Colorado Strain Research Articles

172 ANTIVIRAL ACTION OF PROPOLIS AQUEOUS EXTRACT DURING MATURATION OF INFECTED BOVINE OOCYTES

The objective of this study was to evaluate the decrease of virus replication in BoHV-1 (Colorado strain, 106.5 TCID50/mL) after the treatment using propolis aqueous extract (PAE) during in vitro maturation of infected bovine oocytes (24 h). Cow ovaries were obtained from a local slaughterhouse (Nelore breed) and transported to the laboratory. Cumulus-oocyte complexes (COC) were aspirated from follicles and separated into 4 groups (number of replicates for all groups = 6), which were exposed to 20 μL of sterile physiological solution (SPS), 100 μL of the in vitro maturation (IVM) medium [G1 (control), n = 609]; 10 μL of BoHV-1 (106.5 TCID50/mL) virus, 100 μL IVM medium, and 10 μL of SPS (G2, n = 786); 10 μL of PAE in 0.001% in SPS, 100 μL IVM medium, and 10 μL of SPS (G3, n = 819); 10 μL of PAE extract in 0.001% in SPS, 10 μL of BoHV-1 (106.5 TCID50/mL) virus and 100 μL of the IVM medium (G4, n = 734). All groups were kept for 24 h at 38.5°C, 5% CO2 in air. After the IVM, we analysed COC expansion and the presence of a polar body by optical microscope as well as viral replication by titration (Reed and Muench test) after 72 h of co-culture with Madin-Darby bovine kidney (MDBK) cells. The G1, G3, and G4 showed steady expansion of the cumulus cells and ooplasm with uniform appearance. The G2 did not have expansion of the cumulus cells. In contrast, the cytoplasm showed degenerative appearance and an absence of maturity in numerous oocytes. The maturation rates were as follows: G1 = 79% (482/609), G2 = 51% (407/786), G3 = 80% (662/819), and G4 = 76% (565/734). The differences among groups in maturation rates were compared using the chi-squared test (α = 5%) and the average titrations using the Mann–Whitney test (α = 5%). There was a significant difference (P < 0.01) among G1 and G2 evincing the interference of the virus maturation. The extract did not affect maturation as there was no difference among G1 and G3 (P = 0.43). The main step was finding no significant difference between the groups G1 and G4, (P = 17) proving that the extract interferes with viral replication. The titration after co-culturing the oocytes in MDBK demonstrated that G4 (average titrations = 1.63 × 103 titration) showed a lower rate of viral replication, the Mann-Whitney test, when compared to group G2 (average titrations = 6.04 × 107) which has not been subjected to treatment with PAE (P = 0.02). These results indicate that the propolis aqueous extract reduces the rate of viral replication without interfering with the maturation of oocytes and, therefore, it can be a conclusion that the analysis of the action of the molecules of this extract (by proteomics, for example) and future studies should be directed towards identifying the effect of extract on antiviral activity during the assessment of oocyte competence and embryo development.

Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous Colorado strain and the heterologous Minnesota strain.

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2'-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2'-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV), human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus, and highly lethal emerging pathogens such as Nipah virus and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategies for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2'-O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacking 2'-O MTase were not only highly attenuated in turkeys but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other animal and human paramyxoviruses for rationally designing live attenuated vaccines.

152 EVALUATION OF POMEGRANATE (PUNICA GRANATUM) AS REPLICATION INHIBITOR OF BoHV-1 COLORADO STRAIN DURING IN VITRO MATURATION OF INFECTED BOVINE OOCYTES

The widespread use of biotechnologies in animal breeding in Brazil has enabled greater control over the transmission of pathogens. Nevertheless, disease transmission continues to be a significant concern and justifies the search for better control agents. The objective of this study was to evaluate whether the detrimental effects of the experimental infection of oocytes with BoHV-1 (Colorado strain, 108 TCID 50 mL–1) during in vitro maturation (24 h) could be reduced by the viral inhibitor ethanolic extract of Punica granatum (PG). Cattle ovaries were obtained from a local slaughterhouse (unknown breed), and transported to the laboratory. Cumulus–oocyte complexes (COC) were aspirated from follicles and allocated into four groups, which were exposed to: 10 µL of sterile physiological solution (G1 [control], n = 125); 10 µL of PG extract in 0.01% sterile saline solution (G2, n = 149); 10 µL of BoHV-1 virus (G3, n = 151); or 10 µL of PG extract in 0.01% and 10 µL of BoHV-1 virus (G4, n = 144). All groups were matured in 100 mL of IVM for 24 h at 37.5°C, 5% CO2 in air. After in vitro maturation, we evaluated COC expansion and presence of a polar body by optical microscope, and viral replication by titration (Reed and Muench test) after 72 h co-culture with Madin-Darby bovine kidney (MDBK) cells. Differences among groups in maturation rates were compared by chi-square test, and in titration by t-test. The G1, G2, and G4 showed steady expansion of the cumulus cells and ooplasm with uniform appearance. The G3 did not show expansion of the cumulus cells and ooplasma showed degenerative appearance. The maturation rates were as follows: G1 52% (65/125); G2 45.63% (68/149); G3 27.81% (42/151); G4 41.66% (60/144). We used the χ2 test (P ≤ 0.05) for the rate of maturation and the t-test (P ≤ 0.05) for the titration data. A reduction (P ≤ 0.05) in maturation rate was observed in G3, when compared to the G1 [control]. Oocytes exposed to BoHV-1 virus and matured in the presence of PG extract (G4) had lower (P ≤ 0.05) rates of viral replication than those matured in the absence of PG (G3). These results support the conclusion that PG extract reduces the viral rate of replication without interfering in oocyte maturation, and may be an alternative to sanitary control protocols.

154 EVALUATION OF THE EFFECTIVENESS OF TREATMENT WITH TRYPSIN IN MURINES EMBRYOS EXPERIMENTALLY EXPOSED TO BOVINE HERPES VIRUS TYPE-1 (BoHV-1) BY THE NESTED-PCR TECHNIQUE

The cryopreservation of bovine embryos has allowed an increase in their commercialization causing concern about the transmission of pathogens through the production and transfer of in vitro embryos. The International Society of Embryo Transfer (IETS) suggests the treatment of embryos with trypsin or antibiotics in alternating washes with culture medium in order to remove and/or inactivate infectious agents that may interfere with the final product. The aim of this study was to evaluate the effectiveness of trypsin treatment in the elimination and/or removal of bovine herpes virus type-1 (BoHV-1), Colorado strain, in murine embryos. Viral detection was made by n-PCR and cytopathic effect in Madin Darby bovine kidney (MDBK) cells. Six- to 8-wk-old female mice (Swiss) were superovulated and mated with fertile males of the same strain. After 24 h, the zygotes (n = 262) were divided into 3 groups: control group submitted to sequential wash (CSW), the group exposed to the virus (30 μL; 106.5 virus mL–1) and submitted to sequential wash (ESW), and the group exposed to the virus and submitted to the trypsin treatment (ETT). All the groups of zygotes and the last sequential wash drops were tested by nested-PCR and inoculated in MDBK cells to allow observation of cytopathic effects. All groups except for CSW showed positive results for the nested-PCR for both zygotes and for the last wash drops. A cytopathic effect was observed in all groups except for CSW, demonstrating the viability of the virus after treatment. These results demonstrated that trypsin treatment was not effective in eliminating or removal of BoHV-1. The need for such studies is fundamentally important when considering the potential risk of pathogen transmission by reproductive biotechnologies.

148 RISK OF TRANSMISSION OF BOVINE HERPESVIRUS (BHV-1) BY INFECTED SEMEN TO RECIPIENTS AND EMBRYOS

Bovine herpesvirus-1 (BHV-1) causes a variety of economically important respiratory and reproductive problems, the latter including vulvovaginitis, endometritis and infertility. For that reason, several countries have eradicated the disease and others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BHV-1 transmission through contaminated semen used for artificial insemination, there is no available evidence to indicate whether the resulting embryos, when used for embryo transter (ET), can lead to the transmission of BHV-1 to recipients and offspring. For this experiment, bull semen contaminated in vitro with BHV-1 at 102 TCID50 mL–1 (Colorado strain) and then cryopreserved was used for insemination (2 times at estrus) of BHV-1 seronegative, superovulated heifers (N = 18). Embryos were collected postmortem 7 days post-insemination and were washed according to the IETS recommendations (however without trypsin treatment) or left unwashed. On 4 occasions, washed embryos were transferred to BHV-1 seronegative recipients. The remaining embryos and other samples collected from the reproductive tract were tested for BHV-1 presence using the standard virus isolation test. In total, out of 144 unfertilized oocytes and embryos collected, 9 were ET quality. Most of the embryos were degenerated (N = 79) or unfertilized (N = 56). The 4 heifers, which each received a single morula-stage embryo, maintained seronegative status, but did not become pregnant. BHV-1 was detected in 43% (23/53) unwashed and 0% (0/57) of washed embryos, 78% (14/18) of follicular fluid samples, 89% (16/18) of oviductal epithelial cells, 78% (14/18) of endometrium, and 89% (16/18) of corpora lutea tissues. Results herein suggest that BHV-1 can be transmitted by infected semen to embryo donors. The resulting unwashed embryos may remain infectious. However, whether BHV-1 uninfected offspring can be produced by ET of BHV-1 contaminated embryos that are washed according to the IETS guidelines, remains to be determined.

Thin Layer and High Performance Column Liquid Chromatographic Analysis of Selected Carboxylic Acids in Standards and from Helisoma trivolvis (Colorado Strain) Snails

Separations and detections of acetic, fumaric, lactic, malic, pyruvic, and succinic acid standards were compared using previously reported thin layer chromatography (TLC) and high performance TLC (HPTLC) systems, which consisted mainly of silica gel or cellulose layers and acid‐base indicator or aniline‐aldose detection reagents. The best results were obtained with an HPTLC cellulose layer, triple development with n‐propanol‐2 M ammonium hydroxide mobile phase, and aniline‐xylose reagent, but resolution and sensitivity were not adequate for determination of these acids in Helisoma trivolvis (Colorado strain) snails. Analysis of snail digestive gland‐gonad complex (DGG) samples was carried out using extraction with 50% Locke's solution, cleanup of the extract by anion exchange solid phase extraction (SPE), and ion exclusion high performance liquid chromatography with ultraviolet detection. Hemolymph was applied directly to the SPE column. Acetic, fumaric, malic, and pyruvic acids were detected at concentrations ranging from 24 to 200 ppm in the DGG and less than 154 to 16,000 ppm in hemolymph.

The concentration of calcium carbonate in shells of freshwater snails

The range of concentration of calcium carbonate in the shells of various freshwater gastropods was determined using ion chromatography. Individuals of Helisoma trivolvis (wild) were collected from three ponds in Pennsylvania and New Jersey; individuals of Physa sp. were collected from one pond in New Jersey; individuals of H. trivolvis (Colorado strain) and Biomphalaria glabrata (NMRI strain) were raised in the laboratory; and individuals of Pomacea bridgesii were purchased commercially. The concentrations of calcium carbonate (mean % by dry weight) of the shells were as follows: H. trivolvis (wild), 97.0; Physa sp., 97.8; H. trivolvis (CO), 97.6; B. glabrata, 98.8; P. bridgesii, 98.2. Our data support and validate the previous claim that snail shells are comprised of 95–99.9% calcium carbonate.

238 EFFICACY OF RECOMBINANT TRYPSIN AGAINST BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VITRO-DERIVED PORCINE EMBRYOS

TrypLETM (Invitrogen, Carlsbad, CA, USA) is a recombinant, fungal, trypsin-like protease that is used as a substitute for porcine-origin trypsin in cell culture procedures. It is stable at room temperature and does not present the same risk of contamination as animal-origin trypsin. Previously, TrypLE SelectTM (10X) was shown to remove bovine herpesvirus-1 (BHV-1) from Day 7 in vivo-derived embryos (Marley et al. 2006 Reprod. Fert. Dev. 18, 213–214). The objective of this study was to determine if the same treatment would effectively remove BHV-1 from Day 7 zona pellucida-intact, in vitro-derived porcine embryos. Day 7 in vitro-derived morulae and blastocysts and non-fertile or degenerate embryos (NFD) were washed according to the International Embryo Transfer Society protocol. One group of 10 NFD was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106–108 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 10 NFD was washed and served as the positive control. The remaining developed embryos were divided into groups of 10 and washed and treated as described in Table 1. Following treatment, the embryos were sonicated in groups of 5 and assayed by virus isolation. The negative control embryos, as well as the embryos treated with porcine-origin trypsin, TrypLE Select (10X) for 7 min, and TrypLE Select (10X) diluted 1 : 2 for 10 min, were negative for virus. The positive control embryos in addition to the other treatments were positive on virus isolation (Table 1). Although, TrypLE Select (10X) does have some antiviral effect when used for 10 min, it was not completely effective, as shown by the positive virus isolation results of one group of 10 embryos. The groups treated with TrypLE Select (10X) diluted 1 : 2 for 10 min were negative for virus; however, if a larger sample size had been tested, positive groups might have occurred. Though using a recombinant trypsin product would be beneficial over using an animal-origin product, it is not known if TrypLE Select (10X) would render a single IVF embryo free of infectious virus. Further research would also need to be performed to assess the viability of embryos following treatment with TrypLE Select (10X). In addition, other recombinant trypsin products need to be evaluated to determine their efficacy against BHV-1 associated with IVF embryos. Table 1.Effect of recombinant trypsin-like proteases on BHV-1 virus in porcine embryos

Determination of the Effects of Estivation and Starvation on Neutral Lipids and Phospholipids in Biomphalaria glabrata (NMRI Strain) and Helisoma trivolvis (Colorado Strain) Snails by Quantitative High Performance Thin Layer Chromatography‐Densitometry

High performance thin layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in Biomphalaria glabrata (NMRI strain) and Helisoma trivolvis (Colorado strain) snails during estivation and starvation. Control snails were maintained on a diet of Romaine lettuce leaves. HPTLC analyses showed a significant (ANOVA, P<0.05) decrease in triacylglycerols in estivated and starved snails compared to the controls and a significant increase in steryl esters in estivated and starved snails compared to the controls.

Effects of Estivation on Lutein and β‐Carotene Concentrations in Biomphalaria glabrata (NMRI Strain) and Helisoma trivolvis (Colorado Strain) Snails as Determined by Quantitative High Performance Reversed Phase Thin Layer Chromatography

High performance thin layer chromatrography (HPTLC) on C‐18 chemically bonded silica gel layers with petroleum ether‐acetonitrile‐methanol (1:2:2) mobile phase and visible mode slit‐scanning densitometry was used to examine the effects of estivation on β‐carotene and lutein in the medically and economically important snails Biomphalaria glabrata (NMRI strain) and Helisoma trivolvis (Colorado strain). Snails were estivated for 2 weeks in moist chambers at 24°C and a relative humidity of 98%. Controls were maintained at 24°C for the same period in aerated aquaria containing artificial spring water and fed Romaine lettuce leaves ad libitum. As determined by HPTLC analysis, estivation had no significant effect on the weight percentage of β‐carotene in the whole body or digestive gland‐gonad complex (DGG) of both snail species. A significant reduction (Student's t‐test, P<0.5) was found in the percentage of lutein in the DGG of H. trivolvis. Whereas published and unpublished studies have demonstrated marked reductions of carbohydrates and lipids in estivated pulmonate snails, the effects of estivation on lipophilic pigments in these snails does not appear to be as pronounced.

212 TRYPLE SELECT (10X) EFFECTIVELY REMOVES BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VIVO-DERIVED EMBRYOS

Porcine-origin trypsin will effectively remove bovine herpesvirus-1 (BHV-1) from in vivo-derived embryos. It is not known if TrypLE (Invitrogen, Carlsbad, CA, USA) could be used to remove BHV-1, but this recombinant porcine sequence trypsin-like protease would be an attractive alternative because it is highly stable at room temperature and does not pose the same threat for contamination as animal-origin trypsin. Thus, the objective of this study was to determine if TrypLE Express (1X) for 1.5 min of exposure or TrypLE Select (10X) for 10 min of exposure would be effective at removing BHV-1 from Day 7 zona pellucida-intact, in vivo-derived embryos after they had been exposed to the virus. Day 7 bovine in vivo-derived morulae and blastocysts and non-fertile degenerate (NFD) embryos were collected and shipped overnight to our facility. Upon arrival, the zona pellucida intact embryos were washed according to the International Embryo Transfer Society protocol. Developed embryos were washed separately from NFD embryos. One group of 5 or 10 NFD or developed embryos was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 5 or 10 NFD or developed embryos was washed and served as the positive control. One group of 10 developed embryos was washed and treated with porcine origin trypsin. The remaining developed embryos were divided into groups of 5 or 10 and washed and treated with TrypLE Express for 1.5 min or TrypLE Select (10X) for 10 min. Following treatment, the embryos were sonicated in groups of 5 or 10 and assayed by virus isolation. The negative control embryos, porcine origin trypsin treated embryos, and TrypLE Select treated embryos were negative for virus. The positive control embryos and the TrypLE Express treated embryos were positive on virus isolation (Table 1). When it was determined that TrypLE Express was not effective at 1.5 min, TrypLE Select (10X) was used for 10 min. These preliminary results indicate that use of TrypLE Select (10X) for 10 min is effective for removal of BHV-1 associated with Day 7, zona pellucida-intact, in vivo-derived embryos. In addition, TrypLE Select has the advantage of being an animal-origin-free product. However, use of TrypLE Express (1X) for 1.5 min was not effective. Because it is not practical to expose embryos to trypsin for 10 min, further research is needed to determine the ideal treatment concentration and time that will effectively remove BHV-1 without harming in vivo-derived bovine embryos. Table 1. Virus isolation results

Evaluation of Five Different Antigens in Enzyme-Linked Immunosorbent Assay for the Detection of Avian Pneumovirus Antibodies

Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.

211 LACTOFERRIN INHIBITS BOVINE HERPESVIRUS-1 IN CELL CULTURE AND ALLOWS NORMAL DEVELOPMENT OF IN VITRO-PRODUCED EMBRYOS

Lactoferrin is an iron-binding glycoprotein found in milk, saliva, tears, and other exocrine secretions. It is known to have in vitro antiviral effects against human, feline, and canine herpesviruses. In addition, lactoferrin is known to be safe in cell culture. Bovine herpesvirus-1 (BHV-1) is a likely contaminant of in vitro embryo production. Further, trypsin treatment is not completely effective in removing the virus from these embryos. We hypothesized that a nontoxic concentration of lactoferrin might prevent replication of BHV-1 within in vitro embryo production systems. Thus, the specific objectives of this research were to determine if lactoferrin from bovine milk would inhibit BHV-1 in cell culture and to determine if in vitro-produced embryos could develop normally when cultured in lactoferrin. Two-fold dilutions of lactoferrin (from 10 to 0.625 mg/mL) were added to Madin Darby bovine kidney cells, followed in 15 min by the addition 104 PFU/mL of BHV-1 (Colorado strain). Samples of cell lysate were taken at Day 2 and virus was quantified by plaque assay. The percent of virus inhibited by the antiviral agent at each concentration was determined by comparison to equivalent samples from temporal control cultures in which no compound was added before or after inoculation (Percentage of virus inhibited = [Quantity of virus in the control sample - Quantity of virus in the compound sample]/Quantity of virus in the control sample � 100). Next, the effect of lactoferrin was determined on in vitro-produced embryos. Cumulus oocyte complexes were received from an abattoir, matured in transit, placed in fertilization drops for 6 h, and then placed in culture drops containing lactoferrin (10, 5, and 2.5 mg/mL). At Day 3.5, embryos &gt; 4 cell stage were placed into fresh culture drops containing lactoferrin. On Day 7.5, blastocyst development was noted and the developed embryos were stained to count viable cells. Blastocyst development rate and nucleated cell count of the treated embryos were compared to those of the controls using Chi square test, and ANOVA and Tukey-Kramer HSD, respectively. Lactoferrin (10 mg/mL) inhibited 2 to 5 logs of virus. At concentrations of 5 and 2.5 mg/mL, 1 to 3 logs of virus were inhibited, and concentrations of 1.25 and 0.625 mg/mL inhibited 0 to 2 logs of virus. Lactoferrin did not affect the nucleated cell count of the treated embryos. In addition, unlike 10 and 5 mg/mL, 2.5 mg/mL of lactoferrin did not affect blastocyst development. These preliminary results indicate that lactoferrin from bovine milk can significantly inhibit BHV-1 in cell culture. Furthermore, supplementation of in vitro culture with 2.5 mg/mL of lactoferrin does not affect blastocyst development or cell count of in vitro-produced embryos.

Comparison of the full-length genome sequence of Avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3′ end and a 45 nt trailer at its 5′ end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.

Effects of glucose on survival, infectivity and linear movement of the cercariae of Echinostoma caproni

AbstractThe effects of glucose in artificial spring water (ASW) on the survival, infectivity, and linear movement of Echinostoma caproni cercariae were studied. Cercariae maintained at 23°C in 1% glucose in ASW (ASWG) or ASW alone, reached 50% survival at 26 and 23 h, respectively. All cercariae in ASWG and ASW were dead by 50 and 32 h, respectively. Infectivity to juvenile Helisoma trivolvis (Colorado strain) snails was significantly less for cercariae aged 16 h in ASWG compared to cercariae aged 16 h in ASW. Linear movement, i.e. the ability of cercariae to traverse a 1-cm radius, ceased at 16 and 20 h for cercariae maintained in ASWG and ASW, respectively. Glucose added to ASW extended the survival time of E. caproni cercariae but decreased their ability to infect snails or move in a linear direction.

Effects of glucose on survival, infectivity and linear movement of the cercariae of Echinostoma caproni.

The effects of glucose in artificial spring water (ASW) on the survival, infectivity, and linear movement of Echinostoma caproni cercariae were studied. Cercariae maintained at 23 degrees C in 1% glucose in ASW (ASWG) or ASW alone, reached 50% survival at 26 and 23 h, respectively. All cercariae in ASWG and ASW were dead by 50 and 32 h, respectively. Infectivity to juvenile Helisoma trivolvis (Colorado strain) snails was significantly less for cercariae aged 16 h in ASWG compared to cercariae aged 16 h in ASW. Linear movement, i.e. the ability of cercariae to traverse a 1-cm radius, ceased at 16 and 20 h for cercariae maintained in ASWG and ASW, respectively. Glucose added to ASW extended the survival time of E. caproni cercariae but decreased their ability to infect snails or move in a linear direction.

Effects of snail size and diet on encystment of Echinostoma caproni cercariae in juvenile Helisoma trivolvis (Colorado strain) and observations on survival of infected snails.

The effects of snail size and diet on encystment of Echinostoma caproni cercariae in juvenile Helisoma trivolvis (Colorado strain) snails were studied. Encystment in neonatal (<1-mm shell diameter) and juvenile (2- to 3-mm shell diameter) snails was compared 24 hr postinfection (PI) after individual exposure of snails of each size to 1, 5, 10, 25, or 50 cercariae. Significantly more cysts were recovered from juvenile snails exposed to 10, 25, or 50 cercariae than from neonatals with comparable exposure. The maximum number of cysts recovered from juveniles exposed to 50 cercariae was 42, compared with a maximum of 15 cysts in neonatals comparably exposed. Size of H. trivolvis was a major factor in determining cyst burden in this planorbid. A diet of either Romaine lettuce leaf or hen's egg yolk did not have a significant effect on the number of cysts recovered at 24 hr PI from juvenile snails exposed to 25 or 75 cercariae. Survival of infected versus uninfected neonatals was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 6 and 7 days PI when compared with uninfected controls.

High-performance thin-layer chromatographic analysis of lipids in juvenileHelisoma trivolvis(Colorado strain) maintained on a hen’s egg yolk diet

High-performance thin-layer chromatographic analysis of lipids in juvenile<i>Helisoma trivolvis</i>(Colorado strain) maintained on a hen’s egg yolk diet

Effects of temperature on survival, infectivity and in vitro encystment of the cercariae of Echinostoma caproni.

The effects of temperature on survival, infectivity and in vitro encystment of Echinostoma caproni cercariae in artificial spring water (ASW) were studied. Effects of aging cercariae in ASW at various temperatures showed that at 23 degrees C cercariae achieved 50% survival in 24 h, compared to 92 h at 12 degrees C. Cercariae aged in ASW at 28 and 37.5 degrees C showed 50% survival at 16 and 10 h, respectively. Cercariae aged at different temperatures for various times were used to infect juvenile Helisoma trivolvis (Colorado strain) snails maintained in ASW at 23 degrees C. Index of infectivity was based on counting encysted metacercariae in the snails at 8 to 12 h post-infection. Cercariae aged at 23, 28 and 37.5 degrees C showed 50% encystment at 6, 8 and 4 h, respectively. Cercariae aged at 4 degrees C showed 50% encystment in 10 h and cercariae aged at 12 degrees C showed 50% encystment beyond 16 h. Cercariae showed maximal longevity and infectivity in snails when aged at 12 degrees C in ASW. For E. caproni, as in other digeneans, the infective period of cercariae is markedly shorter than the maximal life-span at any given temperature. Studies on in vitro encystment of E. caproni cercariae in Locke's solution:ASW (1:1) showed that encystment was optimal at 23 degrees C (78% encystment) and that it declined to 44% at 28 degrees C and became almost nil (0.02%) at 12 or 37.5 degrees C.

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses.

A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted M(r) of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.