High genetic merit dairy heifers grazing low quality forage had similar weight gain and urinary nitrogen excretion to those of low genetic merit heifers.

Climate variability and increasing drought events have become significant concerns in recent years. However, there is limited published research on body weight (BW) change of dairy heifers with different genetic merit when grazing on drought impacted pastures in southern Australia. Achieving target body weight (BW) is vital for dairy heifers, especially during critical stages like mating and calving. This study aimed to assess dry matter (DM) intake, BW change, urinary nitrogen excretion, and grazing behaviours of high vs. low genetic dairy heifers grazing pasture during a 43-day experimental period in a drought season. Forty-eight Holstein Friesian heifers grazed on ryegrass-dominant pasture and were divided into two groups based on their high and low Balanced Performance Index (HBPI and LBPI, respectively). Each group was further stratified into six plots, with similar BW, resulting in four heifers per replication group. Data from the five measurement days were averaged for individual cows to analyse the dry matter intake, nitrogen intake and nitrogen excretion. The statistical model included the treatment effect of BPI (H and L) and means were analysed using ANOVA. The pasture quality was poor, with metabolizable energy 9.3 MJ/Kg DM and crude protein 5.9% on a DM basis. Nitrogen intake and urinary nitrogen excretion were significantly higher (p < 0.05) in HBPI compared to the LBPI. However, despite these differences, the study did not find any advantages of having HBPI heifer grazing on low quality forage in terms of BW performance.

The effect of pasture quantity temporal variation on milking robot utilization.

In pasture-based automatic milking systems (AMS), a decrease in robot utilization (RU) often occurs in the early morning hours. Novel feeding strategies that encourage voluntary cow traffic throughout 24 h could help mitigate this problem. We determined the effect of 3 distinct pasture allocation methods on RU patterns throughout a 24-h period. The experiment was conducted at the University of Melbourne's Dookie research farm in northern Victoria, Australia. Three Lely Astronaut A3 robotic milking units (Lely, Maassluis, the Netherlands) milked 133 cows, grazing pasture, with concentrate offered at milking in the robots. The farm operated a system of 3-way grazing, with active access to each pasture allocation: 2030-0400 h (allocation A), 0400-1330 h (allocation B), and 1330-2030 h (allocation C). Treatments varied in the quantity of feed offered per hour of active access to each of the 3 pasture allocations. The control treatment offered the same proportion of feed (corrected for active access time) in all 3 pasture allocations (allocation A = 31.3%, B = 39.6%, and C = 29.2%). The day treatment offered the largest proportion of feed during the day (allocation A = 20%, B = 40%, and C = 40%), following the cows' diurnal pattern of feeding activity. The night treatment offered the largest proportion of feed at night (allocation A = 42%, B = 40%, and C = 18%). Due to the nature of pasture-based AMS, treatments could not be applied simultaneously. Therefore, treatments were applied to the entire herd and repeated twice over 42 d, lasting 7 d/treatment, with the first 3 d for habituation, followed by 4 d of data collection. Robot utilization (milkings/h) varied throughout 24 h between treatments, with the night treatment recording greater RU at 0800, 1800, and 1900 h and lower RU between 2100 to 0100 h, compared with the day treatment. The proportion of the herd milking between 0000 and 0600 h was greater for the control (43.3%) and day (45.3%) treatments compared with the night treatment (25.8%). Herd-average daily pasture intake was similar (10.5 kg of dry matter) for all treatments. This experiment is the first to demonstrate the manipulation of RU by varying the quantity of pasture offered. However, the use of variable allocation alone did not eliminate the decrease in RU between 0000 and 0600 h, with the timing of allocation also likely to play a role. We recommend a further research focus on combining both timing and quantity of pasture allocated to improve RU in pasture-based AMS.

A model of milk production in lactating dairy cows in relation to energy and nitrogen dynamics.

A generic daily time-step model of a dairy cow, designed to be included in whole-system pasture simulation models, is described that includes growth, milk production, and lactation in relation to energy and nitrogen dynamics. It is a development of a previously described animal growth and metabolism model that describes animal body composition in terms of protein, water, and fat, and energy dynamics in relation to growth requirements, resynthesis of degraded protein, and animal activity. This is further developed to include lactation and fetal growth. Intake is calculated in relation to stage of lactation, pasture availability, supplementary feed, and feed quality. Energy costs associated with urine N excretion and methane fermentation are accounted for. Milk production and fetal growth are then calculated in relation to the overall energy and nitrogen dynamics. The general behavior of the model is consistent with expected characteristics. Simulations using the model as part of a whole-system pasture simulation model (DairyMod) are compared with experimental data where good agreement between pasture, concentrate and forage intake, as well as milk production over 3 consecutive lactation cycles, is observed. The model is shown to be well suited for inclusion in large-scale system simulation models.

Forage-based dairying in a water-limited future: use of models to investigate farming system adaptation in southern Australia.

The irrigated dairy industry in southern Australia has experienced significant restrictions in irrigation water allocations since 2005, consistent with climate change impact predictions for the region. Simulation models of pasture growth (DairyMod), crop yield (Agricultural Production Systems Simulator, APSIM), and dairy system management and production (UDDER) were used in combination to investigate a range of forage options that may be capable of sustaining dairy business profitability under restricted water-allocation scenarios in northern Victoria, Australia. A total of 23 scenarios were simulated and compared with a base farm system (100% of historical water allocations, grazed perennial ryegrass pasture with supplements; estimated operating surplus $A2,615/ha at a milk price of $A4.14/kg of milk solids). Nine simulations explored the response of the base farm to changes in stocking rate or the implementation of a double cropping rotation on 30% of farm area, or both. Five simulations explored the extreme scenario of dairying without any irrigation water. Two general responses to water restrictions were investigated in a further 9 simulations. Annual ryegrass grazed pasture, complemented by a double cropping rotation (maize grown in summer for silage, followed by either brassica forage crop and annual ryegrass for silage in winter and spring) on 30% of farm area, led to an estimated operating surplus of $A1746/ha at the same stocking rate as the base farm when calving was moved to autumn (instead of late winter, as in the base system). Estimated total irrigation water use was 2.7ML/ha compared with 5.4ML/ha for the base system. Summer-dormant perennial grass plus double cropping (30% of farm area) lifted operating surplus by a further $A100/ha if associated with autumn calving (estimated total irrigation water use 3.1ML/ha). Large shifts in the forage base of dairy farms could sustain profitability in the face of lower, and fluctuating, water allocations. However, changes in other strategic management policies, notably calving date and stocking rate, would be required, and these systems would be more complex to manage. The adaptation scenarios that resulted in the highest estimated operating surplus were those where at least 10 t of pasture or crop DM was grazed directly by cows per hectare per year, resulting in grazed pasture intake of at least 2 t of DM/cow, and at least 60% of all homegrown feed that was consumed was grazed directly.

Expression and function of microRNAs encoded by Kaposi's sarcoma-associated herpesvirus.

microRNAs (miRNAs) are widely used by animal and plant cells to posttranscriptionally regulate cellular gene expression, but this regulatory mechanism can also be used by pathogenic viruses for the same purpose. It is now well established that numerous miRNAs are expressed by a wide range of pathogenic herpesviruses, although their mRNA targets and role in the viral life cycle remain unclear. Here, we discuss what is currently known about the expression and function of the 12 miRNAs that are expressed by the pathogenic gamma herpesvirus Kaposi's sarcoma-associated herpesvirus in latently infected human B cells.

The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML.

AIMS: To determine the complete nucleotide sequence of the bacteriophage VHML and establish a hypothesis for the virulence conversion caused by VHML infection of Vibrio harveyi. METHODS AND RESULTS: The complete nucleotide sequence of VHML was determined (43,193 bp) and used to identify putative genes. The translated products of these genes were compared with reported sequences to assign hypothetical functions. All anticipated structural genes and putative genes for lysogeny were identified. In addition, we found a complete N6-adenine methyltransferase (Dam) gene that appeared to have an essential site for ADP-ribosylating toxins at the C-terminal of the translated product. CONCLUSIONS: Virulence conversion of V. harveyi by VHML may be associated with Dam transcriptional regulation. The Dam gene may also encode for a toxin component similar to ADP-ribosylating toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript lays the foundation for understanding the virulence of toxin-producing V. harveyi. Further research into aspects discussed here will lead to a greater comprehension regarding the invertebrate disease vibriosis and its control in the farming of these animals.

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans.

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin-embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experimental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.

Two closely related human nuclear export factors utilize entirely distinct export pathways.

Nuclear mRNA export mediated by the human protein TAP requires a carboxy-terminal domain that directly interacts with components of the nuclear pore complex. Here we demonstrate that NXF3, a human RNA binding protein related to TAP, lacks this domain yet retains the ability to export tethered RNA transcripts and to shuttle between the nucleus and the cytoplasm. NXF3 contains a novel Crm1-dependent nuclear export signal that compensates in cis for the loss of the nuclear pore targeting domain. NXF3-dependent RNA export is therefore blocked by Crm1-specific inhibitors that do not affect TAP function. Thus, while the related TAP and NXF3 proteins are both capable of mediating nuclear RNA export, they do so via unrelated export pathways.

Molecular basis for cell tropism of CXCR4-dependent human immunodeficiency virus type 1 isolates.

Laboratory isolates of human immunodeficiency virus type 1 (HIV-1) that utilize CXCR4 as a coreceptor infect primary human macrophages inefficiently even though these express a low but detectable level of cell surface CXCR4. In contrast, infection of primary macrophages by primary CXCR4-tropic HIV-1 isolates is readily detectable. Here, we provide evidence suggesting that this difference in cell tropism results from a higher requirement for cell surface CXCR4 for infection by laboratory HIV-1 isolates. Transfected COS7 cells that express a high level of CD4 but a low level of CXCR4 were infected significantly more efficiently by two primary CXCR4-tropic HIV-1 isolates compared to the prototypic laboratory HIV-1 isolate IIIB. More importantly, overexpression of either wild-type or signaling-defective CXCR4 on primary macrophages dramatically enhanced the efficiency of infection by the laboratory HIV-1 isolate yet only modestly enhanced infection by either primary CXCR4-tropic virus. Overexpression of CD4 had, in contrast, only a limited effect on macrophage infection by the laboratory HIV-1, although infection by the primary isolates was markedly enhanced. We therefore conclude that the laboratory CXCR4-tropic HIV-1 isolate exhibits a significantly higher CXCR4 requirement for efficient infection than do the primary CXCR4-tropic isolates and that this difference can explain the poor ability of the laboratory HIV-1 isolate to replicate in primary macrophages. More generally, we propose that the cell tropisms displayed by different strains of HIV-1 in culture can largely be explained on the basis of differential requirements for cell surface CD4 and/or coreceptor expression levels.

The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression.

It has been suggested that sequences located within the 5' noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete approximately 151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5' noncoding region but that is inactive when inverted or when placed in the 3' noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5' splice site positioned at +51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3' to this splice site. Analysis of total cellular gag and pol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave the pol 5' splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5' noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3' noncoding sequence. Neither the R element nor the introduced 5' intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in cis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3' noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847--4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.

Using viral species specificity to define a critical protein/RNA interaction surface.

The Tap protein mediates the sequence-specific nuclear export of mRNAs bearing the retroviral constitutive transport element (CTE) and also plays a critical role in the sequence nonspecific export of cellular mRNAs. Previously, we have demonstrated that CTE function displays species specificity, that is, the CTE functions in human but not quail cells. Here, we demonstrate that quail Tap fails to support CTE function because it cannot bind the CTE. However, changing a single residue in quail Tap, glutamine 246, to arginine, the residue found in human Tap, rescues both CTE function and CTE binding. This residue, which is located on the exterior of a recently reported molecular structure of Tap, defines a surface on Tap that is critical for CTE binding. These data emphasize the potential importance of cross-species genetic complementation in the identification and characterization of cellular factors that are critical for different aspects of viral replication.

A new entry route for HIV.

Identification of HIV-1 variants capable of entering T cells via the CD8 receptor suggests a new mode of viral pathogenesis. But are these variants rare, aberrant viruses or a real problem?

The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region.

Human endogenous retrovirus K (HERV-K) is the name given to an approximately 30-million-year-old family of endogenous retroviruses present at >50 copies per haploid human genome. Previously, the HERV-K were shown to encode a nuclear RNA export factor, termed K-Rev, that is the functional equivalent of the H-Rev protein encoded by human immunodeficiency virus type 1. HERV-K was also shown to contain a cis-acting target element, the HERV-K Rev response element (K-RRE), that allowed the nuclear export of linked RNA transcripts in the presence of either K-Rev or H-Rev. Here, we demonstrate that the functionally defined K-RRE coincides with a statistically highly significant unusual RNA folding region and present a potential RNA secondary structure for the approximately 416-nt K-RRE. Both in vitro and in vivo assays of sequence specific RNA binding were used to map two primary binding sites for K-Rev, and one primary binding site for H-Rev, within the K-RRE. Of note, all three binding sites map to discrete predicted RNA stem-loop subdomains within the larger K-RRE structure. Although almost the entire 416-nt K-RRE was required for the activation of nuclear RNA export in cells expressing K-Rev, mutational inactivation of the binding sites for K-Rev resulted in the selective loss of the K-RRE response to K-Rev but not to H-Rev. Together, these data strongly suggest that the K-RRE, like the H-RRE, coincides with an extensive RNA secondary structure and identify specific sites within the K-RRE that can recruit either K-Rev or H-Rev to HERV-K RNA transcripts.

Multiple blocks to human immunodeficiency virus type 1 replication in rodent cells.

The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.

Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm.

Mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor initiates most hereditary and sporadic colon carcinomas. Although APC protein is located in both the cytoplasm and the nucleus, the protein domains required to maintain a predominantly cytoplasmic localization are unknown. Here, we demonstrate that nuclear export of APC is mediated by two intrinsic, leucine-rich, nuclear export signals (NESs) located near the amino terminus. Each NES was able to induce the nuclear export of a fused carrier protein. Both APC NESs were independently able to interact with the Crm1 nuclear export factor and substitute for the HIV-1 Rev NES to mediate nuclear mRNA export. Both APC NESs functioned within the context of APC sequence: an amino-terminal APC peptide containing both NESs interacted with Crm1 and showed nuclear export in a heterokaryon nucleocytoplasmic shuttling assay. Also, mutation of both APC NESs resulted in the nuclear accumulation of the full-length, approximately 320-kDa APC protein, further establishing that the two intrinsic APC NESs are necessary for APC protein nuclear export. Moreover, endogenous APC accumulated in the nucleus of cells treated with the Crm1-specific nuclear export inhibitor leptomycin B. Together, these data indicate that APC is a nucleocytoplasmic shuttle protein whose predominantly cytoplasmic localization requires NES function and suggests that APC may be important for signaling between the nuclear and cytoplasmic compartments of epithelial cells.

Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K.

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the approximately 25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

Analysis of cellular factors that mediate nuclear export of RNAs bearing the Mason-Pfizer monkey virus constitutive transport element.

There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)(+) RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin beta-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.