012; Boisvert et al., 2010; Nam et al., 2006). A recent sialylated glycan microarray revealed that MVM binds to diverse sialylated derivatives, but there was different recognition between MVMp and MVMi (Halder et al., 2014). For example, MVMp, but not MVMi, binds to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives (Halder et al., 2014). Despite this difference, MVM attachment to sialic acid induces virion internalization by clathrin-dependent endocytosis (Linser et al., 1977, 1979; Vihinen-Ranta and Parrish, 2006) (reviewed by Vihinen-Ranta and Parrish, 2006), and possibly other endocytic pathways as has been recently shown for other parvoviruses (Bantel-Schaal et al., 2009; Boisvert et al., 2010; Nonnenmacher and Weber, 2011). Following endocytosis, MVM escapes from endocytic compartments into the cytosol by means of the enzymatic action of a phospholipase A2 (PLA2) domain in the unique region of VP1 (Farr et al., 2005). The virions that escape the endosomes then enter the nucleus where they wait for the host cell to transition into the S-phase, to initiate viral DNA replication (Cotmore and Tattersall, 2006).

br Introduction Hepatitis C Virus HCV affects more than million

Hepatitis C Virus (HCV) affects more than 170 million people worldwide, or 3% of the world population (Perz et al., 2006), with 4 million new cases and more than 300,000 deaths per year (Bukh, 2012). Clinical conditions of the disease range from an asymptomatic carrier state to persistent infections. Of those individuals infected, 70% will develop chronic HCV infections, and 20% of chronic infections will progress to cirrhosis and terminal hepatocellular carcinoma (Bradley, 2000; Lauer and Walker, 2001; Seeff, 2002; Hoofnagle, 1997). There are currently no vaccines available to the public to prevent HCV due to the high genetic variability of the virus and its ability to escape host immune defenses (Di Lorenzo et al., 2011).
The current standard of care (SOC) treatments may include a combination of pegylated interferon-α and ribavirin (Christie and Chapman, 1999) and direct acting antivirals such as Sofosbuvir and Simeprevir (Belousova et al., 2015). The drug combination has unfavorable side effects and may ultimately lead to drug resistance and relapse. One of the main reasons may be attributed to the generation of quasispecies genome meclofenoxate common for HCV infections, a phenomenon that results in infection by a swarm of microvariants derived from a predominant “master sequence” within an individual host (Bukh et al., 1995). Quasispecies are more prominent in the setting of persistent infections and may be responsible for drug treatment failures (Farci et al., 2000; Domingo and Gomez, 2007). Quasispecies result from the high error rate of the non-proofreading HCV RNA-dependent RNA polymerase (RdRp) leading to continuous production of mutated virus sequences which is one mechanism the virus employs to escape immune system defense (Carmichael, 2002). This warrants a continued intensive search for alternative antiviral approaches to combating HCV.
HCV is a plus-strand RNA virus of the Hepacivirus genus, having a 9600 nt long genome encoding a single ORF flanked by highly conserved 5′ and 3′ untranslated regions (UTRs) (Takamizawa et al., 1991). The ORF encodes a single polyprotein that is modified post-translationally by both cellular and viral proteases to produce 3 structural (C, E1, E2) and 7 non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (Fauvelle et al., 2013). The 5′ UTR of the viral RNA contains an internal ribosome entry site (IRES) that is highly conserved among most known HCV quasispecies (Brown et al., 1992). The 5′ UTR of HCV facilitates viral replication and mediates cap-independent viral protein translation by acting as a scaffold and recruiting multiple protein factors during the initiation of translation upon early infection (Rosenberg, 2001; Kieft et al., 1999; Friebe and Bartenschlager, 2002). Because the IRES serves a crucial function for viral infection and propagation and is therefore highly conserved, it represents an ideal target for anti-HCV approaches employing nucleic acid homologies such as trans-splicing group I introns (Ryu et al., 2003).
Trans-splicing group I introns derived from the cis-splicing group I intron of Tetrahymena thermophila mediate RNA splicing through two successive transesterification steps (Cech, 1991). First, the intron recognizes a specific uracil on the target RNA during complementary base pairing with the surrounding sequence. The target RNA is then cleaved at that uracil, and the intron-attached 3′ exon is cleaved from the group I intron and appended onto the cleaved target RNA to create a product RNA. If that product is capable of translation it will express a new protein encoded by the sequence of the 3′ exon (Long et al., 2003). Group I introns have been used successfully in a number of anti-viral applications including targeting of Dengue Fever virus (Carter et al., 2010), HCV (Ryu et al., 2003), and HIV (Kohler et al., 1999) genomes, and in posttranscriptional gene manipulations including the restoration of wild-type p53 activity in three cancerous cell lines (Shin et al., 2004) and the repair of sickle β-globin mRNAs in erythrocyte precursors (Lan et al., 1998).

Because of its potent antiretroviral activity against a wide

Because of its potent antiretroviral activity against a wide range of retroviruses, A3G is the best-characterized member of the A3 family. A3G has two Z domains (Z2-Z1) with each one containing a seemingly intact catalytic site, however only its C-terminal Z1 domain (CTD) is catalytically active (Hache et al., 2005; Navarro et al., 2005). The N-terminal domain (NTD) of the enzyme has been ascribed various other functions such as binding to HIV-1 Vif and virion encapsidation (Huthoff et al., 2009; Huthoff and Malim, 2007; Lavens et al., 2010; Mangeat et al., 2004; Schrofelbauer et al., 2004). Residues located in the NTD have also been implicated in nucleic cetrimonium bromide binding, especially RNA (Bach et al., 2008; Bélanger et al., 2013; Bulliard et al., 2009; Huthoff et al., 2009; Iwatani et al., 2006; Khan et al., 2007; Shindo et al., 2012).
In addition to its cytosine deaminase activity, it is well established that A3G also has the ability, in certain experimental conditions, to hinder retroviral infection by means that are independent of deamination (Bélanger et al., 2013; Bishop et al., 2006, 2008; Guo et al., 2007; Iwatani et al., 2007; Li et al., 2007; Lu et al., 2015; Luo et al., 2007; Mariani et al., 2003; Mbisa et al., 2010; Newman et al., 2005; Wang et al., 2012). In particular, several groups have demonstrated defects in tRNA3primer annealing and strand transfers during replication, which consequently lead to decreased levels of early and late reverse viral transcript accumulation as well as reduced proviral integration. Overall, these restriction mechanisms are thought to occur as a result of the direct interaction of A3G with the viral reverse transcriptase (RT) and integrase (IN), and possibly also through an alteration in the processivity of the reverse transcriptase caused by the clamping of A3G onto its ssDNA substrate during reverse transcription (Bishop et al., 2008; Luo et al., 2007; Wang et al., 2012). Recent work from our group has demonstrated that RNA binding by A3G is required for deamination-independent restriction (Bélanger et al., 2013). We mapped the RNA-binding ability of A3G to two tryptophans, W94 and W127, in the non-catalytic NTD of the protein. By individually substituting these two residues to alanine, A3G lost most deamination-independent functions including the inhibitions of late reverse transcript accumulation and proviral integration (Bélanger et al., 2013).

Results and discussion
In a previous study we demonstrated that A3G [W94A] and A3G [W127A] mutants each had a significantly reduced ability to bind RNA, and more specifically, to 7SL, Alu, hY1 and hY3 RNAs (Bélanger et al., 2013). However, weak RNA binding could still be detected with these mutants. Here we were first interested to determine whether the double W94A/W127A substitution could account for all detectable RNA binding by A3G. Lysates from 293T cells transfected with Flag-tagged A3G or mutants were used for Co-IP using anti-Flag conjugated agarose beads followed by RNA isolation. Samples were then subjected to qPCR to measure the relative binding of each APOBEC to RNA. In these conditions, [W94A/W127A] did not bind to any of the RNAs tested above the background level set by the APOBEC2 (A2) negative control (Fig. 1A). A3G has a well-established ability to assemble into large oligomeric or high molecular mass (HMM) ribonucleic complexes (Chiu et al., 2005). In a previous report we showed that the W94A and W127A point mutants displayed a partial ability to assemble into oligomeric structure compared to wild type A3G (Bélanger et al., 2013). Here we analyzed the double mutant in presence and absence of RNase treatment. We could not detect any evidence of assembly into complexes that are RNA-dependent (Fig. 1B). Surprisingly, although RNA binding was lost, cytoplasmic foci believed to be P-bodies, stress granules and other RNA processing bodies that normally associate with A3G were still visible when similar levels of protein expression were compared (Fig. 1C and D) (Gallois-Montbrun et al., 2007; Kozak et al., 2006; Wichroski et al., 2006). This observation thereby indicates that association of A3G with these structures is not dependent on cellular RNA binding, nor is it dependent on protein aggregation or self-assembly (Fig. 1E).