Andes Virus Genome Mutations That Are Likely Associated with Animal Model Attenuation and Human Person-to-Person Transmission.

We performed whole-genome sequencing with bait enrichment techniques to analyze Andes virus (ANDV), a cause of human hantavirus pulmonary syndrome. We used cryopreserved lung tissues from a naturally infected long-tailed colilargo, including early, intermediate, and late cell culture, passages of an ANDV isolate from that animal, and lung tissues from golden hamsters experimentally exposed to that ANDV isolate. The resulting complete genome sequences were subjected to detailed comparative genomic analysis against American orthohantaviruses. We identified four amino acid substitutions related to cell culture adaptation that resulted in attenuation of ANDV in the typically lethal golden hamster animal model of hantavirus pulmonary syndrome. Changes in the ANDV nucleocapsid protein, glycoprotein, and small nonstructural protein open reading frames correlated with mutations typical for ANDV strains associated with increased virulence in the small-animal model. Finally, we identified three amino acid substitutions, two in the small nonstructural protein and one in the glycoprotein, that were only present in the clade of viruses associated with efficient person-to-person transmission. Our results indicate that there are single-nucleotide polymorphisms that could be used to predict strain-specific ANDV virulence and/or transmissibility. IMPORTANCE Several orthohantaviruses cause the zoonotic disease hantavirus pulmonary syndrome (HPS) in the Americas. Among them, HPS caused by Andes virus (ANDV) is of great public health concern because it is associated with the highest case fatality rate (up to 50%). ANDV is also the only orthohantavirus associated with relatively robust evidence of person-to-person transmission. This work reveals nucleotide changes in the ANDV genome that are associated with virulence attenuation in an animal model and increased transmissibility in humans. These findings may pave the way to early severity predictions in future ANDV-caused HPS outbreaks.

Novel viruses in hard ticks collected in the Republic of Korea unveiled by metagenomic high-throughput sequencing analysis.

Ticks are vectors of a wide range of zoonotic viruses of medical and veterinary importance. Recently, metagenomics studies demonstrated that they are also the source of potentially pathogenic novel viruses. During the period from 2015 to 2017, questing ticks were collected by dragging the vegetation from geographically distant locations in the Republic of Korea (ROK) and a target-independent high-throughput sequencing method was utilized to study their virome. A total of seven viruses, including six putative novel viral entities, were identified. Genomic analysis showed that the novel viruses were most closely related to members in the orders Jingchuvirales and Bunyavirales. Phylogenetic reconstruction showed that the Bunyavirales-like viruses grouped in the same clade with other viruses within the Nairovirus and Phlebovirus genera, while the novel Jingchuvirales-like virus grouped together with other viruses within the family Chuviridae. Real-time RT-PCR was used to determine the geographic distribution and prevalence of these viruses in adult ticks. These novel viruses have a wide geographic distribution in the ROK with prevalences ranging from 2% to 18%. Our study expands the knowledge about the composition of the tick virome and highlights the wide diversity of viruses they harbor in the ROK. The discovery of novel viruses associated with ticks in the ROK highlights the need for an active tick-borne disease surveillance program to identify possible reservoirs of putative novel human pathogens.

"Super-Spreaders" and Person-to-Person Transmission of Andes Virus in Argentina.

BACKGROUND: From November 2018 through February 2019, person-to-person transmission of Andes virus (ANDV) hantavirus pulmonary syndrome occurred in Chubut Province, Argentina, and resulted in 34 confirmed infections and 11 deaths. Understanding the genomic, epidemiologic, and clinical characteristics of person-to-person transmission of ANDV is crucial to designing effective interventions. METHODS: Clinical and epidemiologic information was obtained by means of patient report and from public health centers. Serologic testing, contact-tracing, and next-generation sequencing were used to identify ANDV infection as the cause of this outbreak of hantavirus pulmonary syndrome and to reconstruct person-to-person transmission events. RESULTS: After a single introduction of ANDV from a rodent reservoir into the human population, transmission was driven by 3 symptomatic persons who attended crowded social events. After 18 cases were confirmed, public health officials enforced isolation of persons with confirmed cases and self-quarantine of possible contacts; these measures most likely curtailed further spread. The median reproductive number (the number of secondary cases caused by an infected person during the infectious period) was 2.12 before the control measures were enforced and decreased to 0.96 after the measures were implemented. Full genome sequencing of the ANDV strain involved in this outbreak was performed with specimens from 27 patients and showed that the strain that was present (Epuyén/18-19) was similar to the causative strain (Epilink/96) in the first known person-to-person transmission of hantavirus pulmonary syndrome caused by ANDV, which occurred in El Bolsón, Argentina, in 1996. Clinical investigations involving patients with ANDV hantavirus pulmonary syndrome in this outbreak revealed that patients with a high viral load and liver injury were more likely than other patients to spread infection. Disease severity, genomic diversity, age, and time spent in the hospital had no clear association with secondary transmission. CONCLUSIONS: Among patients with ANDV hantavirus pulmonary syndrome, high viral titers in combination with attendance at massive social gatherings or extensive contact among persons were associated with a higher likelihood of transmission. (Funded by the Ministerio de Salud y Desarrollo Social de la Nación Argentina and others.).

A Model for the Production of Regulatory Grade Viral Hemorrhagic Fever Exposure Stocks: From Field Surveillance to Advanced Characterization of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging human pathogen, endemic in areas of China, Japan, and the Korea (KOR). It is primarily transmitted through infected ticks and can cause a severe hemorrhagic fever disease with case fatality rates as high as 30%. Despite its high virulence and increasing prevalence, molecular and functional studies in situ are scarce due to the limited availability of high-titer SFTSV exposure stocks. During the course of field virologic surveillance in 2017, we detected SFTSV in ticks and in a symptomatic soldier in a KOR Army training area. SFTSV was isolated from the ticks producing a high-titer viral exposure stock. Through the use of advanced genomic tools, we present here a complete, in-depth characterization of this viral stock, including a comparison with both the virus in its arthropod source and in the human case, and an in vivo study of its pathogenicity. Thanks to this detailed characterization, this SFTSV viral exposure stock constitutes a quality biological tool for the study of this viral agent and for the development of medical countermeasures, fulfilling the requirements of the main regulatory agencies.

Modeling mosquito-borne and sexual transmission of Zika virus in an enzootic host, the African green monkey.

Mosquito-borne and sexual transmission of Zika virus (ZIKV), a TORCH pathogen, recently initiated a series of large epidemics throughout the Tropics. Animal models are necessary to determine transmission risk and study pathogenesis, as well screen antivirals and vaccine candidates. In this study, we modeled mosquito and sexual transmission of ZIKV in the African green monkey (AGM). Following subcutaneous, intravaginal or intrarectal inoculation of AGMs with ZIKV, we determined the transmission potential and infection dynamics of the virus. AGMs inoculated by all three transmission routes exhibited viremia and viral shedding followed by strong virus neutralizing antibody responses, in the absence of clinical illness. All four of the subcutaneously inoculated AGMs became infected (mean peak viremia: 2.9 log10 PFU/mL, mean duration: 4.3 days) and vRNA was detected in their oral swabs, with infectious virus being detected in a subset of these specimens. Although all four of the intravaginally inoculated AGMs developed virus neutralizing antibody responses, only three had detectable viremia (mean peak viremia: 4.0 log10 PFU/mL, mean duration: 3.0 days). These three AGMs also had vRNA and infectious virus detected in both oral and vaginal swabs. Two of the four intrarectally inoculated AGMs became infected (mean peak viremia: 3.8 log10 PFU/mL, mean duration: 3.5 days). vRNA was detected in oral swabs collected from both of these infected AGMs, and infectious virus was detected in an oral swab from one of these AGMs. Notably, vRNA and infectious virus were detected in vaginal swabs collected from the infected female AGM (peak viral load: 7.5 log10 copies/mL, peak titer: 3.8 log10 PFU/mL, range of detection: 5-21 days post infection). Abnormal clinical chemistry and hematology results were detected and acute lymphadenopathy was observed in some AGMs. Infection dynamics in all three AGM ZIKV models are similar to those reported in the majority of human ZIKV infections. Our results indicate that the AGM can be used as a surrogate to model mosquito or sexual ZIKV transmission and infection. Furthermore, our results suggest that AGMs are likely involved in the enzootic maintenance and amplification cycle of ZIKV.

Person-to-Person Transmission of Andes Virus in Hantavirus Pulmonary Syndrome, Argentina, 2014.

Andes virus is unique among hantaviruses because it can be transmitted from person to person. This mechanism was previously supported by epidemiologic data and genetic evidence based only on partial sequences. We used full-length virus sequencing to confirm person-to-person transmission of this virus in a cluster of 3 cases in Argentina in 2014.

Target-independent high-throughput sequencing methods provide evidence that already known human viral pathogens play a main role in respiratory infections with unexplained etiology.

Despite the advanced PCR-based assays available, a fraction of the pediatric respiratory infections remain unexplained every epidemic season, and there is a perception that novel viruses might be present in these specimens. We systematically collected samples from a prospective cohort of pediatric patients with respiratory infections, that returned negative results by validated molecular RT-PCR assays, and studied them with a target-independent, high-throughput sequencing-based approach. We also included a matched cohort of children with no symptoms of respiratory infection, as a contrast study population. More than fifty percent of the specimens from the group of patients with unexplained respiratory infections were resolved. However, the higher rate of detection was not due to the presence of novel viruses, but to the identification of well-known viral respiratory pathogens. Our results show that already known viral pathogens are responsible for the majority of cases that remain unexplained after the epidemic season. High-throughput sequencing approaches that use pathogen-specific probes are easier to standardize because they ensure reproducible library enrichment and sequencing. In consequence, these techniques might be desirable from a regulatory standpoint for diagnostic laboratories seeking to benefit from the many advantages of these sequencing technologies.

Ribavirin Had Demonstrable Effects on the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Population and Load in a Patient With CCHF Infection.

The use of ribavirin to treat Crimean-Congo hemorrhagic fever virus (CCHFV) infection has been controversial, based on uncertainties about its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case by deep-sequencing analysis of plasma samples obtained from a CCHFV-infected patient before, during, and after a 5-day regimen of ribavirin treatment. The CCHFV load dropped during ribavirin treatment, and subclonal diversity (transitions) and indels increased in viral genomes during treatment. Although the results are based on a single case, these data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo.

High Infection Rates for Adult Macaques after Intravaginal or Intrarectal Inoculation with Zika Virus.

Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.

Respiratory Infections by Enterovirus D68 in Outpatients and Inpatients Spanish Children.

BACKGROUND: The incidence of enterovirus D68 (EV-D68) and the spectrum of clinical disease in children are not well known in European countries. We have designed a study with the objective of describing the clinical impact of EV-D68 detected in children with respiratory tract infections. METHODS: As a part of a prospective study to identify the etiology and clinical characteristics of viral respiratory infections in children in Spain, we performed the analysis of the cases of EV infections in all children hospitalized in a secondary hospital in Madrid, during the epidemic respiratory season 2012-2013. A second group of samples was corresponded to infants of the same area, with ambulatory respiratory infection or asymptomatic. Phylogenetic EV-D68 analysis was made using the viral protein 1 gene (VP1). Clinical data of EV-D68 patients were compared with those infected by rhinovirus in the same period and population. RESULTS: The study population consisted of 720 patients corresponding to 399 episodes of hospitalization for respiratory causes, 44 episodes of ambulatory respiratory infections and 277 children determined as a healthy control group. A total of 22 patients were positive for EVs (3.05%), and 12 of them were specifically typed as EV-D68 (11/443 respiratory infections, 2.5%). The most frequent diagnosis in the 10 hospitalized children with EV-D68 detection was recurrent wheezing. Hypoxia was present in 70% of cases, but admission in the intensive care unit was not required. No neurological signs or symptoms were observed. One patient had an ambulatory mild bronchiolitis and another was asymptomatic. No differences were found with rhinovirus infections except less duration of hypoxia and fever in EV-D68 group. CONCLUSIONS: EV-D68 infections were detected in 3.05% of respiratory studied samples (2.5% of admissions). The infection was associated with wheezing episodes with hypoxia. No admissions to intensive care unit or neurological symptoms were found.

Human norovirus occurrence and diversity in the Llobregat river catchment, Spain.

Human noroviruses (NoV) were quantified and characterized in an 18 month survey conducted along the Llobregat river catchment in Spain. Sample types included freshwater, untreated and treated wastewater and drinking water. High NoV genome copy numbers were reported, reaching up to 10(6) l(-1) and 10(9) l(-1) in freshwater and raw sewage respectively. In both types of samples, GII NoV genome copies outnumbered those of GI, although without significance. All samples of semi-treated and treated drinking water were negative for NoV. A clear seasonality of NoV occurrence was observed both in river water and sewage samples, with significantly higher genome copy numbers in the cold than in the warm months period. Mean NoV log reduction rates after biological treatment of sewage were 2.2 and 3.1 for GI and GII respectively. A total of 77 NoV strains isolated in the Llobregat river catchment could be phylogenetically characterized, 44 belonging to GI and 33 to GII. The most prevalent genotype was GI.4, followed by GII.4 and GII.21. Several variants of the pandemic GII.4 strain were detected in the environment, corroborating their circulation among the population.

Molecular epidemiology of hepatitis A virus infections in Catalonia, Spain, 2005-2009: circulation of newly emerging strains.

BACKGROUND: In spite of annual vaccination campaigns, hepatitis A cases increased in Catalonia (North-East Spain) in the period 2002-2005 calling for the elucidation of the underlying mechanisms associated to the epidemiological shifts. OBJECTIVE: The molecular characterization of the circulating strains to trace their origin and the study of the effects of vaccination on the incidence of sporadic and outbreak-associated cases. STUDY DESIGN: Forty-eight different hepatitis A virus (HAV) strains isolated from sporadic and outbreaks cases during 2005-2009 in Catalonia were molecularly characterized. RESULTS: Seventeen out of 48 strains were imported from endemic areas through traveling, immigration and food trade, 12 were endemic strains circulating in the men having sex with men (MSM) group and 1 was from a Roman child. The remaining 18 could not be associated to any specific origin and thus were considered autochthonous. Forty-eight percent of the strains belonged to subgenotype IA, 40% to subgenotype IB and 2% to subgenotype IIIA. The remaining 10% belonged to an undetermined subgenotype equidistant from IA and IB. CONCLUSIONS: During the period 2005-2009, the annual attack rates remained around 3.5 and even increased up to 6.5 in the first half of 2009. This increase with respect to the period 1999-2001, in which vaccination campaigns started to be implemented, is explained by an increase in the number of outbreaks. The predominant subgenotypes were IA and IB. However a considerable amount of strains imported from Peru through consumption of contaminated shellfish belonged to an undeterminded subgenotype that may constitute a new candidate subgenotype IC.

Hepatitis a virus vaccine escape variants and potential new serotype emergence.

Six hepatitis A virus antigenic variants that likely escaped the protective effect of available vaccines were isolated, mostly from men who have sex with men. The need to complete the proper vaccination schedules is critical, particularly in the immunocompromised population, to prevent the emergence of vaccine-escaping variants.

Quantification and genotyping of human sapoviruses in the Llobregat river catchment, Spain.

Human sapoviruses (SaVs) were quantified and characterized in an 18-month survey conducted along the Llobregat river catchment area in Spain. Sample types included freshwater, untreated and treated wastewater, and drinking water. All genogroups were recovered, and a seasonal distribution was observed. This is the first report of SaV quantification and genotyping in the environment outside Japan.