Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

Effects of coronavirus disease 2019 on the digestive system and its nutritional implications.

PURPOSE OF REVIEW: There has been a deluge of scientific data since coronavirus disease 2019 (COVID-19) was first reported. The effects of COVID-19 on the digestive system are now increasingly well understood. This article aims to review the current data on the effects of COVID-19 on the digestive system with particular emphasis on preexisting digestive diseases and its implications on nutrition practices. RECENT FINDINGS: Evidence has shown that Severe acute respiratory syndrome coronavirus 2 virus affects the gastrointestinal (GI) tract, pancreas and hepatobiliary system resulting in different GI manifestations. Several preexisting digestive diseases have been investigated. These studies have revealed that these special patient population groups are generally not at an increased risk to contract COVID-19, but are susceptible to develop increasing severity of disease. Aside from medical therapy, optimizing nutritional care has a beneficial role in this group of patients. SUMMARY: GI manifestations of COVID-19 in addition to preexisting digestive diseases have an impact on patient's nutrition. Digestion, absorption and transport of nutrients may be impaired. To date, there are no existing guidelines on the nutritional management of patients for this particular at-risk group. Most nutrition practices are based only on observations and clinical experience. Basic prepandemic nutrition care principles are primarily followed but often individualized based on clinical judgment.

Nuances of Whitefly Vector-Crinivirus Interactions Revealed in the Foregut Retention and Transmission of Lettuce Chlorosis Virus by Two Bemisia tabaci Cryptic Species.

Lettuce infectious yellows virus is the first crinivirus for which the retention of purified virions ingested into the whitefly (Bemisia tabaci New World (NW)) vector's foregut, has been demonstrated to be a requisite for successful virus transmission. This key finding supports the hypothesis that the determinant of foregut retention and transmission is present on the virion itself. However, whether this is also true for other criniviruses has not been established. Here, we provide evidence that lettuce chlorosis virus (LCV) acquired from plants is retained in the foreguts of both the B. tabaci NW and Middle East-Asia Minor 1 (MEAM1) vector species and transmitted upon inoculation feeding. An association between foregut retention and transmission by NW vectors is also observed following the acquisition and inoculation feeding of LCV virions purified using a standard procedure involving 2% or 4% (v/v) Triton™ X-100 (TX-100). However, while virions purified with 2% or 4% TX-100 are also retained in the foreguts of MEAM1 vectors, transmission is observed with the 4% TX-100-purified virions or when more vectors are used for acquisition and inoculation feeding. These results suggest that an intrinsic difference exists between NW and MEAM1 vectors in their interactions with, and transmission of, LCV virions.

Physical and Chemical Barriers in the Larval Midgut Confer Developmental Resistance to Virus Infection in Drosophila.

Insects can become lethally infected by the oral intake of a number of insect-specific viruses. Virus infection commonly occurs in larvae, given their active feeding behaviour; however, older larvae often become resistant to oral viral infections. To investigate mechanisms that contribute to resistance throughout the larval development, we orally challenged Drosophila larvae at different stages of their development with Drosophila C virus (DCV, Dicistroviridae). Here, we showed that DCV-induced mortality is highest when infection initiates early in larval development and decreases the later in development the infection occurs. We then evaluated the peritrophic matrix as an antiviral barrier within the gut using a Crystallin-deficient fly line (Crys-/-), whose PM is weakened and becomes more permeable to DCV-sized particles as the larva ages. This phenotype correlated with increasing mortality the later in development oral challenge occurred. Lastly, we tested in vitro the infectivity of DCV after incubation at pH conditions that may occur in the midgut. DCV virions were stable in a pH range between 3.0 and 10.5, but their infectivity decreased at least 100-fold below (1.0) and above (12.0) this range. We did not observe such acidic conditions in recently hatched larvae. We hypothesise that, in Drosophila larvae, the PM is essential for containing ingested virions separated from the gut epithelium, while highly acidic conditions inactivate the majority of the virions as they transit.

The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori).

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.

The Fatty Acid Lipid Metabolism Nexus in COVID-19.

Enteric symptomology seen in early-stage severe acute respiratory syndrome (SARS)-2003 and COVID-19 is evidence of virus replication occurring in the intestine, liver and pancreas. Aberrant lipid metabolism in morbidly obese individuals adversely affects the COVID-19 immune response and increases disease severity. Such observations are in line with the importance of lipid metabolism in COVID-19, and point to the gut as a site for intervention as well as a therapeutic target in treating the disease. Formation of complex lipid membranes and palmitoylation of coronavirus proteins are essential during viral replication and assembly. Inhibition of fatty acid synthase (FASN) and restoration of lipid catabolism by activation of AMP-activated protein kinase (AMPK) impede replication of coronaviruses closely related to SARS-coronavirus-2 (CoV-2). In vitro findings and clinical data reveal that the FASN inhibitor, orlistat, and the AMPK activator, metformin, may inhibit coronavirus replication and reduce systemic inflammation to restore immune homeostasis. Such observations, along with the known mechanisms of action for these types of drugs, suggest that targeting fatty acid lipid metabolism could directly inhibit virus replication while positively impacting the patient's response to COVID-19.

A vector whitefly endocytic receptor facilitates the entry of begomoviruses into its midgut cells via binding to virion capsid proteins.

Many circulative plant viruses transmitted by insect vectors are devastating to agriculture worldwide. The midgut wall of vector insects represents a major barrier and at the same time the key gate a circulative plant virus must cross for productive transmission. However, how these viruses enter insect midgut cells remains poorly understood. Here, we identified an endocytic receptor complex for begomoviruses in the midgut cells of their whitefly vector. Our results show that two whitefly proteins, BtCUBN and BtAMN, compose a receptor complex BtCubam, for which BtCUBN contributes a viral-binding region and BtAMN contributes to membrane anchorage. Begomoviruses appear to be internalized together with BtCubam via its interaction with the 12-19 CUB domains of BtCUBN via clathrin-dependent endocytosis. Functional analysis indicates that interruption of BtCUBN and BtAMN lead to reduction of virus acquisition and transmission by whitefly. In contrast, CUBN-begomovirus interaction was not observed in two non-competent whitefly-begomovirus combinations. These observations suggest a major role of the specific endocytic receptor in facilitating viral entry into vector midgut cells.

Selection of DNA Aptamers for Subcellular Localization of RBSDV P10 Protein in the Midgut of Small Brown Planthoppers by Emulsion PCR-Based SELEX.

Rice black-streaked dwarf virus (RBSDV), classified under the Reoviridae, Fijivirus genus, caused an epidemic in the eastern provinces of China and other East Asian countries and resulted in severe yield loss in rice and wheat production. RBSDV is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus Fallén) in a persistent manner. In order to provide a stable and cost-effective detection probe, in this study we selected three DNA aptamers (R3, R5 and R11) by an optimized, standardized and time saving emulsion PCR-based SELEX, for the detection of RBSDV outer-shell P10 protein for in situ localization studies in the midgut of SBPH. The specificity of these three DNA aptamers was tested through detection of the P10 protein using an enzyme-linked oligonucleotide assay (ELONA) and aptamer-based dot-blot ELISA. All three DNA aptamers can be used to detect RBSDV P10 protein by immunofluorescent labeling in the midgut of RBSDV-infected SBPH. These data show that the selected aptamers can be used for the detection of RBSDV P10 protein in vitro and in vivo. This is the first report of aptamers being selected for detection of a rice virus capsid protein.

The Ras/ERK signaling pathway couples antimicrobial peptides to mediate resistance to dengue virus in Aedes mosquitoes.

Aedes mosquitoes can transmit dengue and several other severe vector-borne viral diseases, thereby influencing millions of people worldwide. Insects primarily control and clear the viral infections via their innate immune systems. Mitogen-Activated Protein Kinases (MAPKs) and antimicrobial peptides (AMPs) are both evolutionarily conserved components of the innate immune systems. In this study, we investigated the role of MAPKs in Aedes mosquitoes following DENV infection by using genetic and pharmacological approaches. We demonstrated that knockdown of ERK, but not of JNK or p38, significantly enhances the viral replication in Aedes mosquito cells. The Ras/ERK signaling is activated in both the cells and midguts of Aedes mosquitoes following DENV infection, and thus plays a role in restricting the viral infection, as both genetic and pharmacological activation of the Ras/ERK pathway significantly decreases the viral titers. In contrast, inhibition of the Ras/ERK pathway enhances DENV infection. In addition, we identified a signaling crosstalk between the Ras/ERK pathway and DENV-induced AMPs in which defensin C participates in restricting DENV infection in Aedes mosquitoes. Our results reveal that the Ras/ERK signaling pathway couples AMPs to mediate the resistance of Aedes mosquitoes to DENV infection, which provides a new insight into understanding the crosstalk between MAPKs and AMPs in the innate immunity of mosquito vectors during the viral infection.

Involvement of the digestive system in covid-19. A review. / Afectación del aparato digestivo en la covid-19. Una revisión sobre el tema.

The SARS-CoV-2 pandemic is leading to high mortality and a global health crisis. The primary involvement is respiratory; however, the virus can also affect other organs, such as the gastrointestinal tract and liver. The most common symptoms are anorexia and diarrhea. In about half of the cases, viral RNA could be detected in the stool, which is another line of transmission and diagnosis. covid19 has a worse prognosis in patients with comorbidities, although there is not enough evidence in case of previous digestive diseases. Digestive endoscopies may give rise to aerosols, which make them techniques with a high risk of infection. Experts and scientific organizations worldwide have developed guidelines for preventive measures. The available evidence on gastrointestinal and hepatic involvement, the impact on patients with previous digestive diseases and operating guidelines for Endoscopy Units during the pandemic are reviewed.

The α-tubulin of Laodelphax striatellus mediates the passage of rice stripe virus (RSV) and enhances horizontal transmission.

Rice stripe virus (RSV, genus Tenuivirus, family Phenuiviridae) is the causal agent of rice stripe disease transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent propagative manner. The midgut and salivary glands of SBPH are the first and last barriers to the viral circulation and transmission processes, respectively; however, the precise mechanisms used by RSV to cross these organs and transmit to rice plants have not been fully elucidated. We obtained the full-length cDNA sequence of L. striatellus α-tubulin 2 (LsTUB) and found that RSV infection increased the level of LsTUB in vivo. Furthermore, LsTUB was shown to co-localize with RSV nonstructural protein 3 (NS3) in vivo and bound NS3 at positions 74-76 and 80-82 in vitro. Transient gene silencing of LsTUB expression caused a significant reduction in detectable RSV loads and viral NS3 expression levels, but had no effect on NS3 silencing suppressor activity and viral replication in insect cells. However, suppression of LsTUB attenuated viral spread in the bodies of SBPHs and decreased RSV transmission rates to rice plants. Electrical penetration graphs (EPG) showed that LsTUB knockdown by RNAi did not impact SBPH feeding; therefore, the reduction in RSV transmission rates was likely caused by a decrease in viral loads inside the planthopper. These findings suggest that LsTUB mediates the passage of RSV through midgut and salivary glands and leads to successful horizontal transmission.

In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

COVID-19 and the Digestive System.

The outbreak of novel coronavirus pneumonia in 2019 (Coronavirus disease 2019 [COVID-19]) is now threatening global public health. Although COVID-19 is principally defined by its respiratory symptoms, it is now clear that the virus can also affect the digestive system. In this review, we elaborate on the close relationship between COVID-19 and the digestive system, focusing on both the clinical findings and potential underlying mechanisms of COVID-19 gastrointestinal pathogenesis.

Sex differences in the frequency of gastrointestinal symptoms and olfactory or taste disorders in 1942 nonhospitalized patients with coronavirus disease 2019 (COVID-19).

INTRODUCTION: The coronavirus disease 2019 (COVID 19) is a communicable disease caused by a novel coronavirus. OBJECTIVES: This study aimed to assess self­reported frequency of gastrointestinal symptoms and olfactory or taste disorders in nonhospitalized patients with COVID­19 in Poland. PATIENTS AND METHODS: This cross­sectional survey was conducted between April 17 and 18, 2020, in 4516 nonhospitalized patients with COVID­19 in Poland. The questionnaire included 8 questions related to the health status, symptoms of COVID­19, comorbidities, and smoking status. RESULTS: Completed questionnaires were obtained from 1942 patients with COVID­19 with a response rate of 43%. The median age of the respondents was 50 years; 60.2% were women. Among nonhospitalized patients with COVID­19, 21.3% had hypertension, 4.5% had diabetes, and 3.1% had a chronic respiratory disease. Regular tobacco use was declared by 11.2% of patients with COVID­19. At least one gastrointestinal symptom was reported by 53.6% of patients. Almost half of patients (47%) with COVID­19 reported lack of appetite and 24.2% reported diarrhea. Among 1942 interviewed patients, 54.2% reported at least 1 olfactory or taste disorder and 42.5% reported both alterations. Self­reported olfactory and taste disorders were 49.2% and 47.5%, respectively. Self­reported frequency of gastrointestinal symptoms and olfactory or taste disorders during COVID­19 was significantly higher (P <0.001) in women than men. CONCLUSIONS: This study demonstrated that olfactory and taste disorders are frequent symptoms in patients with mild­to­moderate COVID­19. Moreover, our study indicated sex differences in the frequency of gastrointestinal symptoms and olfactory or taste disorders among nonhospitalized patients with COVID­19.

New understanding of the damage of SARS-CoV-2 infection outside the respiratory system.

Since early December 2019, a number of pneumonia cases associated with unknown coronavirus infection were identified in Wuhan, China, and many additional cases were identified in other regions of China and in other countries within 3 months. Currently, more than 80,000 cases have been diagnosed in China, including more than 3000 deaths. The epidemic is spreading to the rest of the world, posing a grave challenge to prevention and control. On February 12, 2020, the International Committee on Taxonomy of Viruses and the World Health Organization officially named the novel coronavirus and associated pneumonia as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19), respectively. According to the recent research on SARS-CoV-2, the virus mainly infects the respiratory system but may cause damage to other systems. In this paper, we will systematically review the pathogenic features, transmission routes, and infection mechanisms of SARS-CoV-2, as well as any adverse effects on the digestive system, urogenital system, central nervous system, and circulatory system, in order to provide a theoretical and clinical basis for the diagnosis, classification, treatment, and prognosis assessment of SARS-CoV-2 infection.

Addressing multiple gastroenterological aspects of coronavirus disease 2019.

The outbreak of the coronavirus disease 2019 (COVID­19) pandemic has become the biggest challenge for the whole human community since many years. It seems that the proper identification of all people infected with severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) is the best strategy to limit the transmission. However, in a significant proportion of patients, there are no clinical manifestations of the disease, and symptoms may be very mild or atypical. There is a growing body of evidence that digestive manifestations of COVID­19 are frequently reported and may precede typical respiratory symptoms. Moreover, SARS­CoV­2 particles were found in the gastrointestinal epithelial cells, and viral RNA was detected in the feces of patients with COVID­19. These data suggest that gastrointestinal symptoms in COVID­19 are not accidental findings and they may result from direct digestive involvement. Patients with new­onset diarrhea, abdominal pain, nausea, and vomiting without any other evident etiological factors should be tested for SARS­CoV­2 infection. Gastroenterologists and members of other medical specialties should also remember that the current epidemiological situation has changed diagnostic and therapeutic algorithms in the management of several gastrointestinal and liver disorders. This review article summarizes the currently available data on multiple gastroenterological aspects of COVID­19 and provides information on practical recommendations and position statements of the most prominent associations in the field of gastroenterology, which appeared in response to the emergence of the pandemic.

COVID-19 and the digestive system: protection and management during the SARS-CoV-2 pandemic.

The purpose of this rapid review is to provide an update on the impact of SARS-CoV-2 infection on Gastroenterology and Hepatology departments, our patients, and our new way of working. The gastrointestinal tract and the liver are affected by SARS-CoV-2, especially in patients with immunosuppressive therapies. Patients with liver transplantation should be followed closely. Digestive endoscopy is a high-risk procedure for the transmission of SARS-CoV-2. While the pandemic lasts, we must adapt its indications and promote protective measures for patients and healthcare professionals alike. The COVID-19 pandemic has changed our priorities and the way we work, although we do not know what the repercussions will be after normality is reinstated.

Covid-19 and the digestive system.

The novel coronavirus disease is currently causing a major pandemic. It is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the Betacoronavirus genus that also includes the SARS-CoV and Middle East respiratory syndrome coronavirus. While patients typically present with fever and a respiratory illness, some patients also report gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain. Studies have identified the SARS-CoV-2 RNA in stool specimens of infected patients, and its viral receptor angiotensin converting enzyme 2 was found to be highly expressed in gastrointestinal epithelial cells. These suggest that SARS-CoV-2 can actively infect and replicate in the gastrointestinal tract. This has important implications to the disease management, transmission, and infection control. In this article, we review the important gastrointestinal aspects of the disease.

The C-termini of the baculovirus per os infectivity factors 1 and 2 mediate ODV oral infectivity by facilitating the binding of PIF0 and PIF8 to the core of the entry complex.

Oral infection of caterpillars by baculoviruses is initiated by occlusion-derived virus particles (ODVs) that infect midgut epithelium cells. The ODV envelope therefore contains at least ten different proteins, which are called per os infectivity factors (PIFs). Nine of these PIFs form the so-called ODV entry complex that consists of a stable core formed by PIF1, 2, 3 and 4, to which the other PIFs [PIF0, 6, 7, 8 and 9 (ac108)] bind with lower affinity. PIF1 and 2 are not only essential for complex formation, but also mediate ODV-binding to the epithelial brush border, probably via the C-termini. To study the involvement of these PIFs during midgut infection in greater detail, we assessed the oral infectivity and the ability to form the complex of a series of PIF1 and PIF2 C-terminal truncation mutants of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which were constructed in this study. Limited truncation of either PIF1 or 2 already severely impaired the ODV oral infectivity, but did not affect the formation of the core complex. However, the entry complex as a whole was not assembled in these mutants as PIF0 and 8 failed to bind to the core. This suggests that the interactions between the core and the loosely associated PIFs are important for the ODV infectivity and that complex formation complicates the determination of the exact roles of PIF1 and 2 during midgut infection. We also showed that the presence of PIF0, 6 and the ZF-domain of PIF8 are crucial for complex formation.