Search

This progress report describes the first transmission study in a series of four studies. This long-term study aims to determine whether vaccination under field circumstances can protect layer flocks long term against HPAI H5N1 virus (clade 2.3.4.4b) and reducing within flock transmission (R<1). This transmission study was performed at 8-weeks post vaccination with layer hens which were housed under field circumstances. In addition to the main output, which is assessment of the level of transmission, other variables such as chicken survival, virus shedding, humoral and cellular immune responses were studied to assess the effectiveness of vaccination with VAXXITEK HVT+IBD+H5 vaccine in poultry against HPAI H5N1 virus infection. The estimated R for the control group was 1.3 (95% CI 0.58–2.87) and transmission in the VAXXITEK HVT+IBD+H5 could not be quantified as none of the inoculated chickens became infected. For the analysis, a chicken is considered infected when the following parameters applied: • Virus shedding: when virus was detected for 2 days or longer (≥2 days) with a minimum equivalent titer of ≥Log 101.7 eqEID50/ml by PCR in swabs collected from either choana or cloaca. and • If the chicken survived the challenge, additional parameters were: o a positive NP-ELISA result (after 21 days) and/or o showed an increase of ≥3 log2 in the heterologous HI-titer. This definition is consistent with the definition used in our previous study [1]. In the control groups 9/10 inoculated chickens and 7/10 contact chickens met these criteria and were considered infected. The non-infected inoculated chicken in control group A, was positive for virus shedding, but had no positive serological response. After vaccination with the VAXXITEK HVT+IBD+H5 vaccine, none of the chickens became infected (although 2/10 inoculated chickens were positive for virus shedding). No positive serological results were obtained. In addition, prior to inoculation, the vaccinated chickens had a signif

This progress report describes the first transmission study in a series of four studies. This long-term study aims to determine whether vaccination under field circumstances can protect layer flocks long term against HPAI H5N1 virus (clade 2.3.4.4b) and reducing within flock transmission (R<1). This transmission study was performed at 8-weeks post vaccination with layer hens which were housed under field circumstances. In addition to the main output, which is assessment of the level of transmission, other variables such as chicken survival, virus shedding, humoral and cellular immune responses were studied to assess the effectiveness of vaccination with VECTORMUNE® AI vaccine in poultry against HPAI H5N1 virus infection. The estimated R for the control group was 1.3 (95% CI 0.58–2.87) and transmission in the VECTORMUNE® AI could not be quantified as none of the inoculated chickens became infected. For the analysis, a chicken is considered infected when the following parameters applied: • Virus shedding: when virus was detected for 2 days or longer (≥2 days) with a minimum equivalent titer of ≥Log 101.7 eqEID50/ml by PCR in swabs collected from either choana or cloaca. and • If the chicken survived the challenge, additional parameters were: o a positive NP-ELISA result (after 21 days) and/or o showed an increase of ≥3 log2 in the heterologous HI-titer. This definition is consistent with the definition used in our previous study [1]. In the control groups 9/10 inoculated chickens and 7/10 contact chickens met these criteria and were considered infected. The non-infected inoculated chicken in control group A, was positive for virus shedding, but had no positive serological response. After vaccination with the VECTORMUNE® AI vaccine, none of the chickens became infected (although two inoculated chickens were positive for virus shedding). No positive serological results were obtained, (except an increased HI-titer of log2 ≥3 for 2 chickens). In addition, prior to inocula

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021–2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal.Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021–2022 outbreak in the Netherlands included red fox (Vulpes vulpes),polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals weresubmitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal.Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.

In het afgelopen decennium zijn er in Nederland een groot aantal uitbraken geweest van vogelgriep (aviaire influenza, AI) op pluimveebedrijven. Bij een verdenking van vogelgriep in pluimvee worden monsters door de NVWA op locatie genomen en vervolgens door het nationaal referentielaboratorium in Lelystad getest met PCR. Als er veel gelijktijdige verdenkingen zijn, kan een eenvoudige sneltest op locatie helpen om prioriteiten te stellen en zo beschikbare middelen efficiënt in te zetten. Er zijn commerciële sneltesten verkrijgbaar voor de detectie van het influenzavirus in monsters afgenomen bij mensen en pluimvee. Een aantal van deze sneltesten is in de voorgaande studie ‘Inventarisatie van sneltesten voor vogelgriep’ onderzocht op gevoeligheid en gebruiksgemak. In de huidige studie zijn een aantal prestatiekenmerken van drie sneltesten (Clungene, Anigen en Biopanda) uitgebreider onderzocht. De resultaten laten het volgende zien. Voor luchtpijp- en cloaca-monsters van kippen geïnfecteerd met AI (n=16) komt de Clungene sneltest voor 93% overeen met de PCR-resultaten. Voor met vogelgriep geïnfecteerde vleeseenden (n=8) is dat 100% voor luchtpijp-monsters, maar slechts 50% voor cloaca-monsters. Voor de sneltesten van Anigen en Biopanda, die beiden getest zijn met een kleiner aantal geïnfecteerde kippen (n=8) en vleeseenden (n=4), variëren de overeenkomsten met PCR van 71% tot 100% voor kippen, en resp. 100% en 0% voor luchtpijp- en cloaca-swabs van vleeseenden. Aanvullend is voor de Clungene sneltest berekend dat 90% van de PCR-positieve veldmonsters van de afgelopen twee jaar correct als positief zouden zijn geïdentificeerd. Voor keel- en cloaca-swabs van vogelgriep-negatief pluimvee (20 kippen en 20 vleeseenden) is de Clungene sneltest in 94% van de gevallen correct negatief. De Biopanda en Anigen sneltesten, beiden getest met 4 kippen en 4 vleeseenden, zijn correct negatief met 100% van de geteste monsters. Onder laboratoriumcondities tonen alle drie de snelte

Kennis - In de Staat van Zoönosen worden elk jaar bijzondere gevallen van zoönosen uitgelicht. Zo waren er in 2022 meer meldingen van papegaaienziekte (psittacose) dan normaal in de regio rondom Den Haag. De bron hiervoor zijn niet gevonden. Verder blijkt de antibioticaresistente bacterie MRSA het meest voor te komen bij bedrijven met varkens (75 procent). Dit is meer dan bij bedrijven die vleeskuikens, melkvee of vleeskalveren houden. Net als vorig jaar was de opvallendste uitbraak van 2022 vogelgriep (variant H5N1). Het virus was het hele jaar door aanwezig bij pluimvee en wilde vogels in Nederland, maar er zijn geen mensen besmet. In totaal zijn 76 pluimveebedrijven besmet geraakt en geruimd. Verder is het virus veel gevonden bij wilde vogels, die er vaak aan stierven. Ook zijn er wilde zoogdieren besmet, zoals vossen en bunzingen. Ten slotte behandelt de Staat van Zoönosen elk jaar een thema. Dit jaar is dat ‘Nieuwe voedselbronnen en voedselveiligheid’. Co-publicatie van het RIVM, de NVWA, de GGD, WBVR, WFSR, en anderen.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research

The current pandemic reminds us of the shadow of flu caused by avian influenza viruses, which keep circulating and cause outbreaks all over the world. There are two problems with avian influenza viruses (AIVs) that cause flu in poultry: pathogenicity in poultry and possible effects on human health. The high pathogenic avian influenza viruses (HPAIVs) threaten public health directly by high human mortality, and the low pathogenic avian influenza viruses (LPAIVs) play an important role in influenza virus evolution. Due to the mild disease and low death rate, LPAIVs remain circulating in poultry populations, thereby providing a platform and time for the emergence of strains with high transmission, cross host-transmission properties or higher human pathogenicity. More and more human infections, caused by avian influenza virus directly from birds further increase our concerns. The H9N2 subtype LPAIV has attracted attention for its wide range spread in many bird species, as well as for the fact that it has become endemic in commercial poultry in many areas, affecting poultry productivity. As a prevention method, vaccination against the H9N2 subtype virus in poultry was promoted in several countries. However, the persistence of this virus in poultry after vaccination has been reported and it was suggested that the inactivated-virus vaccine might not stop the transmission, but rather trigger the antigenic drift of the virus in poultry. In this thesis, experiments were performed to analyse the effect of the inactivated-virus vaccine on stopping the transmission of H9N2 subtype virus in poultry. In Chapter 2, I showed that the estimated reproduction ratio of H9N2 subtype virus in non-vaccinated and vaccinated chicken was above 1, suggesting that the inactivated-virus vaccine against the H9N2 subtype virus cannot stop the transmission of the homologous virus strain in vaccinated chicken. In Chapter 3, phylogenetic and antigenic analyses of H9N2 subtype virus strains isolated f

Highly pathogenic avian influenza (HPAI) viruses of H5 clade 2.3.4.4 have spread to many countries in Asia, Europe and North America by migratory wild birds, causing outbreaks on hundreds of poultry farms. Strategies to control spread by wild birds appear limited, hence timely characterization of novel viruses is important to limit the risk for the poultry sector and human health. In this study we characterize three recent viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the H5N8 group A and B viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent virus, the H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with the group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds, and between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, showing that chickens are efficiently infected by (fecal)contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

In 2016/2017, H5N8 highly pathogenic avian infuenza (HPAI) virus of the Goose/Guangdong lineage spread from Asia to Europe, causing the biggest and most widespread HPAI epidemic on record in wild and domestic birds in Europe. We hypothesized that the wide dissemination of the 2016 H5N8 virus resulted at least partly from a change in tissue tropism from the respiratory tract, as in older HPAIV viruses, to the intestinal tract, as in low pathogenic avian infuenza (LPAI) viruses, allowing more efcient faecal-oral transmission. Therefore, we determined the tissue tropism and associated lesions in wild birds found dead during the 2016 H5N8 epidemic, as well as the pattern of attachment of 2016 H5N8 virus to respiratory and intestinal tissues of four key wild duck species. We found that, out of 39 H5N8-infected wild birds of 12 species, four species expressed virus antigen in both respiratory and intestinal epithelium, one species only in resp

Background: Laying hens with access to outdoor ranges are exposed to additional environmental factors and microorganisms, including potential pathogens. Differences in composition of the cloacal microbial community between indoor- and outdoor-housed layers may serve as an indicator for exposure to the outdoor environment, including its pathogens, and may yield insights into factors affecting the chickens’ microbiota community dynamics. However, little is known about the influence of outdoor housing on microbiota community composition in commercial layer flocks. We performed a cross-sectional field study to evaluate differences in the cloacal microbiota of indoor- vs outdoor-layers across farms. Eight layer flocks (four indoor, four outdoor) from five commercial poultry farms were sampled. Indoor and outdoor flocks with the same rearing flock of origin, age, and breed were selected. In each flock, cloacal swabs were taken from ten layers, and microbiota were analysed with 16S rRNA gene amplicon sequencing.Results: Housing type (indoor vs outdoor), rearing farm, farm and poultry house within the farm all significantly contributed to bacterial community composition. Poultry house explained most of the variation (20.9%), while housing type only explained 0.2% of the variation in community composition. Bacterial diversity was higher in indoor-layers than in outdoor-layers, and indoor-layers also had more variation in their bacterial community composition. No phyla or genera were found to be differentially abundant between indoor and outdoor poultry houses. One amplicon sequence variant was exclusively present in outdoor-layers across all outdoor poultry houses, and was identified as Dietzia maris.Conclusions: This study shows that exposure to an outdoor environment is responsible for a relatively small proportion of the community variation in the microbiota of layers. The poultry house, farm, and rearing flock play a much greater role in determining the cloacal microbiot

Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird-associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens.

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between-farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10-year surveillance period, we identified 35 potential cases of between-farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between-farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between-farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p < 0.05). The results further suggest that between-farm transmission was not restricted to holdings of the same poultry type and not related to the housing system. Although separate introductions from the wild bird reservoir cannot be excluded, our study indicates that between-farm transmission occurred in seven of nine virologically analysed cases. Based on these findings, it is likely that between-farm transmission contributes considerably to the incidence of LPAI virus infections in poultry.

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017–2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016–2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm-to-farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long-term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population.

Interspecies transmission of fecal microbiota can serve as an indicator for (indirect) contact between domestic and wild animals to assess risks of pathogen transmission, e.g., avian influenza. Here, we investigated whether oral inoculation of laying hens with feces of wild ducks (mallards, Anas platyrhynchos) resulted in a hen fecal microbiome that was detectably altered on community parameters or relative abundances of individual genera. To distinguish between effects of the duck inoculum and effects of the inoculation procedure, we compared the fecal microbiomes of adult laying hens resulting from 3 treatments: inoculation with wild duck feces (duck), inoculation with chicken feces (auto), and a negative control group with no treatment. We collected cloacal swabs from 7 hens per treatment before (day 0), and 2 and 7 D after inoculation, and performed 16S rRNA amplicon sequencing. No distinguishable effect of inoculation with duck feces on microbiome community (alpha and beta diversity) was found compared to auto or control treatments. At the individual taxonomic level, the relative abundance of the genus Alistipes (phylum Bacteroidetes) was significantly higher in the inoculated treatments (auto and duck) compared to the control 2 D after inoculation. Seven days after inoculation, the relative abundance of Alistipes had increased in the control and no effect was found anymore across treatments. These effects might be explained by the perturbation of the hen's microbiome caused by the inoculation procedure itself, or by intrinsic temporal variation in the hen's microbiome. This experiment shows that a single inoculation of fecal microbiota from duck feces to laying hens did not cause a measurable alteration of the gut microbiome community. Furthermore, the temporary change in relative abundance for Alistipes could not be attributed to the duck feces inoculation. These outcomes suggest that the fecal microbiome of adult laying hens may not be a useful indicator for

During autumn–winter 2016–2017, highly pathogenic avian influenza A(H5N8) viruses caused mass die-offs among wild birds in the Netherlands. Among the ≈13,600 birds reported dead, most were tufted ducks (Aythya fuligula) and Eurasian wigeons (Anas penelope). Recurrence of avian influenza outbreaks might alter wild bird population dynamics.

In October 2016, a severe infection with swine influenza A(H1N1) virus of the Eurasian avian lineage occurred in a child with a previous history of eczema in the Netherlands, following contact to pigs. The patient’s condition deteriorated rapidly and required life support through extracorporeal membrane oxygenation. After start of oseltamivir treatment and removal of mucus plugs, the patient fully recovered. Monitoring of more than 80 close unprotected contacts revealed no secondary cases.

Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H doma

In 2014 zijn vier teelten van chrysant op water gerealiseerd bij Wageningen UR Glastuinbouw. Het onderzoek was gericht op ziekteweerbaar. Gedurende de teelten zijn de volgende lessen geleerd : algemene aspecten van teelt op water, hygiëne en microleven.

Aandacht wordt geschonken aan: voedingswater, micro-leven, rasverschillen, en oud stekmateriaal. Poster van PlantgezondheidEvent 12 maart 2015.

Doelstellingen van dit onderzoek: Effect toetsen van biologische en oxidatieve producten met een contactwerking op jonge gewasscheuten. Effect toetsen van diverse producten die de natuurlijke afweerreacties kunnen versterken tegen Fusarium op jonge planten in een kasproef. Poster van PlantgezondheidEvent 12 maart 2015.

Aim This study examined the effect of temperament (extraversion and negative affect) on sibling relationship quality (warmth and conflict) of 4- to 6-year old children. Method In this cross-sectional study 849 mothers participated by filling out an online questionnaire. They had at least two children, with one child between the ages of 4 and 6. Quality of the sibling relationship was measured by the Parent Report of Early SIBling relationship quality. For temperament the Children’s Behavior Questionnaire - Very Short Form was used. Results Results showed a significant positive effect between temperament (for extraversion and negative affect) and conflict, while no significant effect was found on sibling warmth. Conclusion Higher degrees of extraversion and negative affect were related to more conflict within the sibling relationship. No connection was found between extraversion and negative affect and warmth, presumably because people have a tendency to report negative aspects more easily. This study advances the knowledge of sibling relationships within preschoolers. Early intervention within the sibling relationship can prevent the development of potential problems over time. Implications and future directions are discussed.

HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription. Copyright