Isolation, identification and pathogenic characteristics of tick-derived parainfluenza virus 5 in northeast China.

The number of parainfluenza virus 5 (PIV5) infection cases has increased worldwide over the past six decades; however, factors underlying this increase remain unclear. PIV5 has been emerging or re-emerging in humans and animal species. To date, no information is yet available regarding PIV5 infection in arthropod ticks. Here, we successfully isolated tick-derived PIV5 from the Ixodes persulcatus species designated as HLJ/Tick/2019 in Heilongjiang, China. Phylogenetic analysis revealed that the tick-derived PIV5 is closely related to subclade 2.2.6, which has become the dominant subtype prevalent in dogs, pigs and wildlife across China. Further experiments to understand the importance of this virus as an infectious vector revealed that a ferret animal model experimentally infected with Tick/HLJ/2019 via the oronasal and ocular inoculation routes developed moderate respiratory distress with pneumonia and neurologic tissue damage from inflammation for the first time. Further surveillance of PIV5 in vectors of viral transmission is necessary to enhance our knowledge of its ecology in reservoirs and facilitate the control of re-emerging diseases.

Acute neurologic disease in Porcine rubulavirus experimentally infected piglets.

The objective of this study was to evaluate the clinical disease, humoral response and viral distribution of recent Porcine rubulavirus (PorPV) isolates in experimentally infected pigs. Four, 6-piglet (5-days old) groups were employed (G1-84, G2-93, G3-147, and G4-T). Three viral strains were used for the experimental infection: the reference strain LPMV-1984 (Michoacán 1984) and two other strains isolated in 2013, one in Queretaro (Qro/93/2013) and the other in Michoacán (Mich/147/2013). Each strain was genetically characterized by amplification and sequencing of the gene encoding hemagglutinin-neuroamidase (HN). The inoculation was performed through the oronasal and ocular routes, at a dose of 1×106TCID50/ml. Subsequently, the signs were evaluated daily and necropsies were performed on 3 different days post infection (dpi). We recorded all micro- and macroscopic lesions. Organs from the nervous, lymphatic, and respiratory system were analyzed by quantifying the viral RNA load and the presence of the infectious virus. The presence of the viral antigen in organs was evidenced through immunohistochemistry. Seroconversion was evaluated through the use of a hemagglutination inhibition test. In the characterization of gene HN, only three substitutions were identified in strain Mich/147/2013, two in strain LPMV/1984 (fourth passage) and one in strain Qro/93/2013, with respect to reference strain LPMV-84, these changes had not been identified as virulence factors in previously reported strains. Neurological alterations associated with the infection were found in all three experimental groups starting from 3dpi. Groups G1-84 and G3-147 presented the most exacerbated nervous signs. Group G2-93 only presented milder signs including slight motor incoordination, and an increased rectal temperature starting from day 5 post infection (PI). The main histopathological findings were the presence of a mononuclear inflammatory infiltrate (lymphocytic/monocytic) surrounding the ventricles in the brain and focal interstitial pneumonitis with distention of the alveolar sacs in the lungs. PorPV and RNA distribution were identified in the organs of the nervous, lymphatic, and respiratory systems of the piglets analyzed at different times (days 5, 10, and 15 PI). The viral antigen was detected in the brain and lungs in most of the assessed groups. Seroconversion was evident in groups G1-84 and G2-93. Groups G1-84 and G3-147 were the most clinically affected by the experimental infection. Both strains were isolated in the state of Michoacán. The virulence of the new isolates maintains similar characteristics to those reported more than 30 years ago.

Co-infection of classic swine H1N1 influenza virus in pigs persistently infected with porcine rubulavirus.

Porcine rubulavirus (PorPV) and swine influenza virus infection causes respiratory disease in pigs. PorPV persistent infection could facilitate the establishment of secondary infections. The aim of this study was to analyse the pathogenicity of classic swine H1N1 influenza virus (swH1N1) in growing pigs persistently infected with porcine rubulavirus. Conventional six-week-old pigs were intranasally inoculated with PorPV, swH1N1, or PorPV/swH1N1. A mock-infected group was included. The co-infection with swH1N1 was at 44 days post-infection (DPI), right after clinical signs of PorPV infection had stopped. The pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. In all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. By means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. Viral excretion of PorPV in nasal and oral fluid was recorded at 28 and 52 DPI, respectively. PorPV persisted in several samples from respiratory tissues (RT), secondary lymphoid organs (SLO), and bronchoalveolar lavage fluid (BALF). For swH1N1, the viral excretion in nasal fluids was significantly higher in single-infected swH1N1 pigs than in the co-infected group. However, the co-infection group exhibited an increase in the presence of swH1N1 in RT, SLO, and BALF at two days after co-infection. In conclusion, the results obtained confirm an increase in the clinical signs of infection, and PorPV was observed to impact the spread of swH1N1 in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. In the present study, the interaction of swH1N1 infection is demonstrated in pigs persistently infected with PorPV.

First report of severe parainfluenza virus 4B and rhinovirus C coinfection in a liver transplant recipient treated with immunoglobulin.

We describe the first reported case of severe pneumonia due to coinfection by parainfluenza virus type 4B and rhinovirus C in a liver transplant recipient. The patient responded promptly to intravenous immunoglobulin and timely infection control measures prevented spreading of the infections. This report highlights respiratory viral coinfections as a possible cause of severe morbidity in transplant recipients and the importance of efficient molecular diagnostic technologies with major impact on clinical practice in a transplant center. It also describes a potential therapeutic strategy for such patients.

Respiratory disease in growing pigs after Porcine rubulavirus experimental infection.

The aim of this study was to analyze the pathogenicity and distribution of Porcine rubulavirus (PorPV) in the respiratory tract of experimentally infected pigs. Nine 6-week-old pigs were infected with PorPV and examined clinically. Blood, nasal swab, and tissue samples were collected on different days post-infection (DPI). The humoral immune responses and viral loads were evaluated. The infected pigs exhibited an increase in the respiratory clinical signs. In addition, the excretion of PorPV was extended to 23 DPI in the nasal fluid. The distribution of PorPV in the respiratory tract tissues was extended until the end of the experiment; soft palate tonsil and lymph nodes exhibited high viral loads. The major microscopic lesions observed in the lungs corresponded to interstitial pneumonia and hyperplasia of the associated lymphoid tissue. In conclusion, PorPV infection causes a pneumonic disease characterized by a prolonged virus excretion and high viral load in the lymphoid tissues.

Persistence of porcine rubulavirus in experimentally infected boars.

Porcine rubulavirus is the etiological agent of blue eye disease in pigs. In boars, this virus causes orchitis and epididymitis and reduces seminal quality. The objective of this study was to determine the persistence of porcine rubulavirus in experimentally infected boars. Nine 12-month-old boars were infected with 5 ml of the PAC-3 strain of porcine rubulavirus at 1 × 10(5) TCID(50)/ml and held for 142 days post infection (DPI) to evaluate humoral immune response. The virus was isolated in cell cultures and detected by RT-PCR. Infection with porcine rubulavirus produced clinical signs beginning at 5 DPI. Necropsy results showed that 3 boars had lesions in the testicles and epididymes. Histological analysis showed the characteristic lesions in all infected boars. Porcine rubulavirus antibodies were detected in the second week post infection and increased significantly (P<0.05) over time. Isolation of the virus from semen was achieved between 5 DPI and 48 DPI and from the testicles and epididymes between 64 DPI and 142 DPI. Viral RNA was detected in the serum between 2 DPI and 64 DPI and in the semen until 142 DPI. These results confirm that the RNA of the porcine rubulavirus persists in the semen and that this virus remains in the reproductive tract for prolonged periods of infection. Semen of persistently infected boars, therefore, represents an important source of the virus and a risk factor for the spread of blue eye disease in swine populations.

Human parainfluenza virus type 4 infection in Chinese children with lower respiratory tract infections: a comparison study.

BACKGROUND: Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory tract infections (ARTIs). Although HPIV-4 has been associated with mild ARTIs for years, recent investigations have also associated HPIV-4 infection with severe respiratory syndromes and with outbreaks of ARTIs in children. OBJECTIVES: To characterize the role of HPIV-4 and its clinical features in children with acute lower respiratory tract infections (ALRTIs) in Beijing, China. STUDY DESIGN: Nasopharyngeal aspirates were collected from 2009 hospitalized children with ALRTIs between March 2007 and April 2010. RT-PCR and PCR analyses were used to identify HPIV types and other known respiratory viruses. RESULTS: HPIVs were detected in 246 (12.2%) patients, of whom 25 (10.2%) were positive for HPIV-4, 11 (4.5%) for HPIV-2, 51 (20.7%) for HPIV-1, 151 (61.4%) for HPIV-3, and 8 (3.3%) were co-detected with different types of HPIVs. Like HPIV-3, HPIV-4 was detected in spring, summer, and late fall over the study period. Seasonal incidence varied for HPIV-1 and -2. The median patient age was 20 months for HPIV-4 infections and 7-11 months for HPIV-1, -2, and -3 infections, but the clinical manifestations did not differ significantly between HPIV-1, -2, -3, and -4 infections. Moreover, co-detection of HPIV-4 (44%) with other respiratory viruses was lower than that of HPIV-1 (62.7%), HPIV-2 (63.6%), and HPIV-3 (72.7%). CONCLUSIONS: HPIV-4 plays an important role in Chinese paediatric ALRTIs. The epidemiological and clinical characteristics reported here improve our understanding of the pathogenesis associated with HPIV-4.

A prospective study of parainfluenza virus type 4 infections in children attending daycare.

Studies of parainfluenza virus type 4 (PIV-4) have been limited by difficulty in culturing. We prospectively studied a cohort of 225 young children attending daycare followed for 165 child-years, using polymerase chain reaction to detect 12 viruses, including PIV-4. PIV-4 was second only to PIV-3, occurring in 9 of 87 (10%) PIV+ illnesses. PIV-4 illnesses were not more severe and not associated with a specific clinical syndrome.

Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys.

Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32 degrees C and exhibited little productive replication at 37 degrees C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates.

Viral morphogenesis and morphological changes in human neuronal cells following Tioman and Menangle virus infection.

Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10(1)) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.

Virological and immunological characteristics of fatal virus-associated haemophagocytic syndrome (VAHS).

We report three infants and one teenager with fatal virus-associated haemophagocytic syndrome (VAHS). Two infants were admitted to our hospital because of cardio-pulmonary arrest (CPA). The third infant was admitted to our department because of fever and liver dysfunction, and he was diagnosed as combined immunodeficiency with predominant T cell defect. The teenager was diagnosed as systemic lupus erythema (SLE) when she was 10 years old and admitted to our department because of fever and thrombocytopenia . The histological findings for the four patients' organs revealed many haemophagocytic cells . The patients were infected by Parainfluenza virus type 2 (PIV2), Enterovirus (EV), Cytomegalovirus (CMV), and Epstein-Barr virus (EBV), respectively. Their laboratory data revealed elevated levels of ferritin and IL-6, which also suggested virus-associated haemophagocytic syndrome (VAHS). Although aggressive therapies were performed in all cases, the outcome was fatal. Further investigation would be needed to clarify the mechanism of VAHS and an effective therapeutic regimen is needed.

Altered function in CD8+ T cells following paramyxovirus infection of the respiratory tract.

For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in approximately 85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.

Experimental porcine rubulavirus (La Piedad-Michoacan virus) infection in pregnant gilts.

Porcine rubulavirus (La Piedad-Michoacan virus) (PoRV-LPMV) is a member of the Paramyxoviridae family that causes encephalitis in young piglets and infertility in adult sows and boars. Infertility in sows naturally infected by PoRV-LPMV is characterized by an increased number of returns to oestrus, stillbirths and mummified fetuses. In this study, nine seronegative gilts were inoculated intranasally with the PAC-3 strain of PoRV-LPMV at week 6 or 10 of gestation. These animals were then killed at weeks 8 or 15 of gestation (seven gilts) or after natural parturition (two gilts). Four control gilts were mock-infected at gestation week 6 or 10 and killed between 2 and 4 weeks later. Gross lesions of focal congestion and haemorrhage were seen in the placenta and endometrium of one gilt infected at gestation week 6 and one infected at gestation week 10. PoRV-LPMV was isolated, at 2-6 weeks post-inoculation (pi), from lung, tonsils, ovary, placenta, uterus and lymph nodes of three of the gilts infected at gestation week 6 and at 2-3 weeks pi from lung, tonsil and ovary of two gilts infected at gestation week 10. Many of the fetuses of eight infected gilts were smaller than normal and had dermal ecchymoses. Dehydrated or mummified fetuses were present in six of the infected gilts but not in any control animal. PoRV-LPMV was isolated from brain, lung and liver of fetuses from two gilts infected at gestation week 6, and from two infected at gestation week 10. These results indicate that, after experimental infection, PoRV can replicate in tissues of seronegative pregnant gilts, cross the placenta, and cause fetal death and mummification.

Apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with porcine rubulavirus.

In a first experiment, five pigs were inoculated intranasally with porcine rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2(+) and CD8(+) T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4(+) and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2(+), CD8(+) and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

High resistance of human parainfluenza type 2 virus protein-expressing cells to the antiviral and anti-cell proliferative activities of alpha/beta interferons: cysteine-rich V-specific domain is required for high resistance to the interferons.

Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-alpha/beta) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-gamma. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-alpha/beta. HeLa cells expressing the N-terminally truncated V protein show resistance to IFN-alpha/beta, showing that the IFN resistance determinant maps to the cysteine-rich V-specific domain. A complete defect of Stat2 is found in HeLa-CA and HeLa-V cells, whereas the levels of Stat1 expression are not significantly different among HeLa, HeLa-CA, HeLa-P, and HeLa-V cells, indicating that IFN-alpha/beta resistance of HeLa-CA and HeLa-V cells is due to a defect of Stat2. HeLa-SV41V cells show high resistance to all IFNs, and no expression of Stat1 can be detected. Stat2 mRNA is fully detected in HeLa-V cells. Stat2 was scarcely pulse-labeled in the HeLa-V cells, indicating that synthesis of Stat2 is suppressed or Stat2 is very rapidly degraded in HeLa-V cells. The V protein suppresses the in vitro translation of Stat2 mRNA more extensively than that of Stat1 mRNA. An extremely small amount of Stat2 can be detected in HeLa-V cells treated with proteasome inhibitors. The half-life of Stat2 is approximately 3.5 and 2 h in uninfected and hPIV-2-infected HeLa cells, respectively. This study shows that synthesis of Stat2 may be suppressed and Stat2 degradation is also enhanced in hPIV-2-infected HeLa and HeLa-V cells.

Porcine rubulavirus LPMV RNA persists in the central nervous system of pigs after recovery from acute infection.

In order to study persistence of the porcine rubulavirus LPMV, we examined tissue samples collected from pigs 53 days after experimental infection. These pigs survived the initial infection and could clinically be considered to have recovered from the infection. Two of the pigs used in this study were chemically immunosuppressed during the last 4 days before necropsy. No infectious virus or viral antigen could be detected in any tissue using standard methods for virus isolation and detection. However, the presence of viral genomic RNA and mRNA could be demonstrated in the mid brain of the convalescent pig using an optimised RT-nested PCR. Mid brain, forebrain and lung were all shown to contain LPMV RNA in the immunosuppressed convalescent pigs. In addition we examined the P-gene editing in the recovered pigs and conclude that the viral genome is transcriptionally active in these pigs. The relevance of the persistence of LPMV for maintenance and spread within and/or between pig populations is discussed.

Avian paramyxoviruses serotype 3 isolated from captive birds in Israel: clinical signs, pathology, and antigenic characterization.

Thirteen HA agents were isolated in Israel from captive flamingoes (Phoenicopterus ruber), Egyptian geese (Alopochen aegyptiacus) belonging to order Anseriformes, and ibis (Guara rubra) belonging to order Ciconiiformes. The isolation was done from postmortem materials in three cases of severe respiratory disease with high mortality. The isolates were examined serologically and identified as belonging to the serotype 3 of avian paramyxoviruses (APMV-3). The isolates were more close antigenically to the APMV-3b variety (prototype strain--APMV-3/Parakeet/Netherlands/449/75) than to the APMV-3a variety (prototype strain--APMV-3/Turkey/Wisconsin/68). This is the first isolation of APMV-3 from birds belonging to the orders Anseriformes and Ciconiiformes.