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Background and aims Hepatitis E virus (HEV) variants causing human infection predominantly belong to HEV species A (HEV-A). HEV species C genotype 1 (HEV-C1) circulates in rats and is highly divergent from HEV-A. It was previously considered unable to infect humans, but the first case of human HEV-C1 infection was recently discovered in Hong Kong. The aim of this study is to further describe the features of this zoonosis in Hong Kong. Approach and results We conducted a territory-wide prospective screening study for HEV-C1 infection over a 31-month period. Blood samples from 2,860 patients with abnormal liver function (n = 2,201) or immunosuppressive conditions (n = 659) were screened for HEV-C1 RNA. In addition, 186 captured commensal rats were screened for HEV-C1 RNA. Sequences of human-derived and rat-derived HEV-C1 isolates were compared. Epidemiological and clinical features of HEV-C1 infection were analyzed. HEV-C1 RNA was detected in 6/2,201 (0.27%) patients with hepatitis and 1/659 (0.15%) immunocompromised persons. Including the previously reported case, eight HEV-C1 infections were identified, including five in patients who were immunosuppressed. Three patients had acute hepatitis, four had persistent hepatitis, and one had subclinical infection without hepatitis. One patient died of meningoencephalitis, and HEV-C1 was detected in cerebrospinal fluid. HEV-C1 hepatitis was generally milder than HEV-A hepatitis. HEV-C1 RNA was detected in 7/186 (3.76%) rats. One HEV-C1 isolate obtained from a rat captured near the residences of patients was closely related to the major outbreak strain. Conclusions HEV-C1 is a cause of hepatitis E in humans in Hong Kong. Immunosuppressed individuals are susceptible to persistent HEV-C1 infection and extrahepatic manifestations. Subclinical HEV-C1 infection threatens blood safety. Tests for HEV-C1 are required in clinical laboratories.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic. Accurate detection of SARS-CoV-2 using molecular assays is critical for patient management and the control of the COVID-19 pandemic. However, there is an increasing number of SARS-CoV-2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real-time reverse transcription-polymerase chain reaction (RT-PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence-independent single-primer amplification and nanopore whole-genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5' end of the SARS-CoV-2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID-19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real-time RT-PCR assay. The primers and probes are highly specific for SARS-CoV-2. Validation with 101 clinical specimens showed that our nsp1 RT-PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%-97.2%), which was similar to those of N and E gene RT-PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%-100%). The addition of nsp1 for multitarget detection of SARS-CoV-2 can avoid false-negative results due to mutations at the primers/probes binding sites of currently available RT-PCR assays.

Objectives To determine the viral epidemiology and clinical characteristics of patients with and without clinically apparent respiratory tract infection. Methods This prospective cohort study was conducted during the 2018 winter influenza season. Adult patients with fever/respiratory symptoms (fever/RS group) were age- and sex-matched with patients without fever/RS (non-fever/RS group) in a 1:1 ratio. Respiratory viruses were tested using NxTAG™ Respiratory Pathogen Panel IVD, a commercially-available multiplex PCR panel. Results A total of 214 acutely hospitalized patients were included in the final analysis, consisting of 107 with fever/RS (fever/RS group), and 107 age- and sex-matched patients without fever/RS (non-fever/RS group). Respiratory viruses were detected in 34.1% (73/214) of patients, and co-infection occurred in 7.9% (17/214) of patients. The incidence of respiratory virus was higher in the fever/RS group than in the non-fever/RS group (44.9% (48/107) versus 23.4% (25/107), p 0.001). Influenza B virus, enterovirus/rhinovirus and coronaviruses were detected more frequently in the fever/RS group, whereas parainfluenza virus 4B and adenovirus were detected more frequently in the non-fever/RS group. Among the non-fever/RS group, chest discomfort was more common among patients tested positive for respiratory viruses than those without respiratory virus detected (44% (11/25) versus 22% (18/82), p 0.04). Conclusions Respiratory viruses can be frequently detected among hospitalized patients without typical features of respiratory tract infection. These patients may be a source of nosocomial outbreaks.

Background Hepatitis E virus (HEV) variants belonging to Orthohepevirus species A (HEV-A) are the primary cause of human hepatitis E. However, we previously reported that Orthohepevirus species C genotype 1 (HEV-C1), a divergent HEV variant commonly found in rats, also causes hepatitis in humans. Here, we present a clinical-epidemiological investigation of human HEV-C1 infections detected in Hong Kong, with an emphasis on outcomes in immunocompromised individuals. Methods A surveillance system for detecting human HEV-C1 infections was established in Hong Kong. Epidemiological and clinical characteristics of HEV-C1 cases identified via this system between 1 August 2019 and 31 December 2020 were retrieved. Phylogenetic analysis of HEV-C1 strain sequences was performed. Infection outcomes of immunocompromised individuals with HEV-A and HEV-C1 infections were analyzed. Results HEV-C1 accounted for 8 of 53 (15.1%) reverse-transcription polymerase chain reaction (RT-PCR)–confirmed HEV infections in Hong Kong during the study period, raising the total number of HEV-C1 infections detected in the city to 16. Two distinct HEV-C1 strain groups caused human infections. Patients were elderly and/or immunocompromised; half tested negative for HEV immunoglobulin M. Cumulatively, HEV-C1 accounted for 9 of 21 (42.9%) cases of hepatitis E recorded in immunocompromised patients in Hong Kong. Immunocompromised HEV-C1 patients progressed to persistent hepatitis at similar rates (7/9 [77.8%]) as HEV-A patients (10/12 [75%]). HEV-C1 patients responded to oral ribavirin, although response to first course was sometimes poor or delayed. Conclusions Dedicated RT-PCR–based surveillance detected human HEV-C1 cases that evade conventional hepatitis E diagnostic testing. Immunosuppressed HEV-C1–infected patients frequently progress to persistent HEV-C1 infection, for which ribavirin is a suitable treatment option.

Host cell lipids play a pivotal role in the pathogenesis of respiratory virus infection. However, a direct comparison of the lipidomic profile of influenza virus and rhinovirus infections is lacking. In this study, we first compared the lipid profile of influenza virus and rhinovirus infection in a bronchial epithelial cell line. Most lipid features were downregulated for both influenza virus and rhinovirus, especially for the sphingomyelin features. Pathway analysis showed that sphingolipid metabolism was the most perturbed pathway. Functional study showed that bacterial sphingomyelinase suppressed influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, but promoted rhinovirus replication. These findings suggest that sphingomyelin pathway can be a potential target for antiviral therapy, but should be carefully evaluated as it has opposite effects on different respiratory viruses. Furthermore, the differential effect of sphingomyelinase on rhinovirus and influenza virus may explain the interference between rhinovirus and influenza virus infection.

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

Hepatitis E virus (HEV) genotype 4 (HEV-4) is an emerging cause of acute hepatitis in China. Less is known about the clinical characteristics and natural history of HEV-4 than HEV genotype 3 infections in immunocompromised patients. We report transmission of HEV-4 from a deceased organ donor to 5 transplant recipients. The donor had been viremic but HEV IgM and IgG seronegative, and liver function test results were within reference ranges. After a mean of 52 days after transplantation, hepatitis developed in all 5 recipients; in the liver graft recipient, disease was severe and with progressive portal hypertension. Despite reduced immunosuppression, all HEV-4 infections progressed to persistent hepatitis. Four patients received ribavirin and showed evidence of response after 2 months. This study highlights the role of organ donation in HEV transmission, provides additional data on the natural history of HEV-4 infection, and points out differences between genotype 3 and 4 infections in immunocompromised patients.

All hepatitis E virus (HEV) variants reported to infect humans belong to the species Orthohepevirus A (HEV-A). The zoonotic potential of the species Orthohepevirus C (HEV-C), which circulates in rats and is highly divergent from HEV-A, is unknown. We report a liver transplant recipient with hepatitis caused by HEV-C infection. We detected HEV-C RNA in multiple clinical samples and HEV-C antigen in the liver. The complete genome of the HEV-C isolate had 93.7% nt similarity to an HEV-C strain from Vietnam. The patient had preexisting HEV antibodies, which were not protective against HEV-C infection. Ribavirin was an effective treatment, resulting in resolution of hepatitis and clearance of HEV-C viremia. Testing for this zoonotic virus should be performed for immunocompromised and immunocompetent patients with unexplained hepatitis because routine hepatitis E diagnostic tests may miss HEV-C infection. HEV-C is also a potential threat to the blood product supply.

SARS-CoV-2 is more infectious and transmissible in humans than SARS-CoV, despite the genetic relatedness and sharing the same cellular receptor. We sought to assess whether human airway organoids can model SARS-CoV-2 infection in the human airway and elucidate the cellular basis underlying its higher transmissibility. We demonstrate that SARS-CoV-2 can establish a productive infection in human airway organoids, in which ciliated cell and basal cell are infected. Wildtype SARS-CoV-2 carrying a furin cleavage motif exhibits comparable replication kinetics to a mutant virus without the motif. Human airway organoids sustain higher replication of SARS-CoV-2 than SARS-CoV, whereas interferon response is more potently induced in the latter than the former. Overall, human airway organoids can model SARS-CoV-2 infection and recapitulate the disposable role of furin cleavage motif for virus transmission in humans. SARS-CoV-2 stealth growth and evasion of interferon response may underlie pre-symptomatic virus shedding in COVID-19 patients, leading to its high infectiousness and transmissibility.

After 2 months of relative quiescence, a large coronavirus disease 2019 outbreak occurred in Hong Kong in July 2020 after gradual relaxation of social distancing policy. Unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) phylogenetic clusters have been identified among locally acquired cases, with most genomes belonging to cluster HK1, which is phylogenetically related to SARS-CoV-2 reported overseas.

Background & Aims Rat hepatitis E virus (Orthohepevirus species C; HEV-C1) is an emerging cause of viral hepatitis in humans. HEV-C1 is divergent from other HEV variants infecting humans that belong to Orthohepevirus species A (HEV-A). This study assessed HEV-C1 antigenic divergence from HEV-A and investigated the impact of this divergence on infection susceptibility, serological test sensitivity, and vaccine efficacy. Methods Immunodominant E2s peptide sequences of HEV-A and HEV-C1 were aligned. Interactions of HEV-C1 E2s and anti-HEV-A monoclonal antibodies (mAbs) were modeled. Recombinant peptides incorporating E2s of HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) were expressed. HEV-A and HEV-C1 patient sera were tested using antibody enzymatic immunoassays (EIA), antigen EIAs, and HEV-A4 p239/HEV-C1 p241 immunoblots. Rats immunized with HEV-A1 p239 vaccine (Hecolin), HEV-A4 p239 or HEV-C1 p241 peptides were challenged with a HEV-C1 strain. Results E2s sequence identity between HEV-A and HEV-C1 was only 48%. There was low conservation at E2s residues (23/53; 43.4%) involved in mAb binding. Anti-HEV-A mAbs bound HEV-C1 poorly in homology modeling and antigen EIAs. Divergence resulted in low sensitivity of commercial antigen (0%) and antibody EIAs (10–70%) for HEV-C1 diagnosis. Species-specific HEV-A4 p239/HEV-C1 p241 immunoblots accurately differentiated HEV-A and HEV-C1 serological profiles in immunized rats (18/18; 100%) and infected-patient sera (32/36; 88.9%). Immunization with Hecolin and HEV-A4 p239 was partially protective while HEV-C1 p241 was fully protective against HEV-C1 infection in rats. Conclusions Antigenic divergence significantly decreases sensitivity of hepatitis E serodiagnostic assays for HEV-C1 infection. Species-specific immunoblots are useful for diagnosing HEV-C1 and for differentiating the serological profiles of HEV-A and HEV-C1. Prior HEV-A exposure is not protective against HEV-C1. HEV-C1 p241 is an immunogenic vaccine candidate against HEV-C1. Lay summary Rat hepatitis E virus (HEV-C1) is a new cause of hepatitis in humans. Using a combination of methods, we showed that HEV-C1 is highly divergent from the usual cause of human hepatitis (HEV-A). This divergence reduces the capacity of existing tests to diagnose HEV-C1 and also indicates that prior exposure to HEV-A (via infection or vaccination) is not protective against HEV-C1.

A novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-emerged in humans in Wuhan, China, in December 2019 and has since disseminated globally1,2. As of April 16, 2020, the confirmed case count of coronavirus disease 2019 (COVID-19) had surpassed 2 million. Based on full-genome sequence analysis, SARS-CoV-2 shows high homology to SARS-related coronaviruses identified in horseshoe bats1,2. Here we show the establishment and characterization of expandable intestinal organoids derived from horseshoe bats of the Rhinolophus sinicus species that can recapitulate bat intestinal epithelium. These bat enteroids are fully susceptible to SARS-CoV-2 infection and sustain robust viral replication. Development of gastrointestinal symptoms in some patients with COVID-19 and detection of viral RNA in fecal specimens suggest that SARS-CoV-2 might cause enteric, in addition to respiratory, infection3,4. Here we demonstrate active replication of SARS-CoV-2 in human intestinal organoids and isolation of infectious virus from the stool specimen of a patient with diarrheal COVID-19. Collectively, we established the first expandable organoid culture system of bat intestinal epithelium and present evidence that SARS-CoV-2 can infect bat intestinal cells. The robust SARS-CoV-2 replication in human intestinal organoids suggests that the human intestinal tract might be a transmission route of SARS-CoV-2.

In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV., In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

Summary: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported from China in January, 2020. SARS-CoV-2 is efficiently transmitted from person to person and, in 2 months, has caused more than 82 000 laboratory-confirmed cases of coronavirus disease 2019 (COVID-19) and 2800 deaths in 46 countries. The total number of cases and deaths has surpassed that of the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV). Although both COVID-19 and severe acute respiratory syndrome (SARS) manifest as pneumonia, COVID-19 is associated with apparently more efficient transmission, fewer cases of diarrhoea, increased mental confusion, and a lower crude fatality rate. However, the underlying virus–host interactive characteristics conferring these observations on transmissibility and clinical manifestations of COVID-19 remain unknown. Methods: We systematically investigated the cellular susceptibility, species tropism, replication kinetics, and cell damage of SARS-CoV-2 and compared findings with those for SARS-CoV. We compared SARS-CoV-2 and SARS-CoV replication in different cell lines with one-way ANOVA. For the area under the curve comparison between SARS-CoV-2 and SARS-CoV replication in Calu3 (pulmonary) and Caco2 (intestinal) cells, we used Student's t test. We analysed cell damage induced by SARS-CoV-2 and SARS-CoV with one-way ANOVA. Findings: SARS-CoV-2 infected and replicated to comparable levels in human Caco2 cells and Calu3 cells over a period of 120 h (p=0·52). By contrast, SARS-CoV infected and replicated more efficiently in Caco2 cells than in Calu3 cells under the same multiplicity of infection (p=0·0098). SARS-CoV-2, but not SARS-CoV, replicated modestly in U251 (neuronal) cells (p=0·036). For animal species cell tropism, both SARS-CoV and SARS-CoV-2 replicated in non-human primate, cat, rabbit, and pig cells. SARS-CoV, but not SARS-CoV-2, infected and replicated in Rhinolophus sinicus bat kidney cells. SARS-CoV-2 consistently induced significantly delayed and milder levels of cell damage than did SARS-CoV in non-human primate cells (VeroE6, p=0·016; FRhK4, p=0·0004). Interpretation: As far as we know, our study presents the first quantitative data for tropism, replication kinetics, and cell damage of SARS-CoV-2. These data provide novel insights into the lower incidence of diarrhoea, decreased disease severity, and reduced mortality in patients with COVID-19, with respect to the pathogenesis and high transmissibility of SARS-CoV-2 compared with SARS-CoV. Funding: May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Richard Yu and Carol Yu, Michael Seak-Kan Tong, Respiratory Viral Research Foundation, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund, Chan Yin Chuen Memorial Charitable Foundation, Marina Man-Wai Lee, The Hong Kong Hainan Commercial Association South China Microbiology Research Fund, The Jessie & George Ho Charitable Foundation, Perfect Shape Medical, The Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Diseases and Research Capability on Antimicrobial Resistance for the Department of Health of the Hong Kong Special Administrative Region Government, The Theme-Based Research Scheme of the Research Grants Council, Sanming Project of Medicine in Shenzhen, and The High Level-Hospital Program, Health Commission of Guangdong Province, China.

On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID50]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 × 104 RNA copies/ml (range, 2.21 × 102 to 4.71 × 105 RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.

The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the most common causes of hand, foot, and mouth disease. Severe EV-A71 and CV-A16 infections may be associated with life-threatening complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Lipids are known to play critical roles in multiple stages of the virus replication cycle. The specific lipid profile induced upon virus infection is required for optimal virus replication. The perturbations in the host cell lipidomic profiles upon enterovirus infection have not been fully characterized. To this end, we performed ultra-high performance liquid chromatography&ndash, electrospray ionization&ndash, quadrupole&ndash, time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS)-based lipidomics to characterize the change in host lipidome upon EV-A71 and CV-A16 infections. Our results revealed that 47 lipids within 11 lipid classes were significantly perturbed after EV-A71 and CV-A16 infection. Four polyunsaturated fatty acids (PUFAs), namely, arachidonic acid (AA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), were consistently upregulated upon EV-A71 and CV-A16 infection. Importantly, exogenously supplying three of these four PUFAs, including AA, DHA, and EPA, in cell cultures significantly reduced EV-A71 and CV-A16 replication. Taken together, our results suggested that enteroviruses might specifically modulate the host lipid pathways for optimal virus replication. Excessive exogenous addition of lipids that disrupted this delicate homeostatic state could prevent efficient viral replication. Precise manipulation of the host lipid profile might be a potential host-targeting antiviral strategy for enterovirus infection.

Objectives We assessed the immunological response of hospitalized adult patients with rhinovirus infection, including critically-ill patients. Methods The differential white blood cell (WBC) count and the levels of 29 plasma cytokines/chemokines were compared between 50 adult hospitalized patients with rhinovirus infection and 100 age-matched controls with influenza virus infection. Results The demographics and comorbidities were similar between rhinovirus and influenza patients, but severe disease was more common for the rhinovirus cohort. Rhinovirus patients had significantly higher WBC counts than influenza patients, especially for eosinophil ( P = 3.1 × 10 −8 ). The level of the T H 2 cytokine IL-5 was significantly higher among rhinovirus patients, while the levels of 9 other cytokines/chemokines were significantly lower among rhinovirus patients. The levels of CXCL-10 (IP-10), CCL-2 (MCP-1), IFN-α2, IFN-γ, IL-10, and IL-15 remained significantly lower among rhinovirus patients after correction for multiple comparisons. Notably, CXCL-10 had the highest area under the receiver operating characteristic curve (AUC) in differentiating rhinovirus from influenza patients (AUC, 0.918). In the patient subgroup without asthma, the difference in the WBC count and cytokine/chemokine levels between rhinovirus and influenza patients remained statistically significant. Conclusions Rhinovirus infection was characterized by a prominent T H 2 response, even in patients without asthma. CXCL-10 (IP-10) is a potential biomarker in differentiating rhinovirus from influenza infection.

Japanese encephalitis virus (JEV) is a mosquitoborne virus endemic to China and Southeast Asia that causes severe encephalitis in

Background HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar® HHV-6 PCR Kit. Method The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Results Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 copies/ml and a coefficient of determination (R2) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar® HHV-6 PCR Kit (R2 = 0.926; P Conclusions The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar® HHV-6 PCR Kit.

Rhinovirus has been neglected in the past because it was generally perceived as a respiratory virus only capable of causing mild common cold. Contemporary epidemiological studies using molecular assays have shown that rhinovirus is frequently detected in adult and pediatric patients with upper or lower respiratory tract infections. Severe pulmonary and extrapulmonary complications are increasingly recognized. Contrary to popular belief, some rhinoviruses can actually replicate well at 37 °C and infect the lower airway in humans. The increasing availability of multiplex PCR panels allows rapid detection of rhinovirus and provides the opportunity for timely treatment and early recognition of outbreaks. Recent advances in the understanding of host factors for viral attachment and replication, and the host immunological response in both asthmatic and non-asthmatic individuals, have provided important insights into rhinovirus infection which are crucial in the development of antiviral treatment. The identification of novel drugs has been accelerated by repurposing clinically-approved drugs. As humoral antibodies induced by past exposure and vaccine antigen of a particular serotype cannot provide full coverage for all rhinovirus serotypes, novel vaccination strategies are required for inducing protective response against all rhinoviruses.

Emerging infectious diseases in humans are often caused by respiratory viruses such as pandemic or avian influenza viruses and novel coronaviruses. Microbiological testing for respiratory viruses i...

Post-translational modifications of host or viral proteins are key strategies exploited by viruses to support virus replication and counteract host immune response. SUMOylation is a post-translational modification process mediated by a family of ubiquitin-like proteins called small ubiquitin-like modifier (SUMO) proteins. Multiple sequence alignment of 78 representative flaviviruses showed that most (72/78, 92.3%) have a putative SUMO-interacting motif (SIM) at their non-structural 5 (NS5) protein&rsquo, s N-terminal domain. The putative SIM was highly conserved among 414 pre-epidemic and epidemic Zika virus (ZIKV) strains, with all of them having a putative SIM core amino acid sequence of VIDL (327/414, 79.0%) or VVDL (87/414, 21.0%). Molecular docking predicted that the hydrophobic SIM core residues bind to the &beta, 2 strand of the SUMO-1 protein, and the acidic residues flanking the core strengthen the binding through interactions with the basic surface of the SUMO protein. The SUMO inhibitor 2-D08 significantly reduced replication of flaviviruses and protected cells against ZIKV-induced cytopathic effects in vitro. A SIM-mutated ZIKV NS5 failed to efficiently suppress type I interferon signaling. Overall, these findings may suggest SUMO modification of the viral NS5 protein to be an evolutionarily conserved post-translational modification process among flaviviruses to enhance virus replication and suppress host antiviral response.

Previously, we reported the discovery of a novel canine picornavirus (CanPV) in the fecal sample of a dog. In this molecular epidemiology study, CanPV was detected in 15 (1.11%) of 1347 canine fecal samples from Hong Kong and one (0.76%) of 131 canine fecal samples from Dubai, with viral loads 1.06×10(3) to 6.64×10(6) copies/ml. Complete genome sequencing and phylogenetic analysis showed that CanPV was clustered with feline picornavirus (FePV), bat picornavirus (BatPV) 1 to 3, Ia io picornavirus 1 (IaioPV1) and bovine picornavirus (BoPV), and this cluster was most closely related to the genera Enterovirus and Sapelovirus. The Ka/Ks ratios of all the coding regions were

Zika virus (ZIKV) is unique among human-pathogenic flaviviruses by its association with congenital anomalies and trans-placental and sexual human-to-human transmission. Although the pathogenesis of ZIKV-associated neurological complications has been reported in recent studies, key questions on the pathogenesis of the other clinical manifestations, non-vector-borne transmission and potential animal reservoirs of ZIKV remain unanswered. We systematically characterized the differential cell line susceptibility of 18 human and 15 nonhuman cell lines to two ZIKV isolates (human and primate) and dengue virus type 2 (DENV-2). Productive ZIKV replication (⩾2 log increase in viral load, ZIKV nonstructural protein-1 (NS1) protein expression and cytopathic effects (CPE)) was found in the placental (JEG-3), neuronal (SF268), muscle (RD), retinal (ARPE19), pulmonary (Hep-2 and HFL), colonic (Caco-2),and hepatic (Huh-7) cell lines. These findings helped to explain the trans-placental transmission and other clinical manifestations of ZIKV. Notably, the prostatic (LNCaP), testicular (833KE) and renal (HEK) cell lines showed increased ZIKV load and/or NS1 protein expression without inducing CPE, suggesting their potential roles in sexual transmission with persistent viral replication at these anatomical sites. Comparatively, none of the placental and genital tract cell lines allowed efficient DENV-2 replication. Among the nonhuman cell lines, nonhuman primate (Vero and LLC-MK2), pig (PK-15), rabbit (RK-13), hamster (BHK21) and chicken (DF-1) cell lines supported productive ZIKV replication. These animal species may be important reservoirs and/or potential animal models for ZIKV. The findings in our study help to explain the viral shedding pattern, transmission and pathogenesis of the rapidly disseminating ZIKV, and are useful for optimizing laboratory diagnostics and studies on the pathogenesis and counter-measures of ZIKV.

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

Background Zika virus (ZIKV) infection may be associated with severe complications and disseminated via both vector-borne and nonvector-borne routes. Adenovirus-vectored vaccines represent a favorable controlling measure for the ZIKV epidemic because they have been shown to be safe, immunogenic, and rapidly generable for other emerging viral infections. Evaluations of 2 previously reported adenovirus-vectored ZIKV vaccines were performed using nonlethal animal models and/or nonepidemic ZIKV strain. Methods We constructed 2 novel human adenovirus 5 (Ad5)-vectored vaccines containing the ZIKV premembrane-envelope (Ad5-Sig-prM-Env) and envelope (Ad5-Env) proteins, respectively, and evaluated them in multiple nonlethal and lethal animal models using epidemic ZIKV strains. Results Both vaccines elicited robust humoral and cellular immune responses in immunocompetent BALB/c mice. Dexamethasone-immunosuppressed mice vaccinated with either vaccine demonstrated robust and durable antibody responses and significantly lower blood and tissue viral loads than controls (P < .05). Similar findings were also observed in interferon-α/β receptor-deficient A129 mice. In both of these immunocompromised animal models, Ad5-Sig-prM-Env-vaccinated mice had significantly (P < .05) higher titers of anti-ZIKV-specific neutralizing antibody titers and lower (undetectable) viral loads than Ad5-Env-vaccinated mice. The close correlation between the neutralizing antibody titer and viral load helped to explain the better protective effect of Ad5-Sig-prM-Env than Ad5-Env. Anamnestic response was absent in Ad5-Sig-prM-Env-vaccinated A129 mice. Conclusions Ad5-Sig-prM-Env provided sterilizing protection against ZIKV infection in mice.

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

In 2013, a novel betacoronavirus was identified in fecal samples from dromedaries in Dubai, United Arab Emirates. Antibodies against the recombinant nucleocapsid protein of the virus, which we named dromedary camel coronavirus (DcCoV) UAE-HKU23, were detected in 52% of 59 dromedary serum samples tested. In an analysis of 3 complete DcCoV UAE-HKU23 genomes, we identified the virus as a betacoronavirus in lineage A1. The DcCoV UAE-HKU23 genome has G+C contents; a general preference for G/C in the third position of codons; a cleavage site for spike protein; and a membrane protein of similar length to that of other betacoronavirus A1 members, to which DcCoV UAE-HKU23 is phylogenetically closely related. Along with this coronavirus, viruses of at least 8 other families have been found to infect camels. Because camels have a close association with humans, continuous surveillance should be conducted to understand the potential for virus emergence in camels and for virus transmission to humans.

A natural recombinant of coxsackievirus A2 was found in 4 children with respiratory symptoms in Hong Kong, China, during the summer of 2012. Two of these children died. Vigilant monitoring of this emerging recombinant enterovirus is needed to prevent its transmission to other regions.

A broad range of viral and bacterial pathogens can cause acute respiratory tract infection. For rapid detection of a broad respiratory pathogen spectrum, multiplex real-time PCR is ideal. This study evaluated the performance of the new Luminex NxTAG Respiratory Pathogen Panel (NxTAG-RPP) in comparison with the BioFire FilmArray Respiratory Panel (FA-RP) or singleplex real-time PCR as reference. A total of 284 clinical respiratory specimens and 3 influenza A/H7N9 viral culture samples were tested. All clinical specimens were processed and analyzed in parallel using NxTAG-RPP and the reference standard method. The H7N9 viral culture samples were tested using NxTAG-RPP only. Overall, the NxTAG-RPP demonstrated ≥93% sensitivity and specificity for all respiratory targets except human coronavirus OC43 (HCoV-OC43) and HCoV-HKU1. The H7N9 virus was detected by the influenza A virus matrix gene target, while other influenza A virus subtyping gene targets in the panel remained negative. Complete concordance between NxTAG-RPP and FA-RP was observed in 98.8% (318/322) of positive results (kappa = 0.92). Substantial agreement was found for most respiratory targets, but significant differences were observed in human metapneumovirus ( P = 0.001) and parainfluenza virus type 3 ( P = 0.031). NxTAG-RPP has a higher sample throughput than FA-RP (96 samples versus 1 sample per run) while the turnaround times for NxTAG-RPP and FA-RP were 5 h (up to 96 samples) and 1 h (for one sample), respectively. Overall, NxTAG-RPP demonstrated good diagnostic performance for most respiratory pathogens. The high sample throughput with reasonable turnaround time of this new assay makes it a suitable multiplex platform for routine screening of respiratory specimens in hospital-based laboratories.

While picornaviruses are known to infect different animals, their existence in the domestic cat was unknown. We describe the discovery of a novel feline picornavirus (FePV) from stray cats in Hong Kong. From samples from 662 cats, FePV was detected in fecal samples from 14 cats and urine samples from 2 cats by reverse transcription-PCR (RT-PCR). Analysis of five FePV genomes revealed a distinct phylogenetic position and genomic features, with low sequence homologies to known picornaviruses especially in leader and 2A proteins. Among the viruses that belong to the closely related bat picornavirus groups 1 to 3 and the genus Sapelovirus, G+C content and sequence analysis of P1, P2, and P3 regions showed that FePV is most closely related to bat picornavirus group 3. However, FePV possessed other distinct features, including a putative type IV internal ribosome entry site/segment (IRES) instead of type I IRES in bat picornavirus group 3, protein cleavage sites, and H-D-C catalytic triad in 3C pro different from those in sapeloviruses and bat picornaviruses, and the shortest leader protein among known picornaviruses. These results suggest that FePV may belong to a new genus in the family Picornaviridae. Western blot analysis using recombinant FePV VP1 polypeptide showed a high seroprevalence of 33.6% for IgG among the plasma samples from 232 cats tested. IgM was also detected in three cats positive for FePV in fecal samples, supporting recent infection in these cats. Further studies are important to understand the pathogenicity, epidemiology, and genetic evolution of FePV in these common pet animals. © 2012, American Society for Microbiology., link_to_OA_fulltext

Although human coronavirus OC43-OC43 (HCoV-OC43) is the coronavirus most commonly associated with human infections, little is known about its molecular epidemiology and evolution. We conducted a molecular epidemiology study to investigate different genotypes and potential recombination in HCoV-OC43. Twenty-nine HCoV-OC43 strains from nasopharyngeal aspirates, collected from 2004 to 2011, were subjected to RNA-dependent RNA polymerase (RdRp), spike, and nucleocapsid gene analysis. Phylogenetic analysis showed at least three distinct clusters of HCoV-OC43, although 10 unusual strains displayed incongruent phylogenetic positions between RdRp and spike genes. This suggested the presence of four HCoV-OC43 genotypes (A to D), with genotype D most likely arising from recombination. The complete genome sequencing of two genotype C and D strains and bootscan analysis showed recombination events between genotypes B and C in the generation of genotype D. Of the 29 strains, none belonged to the more ancient genotype A, 5 from 2004 belonged to genotype B, 15 from 2004 to 2006 belonged to genotype C, and 1 from 2004 and all 8 from 2008 to 2011 belonged to the recombinant genotype D. Molecular clock analysis using spike and nucleocapsid genes dated the most recent common ancestor of all genotypes to the 1950s, genotype B and C to the 1980s, genotype B to the 1990s, and genotype C to the late 1990s to early 2000s, while the recombinant genotype D strains were detected as early as 2004. This represents the first study to describe natural recombination in HCoV-OC43 and the evolution of different genotypes over time, leading to the emergence of novel genotype D, which is associated with pneumonia in our elderly population. © 2011, American Society for Microbiology., link_to_OA_fulltext

The family Adenoviridae includes a group of icosahedral nonenveloped viruses containing a double-stranded DNA genome. Members of this family are classified into two previously established genera, Mastadenovirus and Aviadenovirus , comprising mammalian and avian isolates, respectively, and three recently accepted genera, Atadenovirus, Siadenovirus , and Ichtadenovirus , comprising a broad range of hosts including mammalian, amphibian, reptile, and fish isolates. Human adenoviruses (HAdVs) are located in the genus Mastadenovirus and include 54 serotypes that can be divided into seven species subgroups (A, B, C, D, E, F, and G). The genomes of serotypes within a species are highly related and show modest divergence between species. 1, 2 Adenoviruses in humans cause a broad spectrum of diseases including respiratory tract infections, acute conjunctivitis, cystitis, gastroenteritis and systemic infections in immunocompromised patients. 1 Polymerase chain reaction (PCR)–based diagnostic assays have made it possible to detect adenoviruses by family, serotype, or species, with good sensitivity. Pan adenovirus family PCR primers are specific for the highly conserved regions in hexon gene of known members of the family Adenovirida e and enable detection of known and previously unrecognized adenoviruses. We show the utility of the pan adenovirus family PCR approach in identification of two distinctive adenoviruses from wild chimpanzees in western Tanzania. M group chimpanzees ( Pan troglodytes schweinfurthii ) at Mahale Mountains National Park in Tanzania are accustomed to humans and tolerate observation from close proximity for extended periods. We previously demonstrated that a human metapneumovirus was the likely causative agent associated with an acute, fatal respiratory disease outbreak in this group. 3

After human coronaviruses OC43, 229E and NL63, human coronavirus HKU1 (HCoV-HKU1) is the fourth human coronavirus discovered. HCoV-HKU1 is a group 2a coronavirus that is still not cultivable. The G + C contents of HCoV-HKU1 genomes are 32%, the lowest among all known coronaviruses with complete genome sequences available. Among all coronaviruses, HCoV-HKU1 shows the most extreme codon usage bias, attributed most importantly to severe cytosine deamination. All HCoV-HKU1 genomes contain unique tandem copies of a 30-base acidic tandem repeat of unknown function at the N-terminus of nsp3 inside the acidic domain upstream of papain-like protease 1. Three genotypes, A, B and C, of HCoV-HKU1 and homologous recombination among their genomes, are observed. The incidence of HCoV-HKU1 infections is the highest in winter. Similar to other human coronaviruses, HCoV-HKU1 infections have been reported globally, with a median (range) incidence of 0.9 (0 - 4.4) %. HCoV-HKU1 is associated with both upper and lower respiratory tract infections that are mostly self-limiting. The most common method for diagnosing HCoV-HKU1 infection is RT-PCR or real-time RT-PCR using RNA extracted from respiratory tract samples such as nasopharyngeal aspirates (NPA). Both the pol and nucleocapsid genes have been used as the targets for amplification. Monoclonal antibodies have been generated for direct antigen detection in NPA. For antibody detection, Escherichia coli BL21 and baculovirus-expressed recombinant nucleocapsid of HCoV-HKU1 have been used for IgG and IgM detection in sera of patients and normal individuals, using Western blot and enzyme-linked immunoassay., published_or_final_version

The recent emergence of Middle East respiratory syndrome coronavirus from the Middle East and the discovery of the virus from dromedary camels have boosted interest in the search for novel viruses in dromedaries. Whilst picornaviruses are known to infect various animals, their existence in dromedaries was unknown. We describe the discovery of a novel picornavirus, dromedary camel enterovirus (DcEV), from dromedaries in Dubai. Among 215 dromedaries, DcEV was detected in faecal samples of four (1.9 %) dromedaries [one (0.5 %) adult dromedary and three (25 %) dromedary calves] by reverse transcription PCR. Analysis of two DcEV genomes showed that DcEV was clustered with other species of the genus Enterovirus and was most closely related to and possessed highest amino acid identities to the species Enterovirus E and Enterovirus F found in cattle. The G+C content of DcEV was 45 mol%, which differed from that of Enterovirus E and Enterovirus F (49–50 mol%) by 4–5 %. Similar to other members of the genus Enterovirus, the 5′ UTR of DcEV possessed a putative type I internal ribosome entry site. The low ratios of the number of nonsynonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (K a/K s) of various coding regions suggested that dromedaries are the natural reservoir in which DcEV has been stably evolving. These results suggest that DcEV is a novel species of the genus Enterovirus in the family Picornaviridae. Western blot analysis using recombinant DcEV VP1 polypeptide showed a high seroprevalence of 52 % among serum samples from 172 dromedaries for IgG, concurring with its much higher infection rates in dromedary calves than in adults. Further studies are important to understand the pathogenicity, epidemiology and genetic evolution of DcEV in this unique group of animals.

We have recently described the discovery of a novel coronavirus, corona virus HKU1 (CoV-HKU1), associated with community-acquired pneumonia. However, the clinical spectrum of disease and the epidemiology of CoV-HKU1 infections in relation to infections with other respiratory viruses are unknown. In this 12-month prospective study, 4,181 nasopharyngeal aspirates from patients with acute respiratory tract infections were subjected to reverse transcription-PCRs specific for CoV-HKU1 and human coronaviruses NL63 (HCoV-NL63), OC43 (HCoV-OC43), and 229E (HCoV-229E). Coronaviruses were detected in 87 (2.1%) patients, with 13 (03%) positive for CoV-HKU1, 17 (0.4%) positive for HCoV-NL63,53 (13%) positive for HCoV-OC43, and 4 (0.1%) positive for HCoV-229E. Of the 13 patients with CoV-HKU1 infections, 11 were children and 8 had underlying diseases. Similar to the case for other coronaviruses, upper respiratory infection was the most common presentation of CoV-HKU1 infections, although pneumonia, acute bronchiolitis, and asthmatic exacerbation also occurred. Despite a shorter duration of fever (mean, 1.7 days) and no difference in maximum temperature in children with CoV-HKU1 infections compared to patients with most other respiratory virus infections, a high incidence of febrile seizures (50%) was noted, which was significantly higher than those for HCoV-OC43 (14%), adenovirus (9%), human parainfluenza virus 1 (0%), and respiratory syncytial virus (8%) infections. CoV-HKU1 and HCoV-OC43 infections peaked in winter, although cases of the former also occurred in spring to early summer. This is in contrast to HCoV-NL63 infections, which mainly occurred in early summer and autumn but were absent in winter. Two genotypes of CoV-HKU1 cocirculated during the study period. Continuous studies over a longer period are warranted to ascertain the seasonal variation and relative importance of the different coronaviruses. Similar studies in other countries are required to better determine the epidemiology and genetic diversity of CoV-HKU1. Copyright © 2006, American Society for Microbiology. All Rights Reserved., published_or_final_version

Background Emerging human picornaviruses, including human parechovirus (HPeV), Aichi virus (AiV) and salivirus (SalV) were found to be associated with gastroenteritis, but their roles in enteric infections are not fully understood. In addition, no report on the circulation of these viruses in Hong Kong is available. The objective of this study was to investigate the prevalence and genetic diversity of HPeV, AiV and SalV in fecal samples from hospitalized children with gastroenteritis in Hong Kong. Methods Fecal samples from hospitalized children with gastroenteritis were subject to detection of HPeV, AiV and SalV by RT-PCR using consensus primers targeted to their 5′UTRs. Positive samples were subject to capsid and/or 3CD region analysis for genotype determination. The epidemiology of HPeV, AiV and SalV infections was analyzed. Results Among 1,708 fecal samples subjected to RT-PCR using primers targeted to 5′UTR of HPeV, AiV and SalV, viruses were detected in 55 samples, with 50 positive for HPeV only, 3 positive for AiV only, 1 positive for both HPeV and AiV, and 1 positive for both HPeV and SalV. Phylogenetic analysis of the partial VP1 gene of the 33 HPeV strains revealed the presence of genotypes of HPeV- 1, 3, 4, 5, 7, 10, among which HPeV-1 was the predominant genotype circulating in our population. The peak activity of HPeV infection was in fall. Of the 3 children with AiV infection, the 3 AiV strains were found to belong to genotype A based on the phylogenetic analysis of their partial VP1 and 3CD regions. The genotype of a SalV strain detected in this study could not be determined. Co-detection of different pathogens was observed in 24 samples (43.6%) of 55 fecal samples positive for HPeV, AiV and SalV. Conclusions HPeV, AiV and SalV were detected in fecal samples of hospitalized children with gastroenteritis in Hong Kong, with the former having the highest prevalence. HPeV-1 was the predominant genotype among HPeVs, while genotype A was the predominant genotype among AiVs in this study.

While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in poultry. We discovered a previously unknown diversity of picornaviruses in chickens in Hong Kong. Picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by reverse transcription-PCR, with their partial 3Dpol gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornaviruses, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (Avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses, especially in the P1 and P2 regions, including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and Duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses that may cross avian species barriers through mutation or recombination.

We report the first complete genome sequence of a feline astrovirus (FAstV), FAstV2 strain 1637F, identified from a domestic cat. The genome is 6,779 nucleotides (nt) in length and consists of three overlapping open reading frames (ORF1a-ORF1b-ORF2). Sequence analysis suggests that FAstV2 represents a new FAstV genotype that is closely related to human astroviruses.

While novel picornaviruses are being discovered in rodents, their host range and pathogenicity are largely unknown. We identified two novel picornaviruses, rosavirus B from the street rat, Norway rat, and rosavirus C from five different wild rat species (chestnut spiny rat, greater bandicoot rat, Indochinese forest rat, roof rat and Coxing's white-bellied rat) in China. Analysis of 13 complete genome sequences showed that “Rosavirus B” and “Rosavirus C” represent two potentially novel picornavirus species infecting different rodents. Though being most closely related to rosavirus A, rosavirus B and C possessed distinct protease cleavage sites and variations in Yn-Xm-AUG sequence in 5’UTR and myristylation site in VP4. Anti-rosavirus B VP1 antibodies were detected in Norway rats, whereas anti-rosavirus C VP1 and neutralizing antibodies were detected in Indochinese forest rats and Coxing's white-bellied rats. While the highest prevalence was observed in Coxing's white-bellied rats by RT-PCR, the detection of rosavirus C from different rat species suggests potential interspecies transmission. Rosavirus C isolated from 3T3 cells causes multisystemic diseases in a mouse model, with high viral loads and positive viral antigen expression in organs of infected mice after oral or intracerebral inoculation. Histological examination revealed alveolar fluid exudation, interstitial infiltration, alveolar fluid exudate and wall thickening in lungs, and hepatocyte degeneration and lymphocytic/monocytic inflammatory infiltrates with giant cell formation in liver sections of sacrificed mice. Since rosavirus A2 has been detected in fecal samples of children, further studies should elucidate the pathogenicity and emergence potential of different rosaviruses., Author Summary We identified two novel picornaviruses, rosavirus B and C, infecting street and wild rats respectively in China. While rosavirus B was detected from Norway rats, rosavirus C was detected from five different wild rat species (chestnut spiny rat, greater bandicoot rat, Indochinese forest rat, roof rat and Coxing's white-bellied rat) by RT-PCR. Anti-rosavirus B antibodies were detected in Norway rats, whereas anti-rosavirus C antibodies were detected in Indochinese forest rats and Coxing's white-bellied rats, supporting potential interspecies transmission of rosavirus C. Genome analysis supported the classification of rosavirus B and C as two novel picornavirus species, with genome features distinct from rosavirus A. Rosavirus C isolated from 3T3 cells causes multisystemic diseases in a mouse model, with viruses and pathologies detected in various organs of infected mice after oral or intracerebral inoculation. Our results extend our knowledge on the host range and pathogenicity of rodent picornaviruses.

We discovered a novel canine picornavirus in fecal, nasopharyngeal, and urine samples from dogs. The coding potential of its genome (5'-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C pro-3D pol-3', where 3C pro is 3' protease and 3D pol is 3D polymerase) is similar to those of other picornaviruses, with putative P1, P2, and P3 sharing 54% to 58%, 60%, and 64% to 67% amino acid identities with bat picornavirus groups 1, 2, and 3. © 2012, American Society for Microbiology., link_to_OA_fulltext

Background Despite recent discovery of the novel human rhinovirus species, HRV-C, little is known about the association of HRV-C in diseases other than respiratory tract infections. Objectives To investigate the presence of HRV-C in fecal samples of children with gastroenteritis. Study design 734 fecal samples from hospitalized children with gastroenteritis were subject to picornavirus detection by RT-PCR of the conserved 5′-NCR. Positive samples were subject to VP4 and 3D pol gene analysis for species determination. The clinical and molecular epidemiology of HRV-C and other picornaviruses was analyzed. Results Picornaviruses were detected in 113 (15.4%) of 734 fecal samples from children with gastroenteritis by RT-PCR of 5′-NCR, with 58 containing potential HRVs and 55 containing other enteroviruses. PCR of the VP4 and 3D pol regions was positive in 21 and 19 samples respectively (both regions positive in 8 samples). Sequencing analysis showed the presence of HRV-C in four samples, and diverse picornaviruses including HRV-A ( n = 2), HEV-A ( n = 2), HEV-B ( n = 2), HEV-C ( n = 21) and HPeV ( n = 2) in other samples, with co-detection of HRV-C and HPeV in one sample. Of the four children with HRV-C detected in fecal samples, three presented with diarrhea in the absence of respiratory symptoms, while one also had acute bronchiolitis. The four HRV-C strains from fecal samples belonged to the existing clade of diverse HRV-C genotypes, indistinguishable from previous respiratory strains. Conclusions HRV-C can be detected in fecal samples of children with gastroenteritis, in the absence of respiratory symptoms. This study also represented the first to detect HPeV in our population.

Although bats are important reservoirs of diverse viruses that can cause human epidemics, little is known about the presence of picornaviruses in these flying mammals. Among 1,108 bats of 18 species studied, three novel picornaviruses (groups 1, 2, and 3) were identified from alimentary specimens of 12 bats from five species and four genera. Two complete genomes, each from the three picornaviruses, were sequenced. Phylogenetic analysis showed that they fell into three distinct clusters in the Picornaviridae family, with low homologies to known picornaviruses, especially in leader and 2A proteins. Moreover, group 1 and 2 viruses are more closely related to each other than to group 3 viruses, which exhibit genome features distinct from those of the former two virus groups. In particular, the group 3 virus genome contains the shortest leader protein within Picornaviridae, a putative type I internal ribosome entry site (IRES) in the 5′-untranslated region instead of the type IV IRES found in group 1 and 2 viruses, one instead of two GXCG motifs in 2A, an L→V substitution in the DDLXQ motif in 2C helicase, and a conserved GXH motif in 3C protease. Group 1 and 2 viruses are unique among picornaviruses in having AMH instead of the GXH motif in 3C pro. These findings suggest that the three picornaviruses belong to two novel genera in the Picornaviridae family. This report describes the discovery and complete genome analysis of three picornaviruses in bats, and their presence in diverse bat genera/species suggests the ability to cross the species barrier. © 2011, American Society for Microbiology., link_to_OA_fulltext

Background. Human bocavirus (HBoV) is a recently discovered parvovirus associated with respiratory tract infections in children. We conducted the first systematic prospective clinical and molecular study using nasopharyngeal aspirates (NPAs) and fecal samples. Methods. NPAs negative for influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, and coronavirus and fecal samples from patients with acute gastroenteritis were included. On the basis of results from a pilot study using 400 NPAs from all age groups, a prospective 12-month study was conducted to detect HBoV in 1200 NPAs and 1435 fecal samples from patients