Aviation passenger screening has been used worldwide to mitigate the translocation risk of SARS-CoV-2. We present a model that evaluates factors in screening strategies used in air travel and assess their relative sensitivity and importance in identifying infectious passengers. We use adapted Monte Carlo simulations to produce hypothetical disease timelines for the Omicron variant of SARS-CoV-2 for travelling passengers. Screening strategy factors assessed include having one or two RT-PCR and/or antigen tests prior to departure and/or post-arrival, and quarantine length and compliance upon arrival. One or more post-arrival tests and high quarantine compliance were the most important factors in reducing pathogen translocation. Screening that combines quarantine and post-arrival testing can shorten the length of quarantine for travelers, and variability and mean testing sensitivity in post-arrival RT-PCR and antigen tests decrease and increase with the greater time between the first and second post-arrival test, respectively. This study provides insight into the role various screening strategy factors have in preventing the translocation of infectious diseases and a flexible framework adaptable to other existing or emerging diseases. Such findings may help in public health policy and decision-making in present and future evidence-based practices for passenger screening and pandemic preparedness.

The corona virus disease-2019 (COVID-19) pandemic began in Wuhan, China, and quickly spread around the world. The pandemic overlapped with two consecutive influenza seasons (2019/2020 and 2020/2021). This provided the opportunity to study community circulation of influenza viruses and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in outpatients with acute respiratory infections during these two seasons within the Bavarian Influenza Sentinel (BIS) in Bavaria, Germany. From September to March, oropharyngeal swabs collected at BIS were analysed for influenza viruses and SARS-CoV-2 by real-time polymerase chain reaction. In BIS 2019/2020, 1376 swabs were tested for influenza viruses. The average positive rate was 37.6%, with a maximum of over 60% (in January). The predominant influenza viruses were Influenza A(H1N1)pdm09 (n = 202), Influenza A(H3N2) (n = 144) and Influenza B Victoria lineage (n = 129). In all, 610 of these BIS swabs contained sufficient material to retrospectively test for SARS-CoV-2. SARS-CoV-2 RNA was not detectable in any of these swabs. In BIS 2020/2021, 470 swabs were tested for influenza viruses and 457 for SARS-CoV-2. Only three swabs (0.6%) were positive for Influenza, while SARS-CoV-2 was found in 30 swabs (6.6%). We showed that no circulation of SARS-CoV-2 was detectable in BIS during the 2019/2020 influenza season, while virtually no influenza viruses were found in BIS 2020/2021 during the COVID-19 pandemic.

We assessed severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostic sensitivity and cycle threshold (Ct) values relative to symptom onset in symptomatic coronavirus disease-2019 (COVID-19) patients from Bavaria, Germany, of whom a subset was repeatedly tested. Locally weighted scatterplot smoothing method was used to assess the relationship between symptom onset and Ct-values. Kaplan−Meier plots were used to visualise the empirical probability of detecting viral ribonucleic acid (RNA) over time and estimate the time until clearance of viral RNA among the repeatedly tested patients. Among 721 reported COVID-19 cases, the viral RNA was detected in specimens taken between three days before and up to 48 days after symptom onset. The mean Ct-value was 28.6 (95% confidence interval (CI) 28.2–29.0) with the lowest mean Ct-value (26.2) observed two days after symptom onset. Up to 7 days after symptom onset, the diagnostic sensitivity of the RT-PCR among repeatedly sampled patients (n = 208) remained above 90% and decreased to 50% at day 12 (95% CI 10.5–21.5). Our data provide valuable estimates to optimise the timing of sampling of individuals for SARS-CoV-2 detection. A considerable proportion of specimens sampled before symptom onset had Ct-values comparable with Ct-values after symptom onset, suggesting the probability of presymptomatic transmission.

Circulation of multiple dengue virus (DENV) serotypes in a locale has resulted in individuals becoming infected with mixed serotypes. This research was undertaken to study the clinical presentation, presence of DENV serotypes and serological characteristics of DENV infected patients with co-infections from three Provinces of Sri Lanka where DENV-1 and -2 predominated during the study. A reverse transcription polymerase chain reaction was performed on 1249 patient samples and 301 were positive for DENV (24.1%). DENV-1 was the predominant serotype detected in 137 (45.51%) followed by DENV-2 in 65 (21.59%), DENV-3 in 59 (19.6%) and DENV-4 in 4 (1.32%) patients with mono-infections. Thirty-three patients (10.96%) had DENV co-infections with two or more serotypes. The highest number of co-infections was noted between DENV-1 and DENV-2 (57.57%) suggesting co-infection is driven by the frequency of the circulating serotypes. Platelet counts were significantly higher in DENV co-infected patients although clinical disease severity or white blood cell count, packed cell volume or viraemia were not significantly different in the co-infected compared to the mono-infected patients. Thus co-infection with multiple DENV serotypes does occur but with the exception of improved platelet counts in co-infected patients, there is no evidence that clinical or laboratory measures of disease are altered.

Due to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

Despite a long history of dog-transmitted human rabies outbreaks in Liberia, West Africa, no reports exist of molecular characterisation of the causative lyssaviruses. This study investigated Rabies lyssavirus (RABV) strains isolated at the dog–human interface in Monrovia, Liberia 2016 and 2017, by reverse transcription polymerase chain reaction, using primers specific for the nucleoprotein (N) gene. Out of 20 specimens (19 dog brain samples and one human saliva) tested as suspected rabies cases, three (15%) were positive. Purified amplicons from all three positive specimens were sequenced in both forward and reverse directions. Phylogenetic analysis was conducted in MEGA7 and PhyML3 to determine their relationship with RABV sequences accessioned in NCBI GenBank. The first of three RABV strains detected clustered with China lineage 2 RABVs of dogs (99% homology to KU963489 and DQ666322). The second strain segregated with Africa lineage 2 RABVs also of dog origin, and the third strain segregated with Africa lineage 3 RABVs of Southern Africa viverrids. Our results show a transcontinental strain of rabies virus co-circulating with Africa lineages in post-conflict Liberia. This finding should stimulate more effective sub-regional planning and execution of one-health actions, towards stepwise surveillance and elimination of rabies in West Africa by 2030.

Significant experimental evidence supports fat as a taste modality; however, the associated peripheral mechanisms are not well established. Several candidate taste receptors have been identified, but their expression pattern and potential functions in human fungiform papillae remain unknown. The aim of this study is to identify the fat taste candidate receptors and ion channels that were expressed in human fungiform taste buds and their association with oral sensory of fatty acids. For the expression analysis, quantitative RT-PCR (qRT-PCR) from RNA extracted from human fungiform papillae samples was used to determine the expression of candidate fatty acid receptors and ion channels. Western blotting analysis was used to confirm the presence of the proteins in fungiform papillae. Immunohistochemistry analysis was used to localise the expressed receptors or ion channels in the taste buds of fungiform papillae. The correlation study was analysed between the expression level of the expressed fat taste receptors or ion channels indicated by qRT-PCR and fat taste threshold, liking of fatty food and fat intake. As a result, qRT-PCR and western blotting indicated that mRNA and protein of CD36, FFAR4, FFAR2, GPR84 and delayed rectifying K+ channels are expressed in human fungiform taste buds. The expression level of CD36 was associated with the liking difference score (R −0·567, β=−0·04, P=0·04) between high-fat and low-fat food and FFAR2 was associated with total fat intake (ρ=−0·535, β=−0·01, P=0·003) and saturated fat intake (ρ=−0·641, β=−0·02, P=0·008).

We have studied the presence and distribution of α-amino-3–hydroxy-5–methyl-4–isoxazolepropionate (AMPA)-selective glutamate receptor subunits (GluR 1, 2, 3, and 4) in the adult cat visual cortical areas 17, 18, 19, and the lateral suprasylvian areas (LSA). Reverse transcription-polymerase chain reaction (RT-PCR) amplification indicated that the genes encoding GluR 1, 2, 3, and 4 are expressed in these areas and Western blot analysis revealed that the size of the corresponding peptides is similar to those described in the rat brain. In situ hybridization (ISH) using digoxigenin-labeled riboprobes showed that mRNAs coding for GluR1 and GluR3 were located in cells in all layers of the areas examined and also in the underlying white matter. GluR1 mRNA was relatively abundant throughout layers II–VI while GluR3 mRNA revealed a more laminated pattern of expression, preferentially labeling cells in layers II, III, V, and VI. The distribution of AMPA-selective receptor subunit peptides was studied by immunohistochemistry using subunit specific antibodies and found to be consistent with ISH results. In addition, we observed that most of the cells strongly labeled by the anti-GluR1 antibody were non-pyramidal neurons and that intense GluR2/3 immunoreactivity was seen preferentially in pyramidal neurons. Interestingly, double-labeling experiments indicated that neurons expressing γ-aminobutyric acid (GABA) as well as the GluR1 subunit were particularly abundant in deeper layers. The GluR4 peptide was predominantly found in a relatively low number of layer III and layer V neurons with either pyramidal or non-pyramidal morphology. Finally, the distribution of neurons expressing the various receptor subunits was similar in all the visual cortical areas studied. These findings indicate a high expression of GluR1–3 subunits in the cat visual cortex and that GluR1 and GluR2/3 subunits are particularly abundant in non-pyramidal and pyramidal neurons, respectively. In addition, the results described here provide a reference for future studies dealing with the effect of visual deprivation on the expression of this receptor type.

The expression of the YUCAA1 gene and the amount of endogenous indole acetic acid (IAA) in rice (Oryza sativa subsp. japonica) plants and rice suspension cells infected by Rice stripe virus (RSV) were investigated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and high-performance liquid chromatography, respectively. The results showed that the expression of the YUCAA1 gene and the amount of endogenous IAA increased at various times (16, 32, 48 and 64 h) after infection of rice suspension cells by RSV. In rice plants infected with RSV, the expression of the YUCAA1 gene and the amount of endogenous IAA increased, in comparison with healthy rice plants, at 4–8 days after infection, and decreased at 12 and 16 days. These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally, the expression of the RSV gene CP increased 2.9 times in rice plants after they were treated with a KPSC buffer to deplete the endogenous auxins, and decreased 45% after treatment with 30 μmol/l IAA. All of these results suggest that auxin may play a role in RSV replication in rice plants.

Global activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

The c-fos and c-jun proto-oncogenes are involved in the regulation of gene expression, cell proliferation, differentiation and tumorigenesis. Embryogenesis, like tumorigenesis, involves dramatic cell growth, cleavage and differentiation processes. Activation of the c-fos and c-jun proto-oncogenes in sheep conceptuses during the period of rapid growth and elongation was examined using reverse transcription and polymerase chain reaction (RT-PCR). The specificity of PCR products was determined by Southern blot hybridisation analysis with a non-radioactive DNA probe. A band corresponding to a 507 bp fragment (predicted amplified c-fos gene cDNA product) was observed in 3 of 9 day-13, 1 of 4 day-14 and 1 of 2 day-16 embryos. Meanwhile, a 400 bp c-jun transcript was aslo detected in 1 or 2 day-12, 3 of 9 day-13 and 2 of 2 day-16 embroyos. These results suggest that mRNA transcripts of c-fos and c-jun proto-oncogenes were specifically expressed during the period of ovine embryonic elongation and may have a possible role in preimplantation embryonic development of sheep.

The treatment of Neospora caninum infection in the bovine host is still at an experimental stage. In contrast to the in vivo situation, a wide range of compounds have been intensively investigated in cell-culture-based assays. Tools to demonstrate efficacy of treatment have remained conventional including morphological and cell biological criteria. In this work, we present a molecular assay that allows the distinction between live and dead parasites. Live parasites can be detected by measuring the mRNA level of specific genes, making use of the specific mRNA available in live cells. The NcGra2 gene of N. caninum, which is known to be expressed in both tachyzoites and bradyzoites, was used to establish a quantitative real-time RT-PCR, for monitoring parasite viability. Validation of the system in vitro was achieved using Neospora-infected cells that had been treated for 2–20 days with 30 μg/ml toltrazuril. NcGRA2-RT-real time PCR demonstrated that a 10-day toltrazuril-treatment exerted parasitostatic activity, as assessed by the presence of NcGRA2-transcripts, whereas after a 14-day treatment period no NcGRA2-transcripts were detected, showing that the parasites were no longer viable. Concurrently, extended culture for a period of 4 weeks in the absence of the drug following the 14-day toltrazuril treatment did not lead to further parasite proliferation, confirming the parasiticidal effect of the treatment. This assay has the potential to be widely used in the development of novel drugs against N. caninum, with a view to distinguishing between parasiticidal and parasitostatic efficacy of given compounds.

Risk-assessment studies of virus-resistant transgenic plants (VRTPs) focussing on recombination of a plant virus with a transgenic sequence of a different virus should include a comparison of recombination frequencies between viruses in double-infected non-transgenic plants with those observed in singly infected transgenic plants to estimate recombination incidence in VRTPs. In this study, the occurrence of recombination events was investigated in non-transgenic plants double-infected with two different potyviruses, as well as in potyviral genomes in singly infected transgenic plants expressing potyvirus sequences. Different potyviruses, namely Potato virus A (PVA), Tobacco vein mottling virus (TVMV), two strains of Potato virus Y (PVY-O, PVY-H) and two strains of Plum pox virus (PPV-NAT, PPV-SK68), were used in three combinations for double infection of a common host. Furthermore, transgenic plants expressing either potyviral coat protein (CP), helicase (CI) or polymerase (NIb) coding sequences (PPV-NAT-CP, PVY-CI, PVY-NIb) were singly-infected with a heterologous potyvirus, which was not targeted by the respective transgenic resistance. To identify recombinant potyviral sequences, a sensitive RT-PCR was developed to detect up to one recombinant molecule out of 106 parental molecules. In 304 mixed infected non-transgenic plants, 92 mixed and 164 single infected transgenic plants screened for recombinant sequences no recombinant potyviral sequence was found. These results indicate that recombination events between different potyviruses in mixed infections and between a potyvirus infecting a potyvirus-resistant transgenic plant are likely to be very infrequent.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. Two serotypes of this double-stranded RNA virus exist but only serotype 1 viruses cause disease in chickens. Detection and strain identification of IBDV is important because antigenic subtypes found within serotype 1 make it necessary to tailor vaccination programs to the antigenic type found in the bird's environment. Because conventional virus isolation and characterization are not practical for routine diagnosis of IBDV, antigen-capture enzyme-linked immunosorbent assay (ELISA) and molecular assays based on reverse transcription–polymerase chain reaction (RT-PCR) technology were developed. Compared with antigen-capture ELISA, RT-PCR assays have greater versatility and are more sensitive and specific. Strain identification has been accomplished using a variety of post-RT-PCR assays, including restriction enzyme digestion of the RT-PCR products. The resulting restriction fragment length polymorphisms (RFLP) are used to differentiate viruses into molecular groups that correlate with antigenic and pathogenic types. Recently, two types of real-time RT-PCR have been used to identify and differentiate strains of IBDV. Both methods use distance-dependent interaction between two dye molecules, known as fluorescence resonance energy transfer (FRET). The dye molecules are attached to one or more nucleotide probes that detect specific nucleotide sequences of the virus. Our laboratory has used a two-probe assay to identify single-nucleotide mutations among IBDV strains. A mutation probe is used in this assay to detect substitution mutations in a region of the viral genome that encodes a neutralizing epitope of the virus. These assays are accurate, reliable and inexpensive compared with conventional RT-PCR because they do not require RFLP or other labor-intensive post-RT-PCR assays to distinguish viral strains.

Penguin blood samples collected at Bird Island, sub-Antarctic South Georgia, and faecal samples taken from penguins at several localities along the Antarctic Peninsula were analysed in order to investigate if influenza A virus is present in penguin populations in the South Atlantic Antarctic region. Serology was performed on the blood samples while the faecal samples were screened by a RT-PCR method directed at the matrix protein gene for determining the presence of influenza A virus. All faecal samples were negative by PCR, but the blood samples gave serologic indications that influenza A virus is present amongst these penguin species, confirming previous studies, although the virus has still not been isolated from any bird in the Antarctic region.

Sox9 is one of the important genes related to sex differentiation and determination. Two partial cDNAs encoding sox9 were derived from brain, testis and ovary of yellow catfish Pelteobagrus fulvidraco using reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis revealed that the ovarian sox9a2 was different from sox9a1 derived from brain and testis. The identity rate of cDNA was 77%, that of amino acids was 84%, and the different nucleotides were distributed in different sites all over the cDNA. This finding confirmed that there were transcripts from two genes. Based on the homology of the amino-acid sequences for sox9 of other animals, the similarity for sox9a1 from yellow catfish and sox9 of other animals averaged 86%. The similarity for sox9a2 reached a mean of 83%. The phylogenetic analysis revealed yellow catfish sox9a1 and sox9a2 were clustered into the fish branch. And yellow catfish sox9a2 was different from the other fish sox9. There was no significant branch including all sox9 derived from testis and brain or including all ovarian sox9. The genetic distance was the same with the results of the traditional taxonomy. RT-PCR analysis indicated that sox9a1 was expressed in male and female yellow catfish forebrain as well as in gonads, whereas sox9a2 was only expressed in ovaries. The function of sox9a2 in female sex formation and ovary development needs to be studied in depth.

Recombination in RNA viruses is considered to play a major role as a driving force in virus variability to counterbalance loss in fitness that can be due to the accumulation of detrimental mutations. Studies on mixed infections are pertinent for understanding the role of recombination in virus evolution. They also provide important baseline information for studying the biosafety of plants expressing viral sequences. To investigate the possibility of RNA recombination occurrence between two sobemoviruses under little or no selection pressure, we co-infected test plants with Cocksfoot mottle virus (CfMV) and Ryegrass mottle virus (RGMoV). CfMV and RGMoV were selected because of their overlapping host range and geographical distribution. First, symptom development of both viruses in barley (Hordeum vulgare) and oat (Avena sativa) was examined. Both viruses generated quite strong infection symptoms in oat, but synergism was not detected. RGMoV was lethal for barley, whereas CfMV infection in barley was nearly symptomless. RT-PCR analysis revealed 100% infection with both viruses in oat but not in barley. Therefore, an RNA recombination study of CfMV and RGMoV was performed in oat. 105 plants were co-inoculated with both viruses and putative recombinational hot spot regions were screened for recombination events by RT-PCR analysis at a sensitivity level down to 0.1–100 pg of viral genomic RNA. No recombination events between the two sobemoviruses were detected.