Distinct clinical features of transplanted children with Parvovirus B19 infection.

BACKGROUND: The immature and suppressed immune response makes transplanted children a special susceptible group to Parvovirus B19 (PVB19). However, the clinical features of transplanted children with PVB19 infection haven't been comprehensively described. METHODS: We searched the medical records of all the transplant recipients who attended the Children's Hospital of Fudan University from 1 Oct 2020 to 31 May 2023, and reviewed the medical literature for PVB19 infection cases among transplanted children. RESULTS: A total of 10 cases of PVB19 infection were identified in 201 transplanted children at our hospital, and the medical records of each of these cases were shown. Also, we retrieved 40 cases of PVB19 infection among transplanted children from the literature, thus summarizing a total of 50 unique cases of PVB19 infection. The median time to the first positive PVB19 DNA detection was 14 weeks post-transplantation. PVB19 IgM and IgG were detected in merely 26% and 24% of the children, respectively. The incidence of graft loss/dysfunction was as high as 36%. Hematopoietic stem cell transplant (HSCT) recipients showed higher PVB19 load, lower HGB level, greater platelet damage, lower PVB19 IgM/IgG positive rates, and more graft dysfunction than solid-organ transplant (SOT) recipients, indicating a more incompetent immune system. CONCLUSIONS: Compared with the published data of transplanted adults, transplanted children displayed distinct clinical features upon PVB19 infection, including lower PVB19 IgM/IgG positive rates, more graft dysfunction, and broader damage on hematopoietic cell lines, which was even more prominent in HSCT recipients, thus should be of greater concern.

Disseminated Human Parvovirus B19 Infection Induced Multiple Organ Dysfunction Syndrome in an Adult Patient With Alcoholic Hepatitis Complicated by Hemolytic Anemia: A Case Report and Literature Review.

Background: Human parvovirus B19 (B19) can cause acute hepatitis and is attributed to the high mortality of alcoholic hepatitis (AH). B19 infection is generally self-healing in previously healthy people, but it can cause fatal effects in some high-risk groups and increase its virulence and infectivity. Disseminated B19 infection-induced multiple organ dysfunction syndrome (MODS) in patients with AH has not been reported yet. Here, we described B19 viremia in an adult patient with AH accompanied by hemolytic anemia (HA), leading to disseminated infection and secondary MODS, as well as self-limiting B19 infections in seven nurses caring for him. Meanwhile, we reviewed the literature on AH and B19 infection. Case Presentation: A 43-year-old male patient with AH accompanied by HA was transferred to the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, on March 31, 2021. After supportive treatment, his transaminase and bilirubin levels were reduced, but his anemia worsened. He received a red blood cell (RBC) infusion on April 9 for hemoglobin (Hb) lower than 6 g/dl. On April 13, he suddenly had a high fever. Under empirical anti-infection, his high fever dropped and maintained at a low fever level; however, his anemia worsened. On April 25, he was transferred to the medical intensive care unit (MICU) due to severe pneumonia, acute respiratory distress syndrome (ARDS), acute aplastic crisis (AAC), and hemophagocytic syndrome (HPS), which were subsequently confirmed to be related to B19 infection. After methylprednisolone, intravenous immunoglobulin (IVIG), empirical anti-infection, and supportive treatment, the lung infection improved, but hematopoietic and liver abnormalities aggravated, and systemic B19 infection occurred. Finally, the patient developed a refractory arrhythmia, heart failure, and shock and was referred to a local hospital by his family on May 8, 2021. Unfortunately, he died the next day. Fourteen days after he was transferred to MICU, seven nurses caring for him in his first two days in the MICU developed self-limiting erythema infectiosum (EI). Conclusions: B19 infection is self-limiting in healthy people, with low virulence and infectivity; however, in AH patients with HA, it can lead to fatal consequences and high contagion.

Virus-Host Interactions of Enteroviruses and Parvovirus B19 in Myocarditis.

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses.

Shrimp Parvovirus Circular DNA Fragments Arise From Both Endogenous Viral Elements and the Infecting Virus.

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

Parvovirus B19 Seroprevalence in Women with Bad Obstetric History in Kirkuk.

BACKGROUND: In the Iraqi community, abnormal pregnancy forms a major social and psychological health problem. The underlying etiology of this health phenomenon was varied and included sets of infections and autoimmune diseases. Globally human parvovirus 19 infection is common and the infection attributes to bad obstetric outcomes. The global maternal parvovirus B19 remote infection rate was within a range of 13.2% to 97.9%, while the range of acute infection was between 0.5% to 97.9%. In Arab countries, the IgG seroprevalence was from 53.3% to 74%, while IgM seroprevalence range was 2.2% to 84%. OBJECTIVE: To evaluate the role of ParvovirusB19 as an etiology of bad obstetric outcome in women in Kirkuk, Iraq. MATERIALS AND METHODS: Descriptive Case Control Study. Women included in the study were recruited from Kirkuk General Hospital and their age ranged from 14 to 48 years. A total of 663 women were included in the study, of them 237 were not pregnant, while 215 were pregnant. Additionally, the study included 211 women with inevitable abortion. Control group (306 women) women with a history of normal pregnancy included (Pregnant= 149; non-pregnant= 157). Clinical and laboratory investigations were conducted on all patients and control groups to exclude other causes. Medical and obstetric data and demographic characteristics were gathered through interviews according to a previously designed questionnaire. ELISA kits were used to determine Parvovirus B19 IgM and IgG antibodies. RESULTS: The overall parvovirus seroprevalence was 93% and with no significant difference between women with normal (89.5%) and those with abnormal (93.1%) pregnancy outcomes. In addition, parvovirus IgM overall seroprevalence was at56.3%. Furthermore, current parvovirus infection was higher in women with BOH (52.6%) than that in women with normal pregnancy (49.7%) outcomes. Parvovirus IgM seroprevalence was 52.6% in women with BOH and 49.7% in women with normal pregnancy, however, the difference was not statistically significant. In contrast, the acute infection with parvovirus was significantly (X2=11.8, P=0.001) lower in women with normal pregnancy (49.7%) than in those with inevitable abortion (64.9%). While the IgG seroprevalence difference was not significant between the two groups, infection seroprevalence was more frequent in housewives, uneducated women, large families, non-smokers, in rural areas, non-animal exposure areas, women with repeated abortion, congenital anomalies and anaemia. CONCLUSION: Parvovirus B19 infection may be with bad obstetric outcomes if occurred during pregnancy and OR confirmed a significant association of the infection with parvovirus with smoking, occupation, crowding index, education, animal exposure and the number of repeated abortion.

Persistence of Human Bocavirus 1 in Tonsillar Germinal Centers and Antibody-Dependent Enhancement of Infection.

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.

Canine parvovirus vaccination and immunisation failures: Are we far from disease eradication?

Despite extensive vaccination, canine parvovirus (CPV) remains a leading infectious cause of canine mortality, especially among juveniles. This review provides an update on CPV vaccine types and vaccination protocols. The design of CPV prevention strategies and vaccination programs with a goal of herd immunity has been hampered by deficiencies of studies that model companion animal viral infections and inform an understanding of the basic reproduction number. However, the most important issue in eradication of CPV disease is represented by immunisation failures including: i) the presence of interfering titres of maternally-derived antibodies; ii) the presence of non-responders; and iii) possible reversion to virulence. In contrast, the role of the CPV variants in immunisation failures is widely debated. Taking into account the reduced circulation of canine distemper virus and canine adenovirus type 1 in countries where extensive vaccination is carried out, more effort should be made to aim for CPV eradication, including antibody testing to determine the optimal time for vaccinations of pups and adults and homogeneous vaccine coverage of dog population.

Assessment of Immunological Response and Impacts on Fertility Following Intrauterine Vaccination Delivered to Swine in an Artificial Insemination Dose.

To protect the health of sows and gilts, significant investments are directed toward the development of vaccines against infectious agents that impact reproduction. We developed an intrauterine vaccine that can be delivered with semen during artificial insemination to induce mucosal immunity in the reproductive tract. An in vitro culture of uterine epithelial cells was used to select an adjuvant combination capable of recruiting antigen-presenting cells into the uterus. Adjuvant polyinosinic:polycytidylic acid (poly I:C), alone or in combination, induced expression of interferon gamma, tumor necrosis factor alpha, and select chemokines. A combination adjuvant consisting of poly I:C, host defense peptide and polyphosphazene (Triple Adjuvant; TriAdj), which previously was shown to induce robust mucosal and systemic humoral immunity when administered to the uterus in rabbits, was combined with boar semen to evaluate changes in localized gene expression and cellular recruitment, in vivo. Sows bred with semen plus TriAdj had decreased γδ T cells and monocytes in blood, however, no corresponding increase in the number of monocytes and macrophages was detected in the endometrium. Compared to sows bred with semen alone, sows bred with semen plus TriAdj showed increased CCL2 gene expression in the epithelial layer. These data suggest that the adjuvants may further augment a local immune response and, therefore, may be suitable for use in an intrauterine vaccine. When inactivated porcine parvovirus (PPV) formulated with the TriAdj was administered to the pig uterus during estrus along with semen, we observed induction of PPV antibodies in serum but only when the pigs were already primed with parenteral PPV vaccines. Recombinant protein vaccines and inactivated PPV vaccines administered to the pig uterus during breeding as a primary vaccine alone failed to induce significant humoral immunity. More trials need to be performed to clarify whether repeated intrauterine vaccination can trigger strong humoral immunity or whether the primary vaccine needs to be administered via a systemic route to promote a mucosal and systemic immune response.

Development of a monoclonal antibody against canine parvovirus NS1 protein and investigation of NS1 dynamics and localization in CPV-infected cells.

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.

Characterization of a recombinant pseudorabies virus expressing porcine parvovirus VP2 protein and porcine IL-6.

BACKGROUND: Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV. METHODS: The recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals. RESULTS: A recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10). CONCLUSIONS: The recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.

[Immune response and gene therapy with adenoassociated viral vectors]. / Respuesta inmune y terapia génica con vectores virales adenoasociados.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient's immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

En los últimos años la terapia génica se ha posicionado como una opción real y segura en el desarrollo de alternativas terapéuticas para la cura y la prevención de diferentes enfermedades. Consiste en la inserción de material genético en un tejido o célula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unión y entrada a la célula diana, la capacidad de transferencia del material genético al núcleo, la habilidad de expresión del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluación como opción terapéutica en un amplio número de enfermedades monogénicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducción del vector e impiden la expresión del gen que transporta, limitando su aplicación clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia génica con vectores AAV. La presencia de NAb depende principalmente de la exposición al virus en la naturaleza y varía drásticamente según edad, localización geográfica y estado de salud de la persona evaluada.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Analysis of recombinant adeno-associated viral vector shedding in sheep following intracoronary delivery.

Differences between mouse and human hearts pose a significant limitation to the value of small animal models when predicting vector behavior following recombinant adeno-associated viral (rAAV) vector-mediated cardiac gene therapy. Hence, sheep have been adopted as a preclinical animal, as they better model the anatomy and cardiac physiological processes of humans. There is, however, no comprehensive data on the shedding profile of rAAV in sheep following intracoronary delivery, so as to understand biosafety risks in future preclinical and clinical applications. In this study, sheep received intracoronary delivery of rAAV serotypes 2/6 (2 × 1012 vg), 2/8, and 2/9 (1 × 1013 vg) at doses previously administered in preclinical and clinical trials. This was followed by assessment over 96 h to examine vector shedding in urine, feces, nasal mucus, and saliva samples. Vector genomes were detected via real-time quantitative PCR in urine and feces up to 48 and 72 h post vector delivery, respectively. Of these results, functional vector particles were only detected via a highly sensitive infectious replication assay in feces samples up to 48 h following vector delivery. We conclude that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles for all excreta samples, except in the case of feces, where functional vector particles are present up to 48 h following vector delivery. These results may be used to inform containment and decontamination guidelines for large animal dealings, and to understand the biosafety risks associated with future preclinical and clinical uses of rAAV.

Adeno-associated Virus (AAV) versus Immune Response.

Decades ago, Friedmann and Roblin postulated several barriers to gene therapy, including tissue targeting, delivery across the blood⁻brain barrier (BBB), and host immune responses. These issues remain pertinent till today. Since then, several advances have been made in elucidating structures of adeno-associated virus (AAV) serotypes, antibody epitopes, and ways to modify antibody-binding sites. AAVs capsid has also been engineered to re-direct tissue tropism, reduce ubiquitination, and promote passage across the BBB. Furthermore, the use of high(er) dose recombinant AAV (rAAV) has been accompanied by a better understanding of immune responses in both experimental animals and early clinical trials, and novel work is being performed to modulate the immune response. While the immune responses to rAAV remains a major challenge in translating experimental drugs to approved medicine, and will likely require more than a single solution, we now better understand the hurdles to formulate and test experimental solutions to surmount them.

Neonatal Cytomegalovirus Palatal Ulceration and Bocavirus Pneumonitis Associated With a Defect of Lymphocyte Cytotoxicity Caused by Mutations in UNC13D.

Single gene defects that impair lymphocyte cytotoxicity can predispose to severe viral infection that normally remains subclinical. The classic severe presentation is hemophagocytic lymphohistiocytosis. Here, we report the case of a neonate who presented with cytomegalovirus palatal ulceration and bocavirus pneumonitis secondary to impaired cytotoxicity caused by biallelic mutations in the UNC13D gene.

Parvoviral disease in childhood cancer: Clinical outcomes and impact on therapy at a tertiary cancer center in India.

BACKGROUND AND AIM: Parvovirus-B19 disease in immunocompromised children can cause myelosuppression and therapeutic delays. We studied the clinical profiles of children having symptoms suggestive of parvoviral disease at our institution, a large tertiary cancer center. METHODS: Children below age 15 years undergoing treatment for malignancies with clinical features suggestive of parvoviral infection, and/or unexplained drop in hemoglobin, and/or prolonged cytopenia were screened for parvovirus infection using DNA-PCR for parvovirus-B19 (PB19) in the peripheral blood. Patients testing positive from September 2014 till February 2017 were studied. RESULTS: Of the 59 patients (36 patients with hematolymphoid malignancies, 23 with solid tumors) screened for suspected parvoviral infections, 27 tested positive. Median age was 9.6 years (2.25-15 years), 18 (66%) had hematolymphoid malignancies, while 7 (33%) had solid tumors. Six patients (26%) were on intensive phases, 16 (60%) patients developed the symptoms during maintenance chemotherapy, and 4 (15%) after completion of therapy. Isolated anemia was the commonest feature seen in 10 patients (37%) while bicytopenia and pancytopenia were noticed in 8 (30%) and 9 (33%) patients respectively. Fifty percent of patients those who received rituximab (3/6) developed persistent parvoviremia (>4 weeks) as compared with 24% (5/21) of those who did not. Two patients (7%) developed hemophagocytic lymphohistiocytosis (HLH). Treatment delay by more than 14 days was encountered in a majority (62%), with 5 patients requiring treatment modification or even suspension. CONCLUSIONS: Parvoviral infection in children who are on or have recently completed chemotherapy can lead to multiple cytopenias and significant treatment delays. Rituximab exposure may lead to persistent parvoviral disease (p < 0.05). HLH, though occasional, can be a serious complication.

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction.

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-ß expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Global Distribution of Human Protoparvoviruses.

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.

Parvovirus B19 and in situ immune response in eczema and psoriasis skin lesions of patients from the Brazilian Amazon region.

Parvovirus B19 (PVB19) is a virus found in the skin that causes asymptomatic infections and can exist in the host for long periods to time. The virus induces a local inflammatory response and is associated with the development of arthritis and other autoimmunes diseases. Parvovirus B19 DNA was investigated by PCR in the skin of 20 patients with psoriasis and 20 patients with eczema. Additionally, immunohistochemistry was used to characterize the expression of cytokines in these lesions. The sociodemographic variables were similar in the two groups studied. Psoriasis vulgaris was the most common clinical type in men (50%) and women (80%) (p = 0.0106). Comorbidities were observed in most patients with psoriasis (75%), with an OR of 14 (p = 0.0068). Another important finding was the high prevalence (50%) of psychiatric disorders in patients with psoriasis (OR = 16, p = 0.0218). Only two patients (10%) with psoriasis were positive for PVB19. Comparison of cytokine expression showed the same cytokine profile in the two groups (p > 0.05). However, expression of TNF-α tended to be higher in psoriasis patients. There was no significant positivity for PVB19 in the two groups studied. Immunohistochemistry showed higher expression of TNF-α in psoriasis lesions compared to the eczema group.

Long-lived immunity to canine core vaccine antigens in UK dogs as assessed by an in-practice test kit.

OBJECTIVES: To determine the utility of an in-practice test kit to detect protective serum antibody against canine distemper virus, canine adenovirus and canine parvovirus type 2 in a sample of the UK dog population. MATERIALS AND METHODS: Serum samples from 486 dogs, last vaccinated between less than 1 month and 124 months previously, were tested with the VacciCheck™ test kit for protective antibodies against distemper, adenovirus and parvovirus type 2. RESULTS: A high proportion of the dogs tested (93·6%) had protective antibody against all three of the core vaccine antigens: 95·7% of the dogs were seropositive against canine distemper virus, 97·3% against canine adenovirus and 98·5% against canine parvovirus type 2. The small number of dogs that were seronegative for one or more of the antigens (n = 31) may have had waning of previous serum antibody or may have been rare genetic non-responders to that specific antigen. CLINICAL SIGNIFICANCE: UK veterinarians can be reassured that triennial revaccination of adult dogs with core vaccines provides long-lived protective immunity. In-practice serological test kits are a valuable tool for informing decision-making about canine core revaccination.