Subgenotype IB Research Articles

Exploring hepatitis A dynamics in Israel, 2019–2022

BackgroundContinuous monitoring of Hepatitis A Virus (HAV) may assist in identifying local outbreaks. The advent of the COVID-19 pandemic, which affected the circulation of numerous pathogens, may have also impacted the scope of HAV infections. AimTo investigate the incidence and environmental dissemination of HAV between 2019 and 2022 in Israel, a country with an anti-HAV vaccination program. MethodsHAV RT-PCR analysis was performed for all HAV cases and for 280 sewage samples collected in 2019–2022. Available amplified HAV fragments from clinical (n = 107) and sewage (n = 27) were also assessed by genotyping and phylogenetic analysis. ResultsIn 2019–2022, 158 individuals and 12.9 % (36/280) of sewage samples were HAV-RNA positive. Median age was 30 years (IQR 20.5–44); approximately half (51.9 %, 82/158) were males. Almost all patients (98.4 %, 124/126) were not vaccinated. Highest numbers were identified in 2019 (84 cases and 30 %, 21/71, positive sewage samples). In 2020, when three COVID-19 related lockdowns were implemented, 24 cases and 4.3 % (3/69) sewage samples were HAV-RNA positive. The number of HAV-RNA positive cases and positive sewage samples remained low in 2021–2022 (31 and 19 cases, 13.2 %, 9/68 and 4.2 %, 3/72 positive sewage samples, respectively). Sub-genotype IB dominated (90.7 %, 97/107 of cases and 81.5 %, 22/27 of sewage samples), and phylogenetic analysis of HAV samples demonstrated small transmission clusters of sequences from Jews, Bedouin Arabs and foreign workers. Sub-genotype IA was identified in 8.4 % (9/107) of cases and in 18.5 % (5/27) of sewage samples. ConclusionCombined clinical and environmental surveillance is optimal for monitoring HAV. In 2020, the circulation of HAV decreased, possibly following COVD-19 health restrictions. In subsequent years, the incidence remained low. Adults in risk-groups for HAV infection should be vaccinated to minimize HAV circulation.

Use of passive samplers as sewage surveillance tool to monitor a hepatitis A outbreak at a school in Amsterdam, the Netherlands, Oct 2022 – March 2023

BackgroundEnteric hepatitis A virus (HAV) infections during childhood are often asymptomatic but may cause severe illness in adults. To improve public health surveillance we assessed the applicability of sewage monitoring during an HAV outbreak at a primary school.MethodsBetween October 19 and December 27, 2022, five symptomatic HAV cases were notified to the Public Health Service Amsterdam; all attended the same primary school. Passive samplers, small absorbent tools, were deployed in sewage near the school from November 14, 2022, to March 22, 2023. The absorbents were subjected to RNA extraction, HAV PCR testing, and, if positive, sequencing. PCR and sequencing were also performed on plasma and feces samples of HAV cases.ResultsIn 22 out of 88 (25%) of sewage samples, HAV RNA was detected. All HAV-RNA-positive sewage samples until 8 February 2023 were subgenotype IB, matching the strain detected in all cases. Another strain of HAV (subgenotype IA) was detected in sewage from 15 February 2023 onwards, without associated cases.ConclusionsPassive sampler-based sewage monitoring is an effective method to rapidly detect HAV shedding linked to diagnosed cases. It detects unnoticed viral infections and allows monitoring of outbreaks. This suggests that passive sampler-based monitoring is a promising tool supporting the public health response during HAV and other outbreaks.

Molecular characterisation of hepatitis A in the Western Cape province, South Africa in 2023

In 2023, passive laboratory-based surveillance showed an increase in hepatitis A virus (HAV) infection. We investigated hepatitis A incidence using the notifiable medical condition surveillance system (NMCSS) data and molecularly characterised positive blood samples from the Western Cape province for 2023. All HAV IgM seropositive cases from the NMCSS from 1 January to 31 October 2023 in South Africa were investigated. HAV RNA from blood samples that had tested positive for HAV IgM from Western Cape was amplified in the VP1/P2B junction and sequenced (3500Xl Genetic Analyser). Sequences were assembled, aligned (Sequencher) and analysed (Aliview 1.27 and MEGA11). Statistical analysis was performed using Excel and the CuSum2 Threshold to determine suspected outbreaks. In 2023, the incidence of HAV IgM was 6.28/100,000 in South Africa, with the highest incidence in Western Cape province (15.86/100,000). Children aged 5 to 14 years were affected the most in the Western Cape. The positive cases in the Western Cape were above the CuSum2 threshold from January to May 2023, with the highest incidence observed in the City of Cape Town Metropolitan (14.8/100,000). Genotyping was successfully performed on 92.7% (139/150) of serum samples, for which the IB sub-genotype was detected. Three primary mutations R63K, R71S and M104I were observed in more than 49% of the samples. Most of the samples sequenced belonged to patients residing in areas close to each other within the City of Cape Town Southern, Western, and Mitchells Plain sub-districts. The CuSum2 threshold method allowed the identification of suspected HAV outbreaks in the districts within the Western Cape in 2023 while genotyping identified clusters of sub-genotype IB. Genotyping could assist with determining the common source of infection during an outbreak, especially if coupled with epidemiological and geographical data. Further active surveillance can assist in investigating the HAV risk factors for targeted public health responses.

Hepatitis A virus subgenotypes in Latvia, 2008–2021

BackgroundIn Latvia outbreaks of the HAV were observed between 2008 and early 2010 and again in 2017–2018. However, the risks of introducing and spreading infection still exist, as the virus spreads easily when personal hygiene is not followed. MethodsTo determine the spread of HAV subgenotypes in the territory of Latvia the VP1/P2A genomic region of HAV was amplified and sequenced for 259 case serum samples. The study carried out a molecular biological investigation and molecular epidemiological investigation. Demographic data (sex, age), disease data (hepatitis symptoms, hospitalization, vaccination) and epidemiology data (part of the outbreak, possible source of infection, recent travel) were collected. Based on the obtained sequences, the phylogenetic tree was built and analyzed for the homology and belonging to different isolated HAV clusters from other countries. ResultsFrom the obtained data, it was concluded that HAV subgenotype IA had 13 clusters and 12 sporadic cases, HAV subgenotype IB had eight clusters and 11 sporadic cases, HAV subgenotype IIIA had one cluster and nine sporadic cases. It was found that the sources of infection among the investigated cases were different, they were mostly associated with contact with a patient with HAV, travel, as well as between persons who inject drugs and men who have sex with men, and the prevalence of HAV similar sequences was observed in different years. It was concluded that patients with HAV subgenotype IA had the longest hospitalization duration and averaged 9.3 days, while patients with subgenotype IB - 7.3 days, subgenotype IIIA - 7.7 days. Analyzing the data on vaccination, it was found that mostly all were not vaccinated or had an unknown vaccination status. ConclusionsAll of this has led to the conclusion that the application of molecular biological methods of the HAV and a careful analysis of epidemiological data can help to better understand the ways of spreading the infection, investigate local outbreaks, detect cases of imported infection and track the recirculation of the virus.

Molecular epidemiology and characterization of endemic, epidemic and imported hepatitis A virus (HAV) strains in Hungary (2003-2022).

Hepatitis A virus (HAV) is one of the most important etiological agents of acute viral hepatitis but comprehensive molecular epidemiological study with chrono-phylogeographical data are not available from Hungary.Between 2003 and 2022, a total of 8,307 HAV infections were registered officially in Hungary of which 400 (4.8%) HAV IgM antibody-positive serum samples were collected countrywide. HAV genomic RNA was successfully detected in 216/400 (54%) sera by RT-PCR subsequently confirmed by sequencing. The complete nucleotide sequences of VP1 region were determined in 32 representative HAV strains. Based on the sequence analysis, 150 (69.4%) strains were characterized as HAV sub-genotype IA and 66 (30.6%) as sub-genotype IB, respectively. Based on the combined epidemiological and molecular data, epidemic, endemic, and imported HAV strains were also characterized. The first two registered countrywide outbreaks started among men-sex-with men (MSM) in 2011 (sub-genotype IA) and 2021 (sub-genotype IB), the continuously circulating endemic/domestic HAV strain (sub-genotype IA) in East Hungary and the travel-related sub-genotype IB strains from Egypt should be highlighted. All HAV strains are deposited in the HAVNET database (https://www.rivm.nl/en/havnet).In this 20-year-long comprehensive molecular epidemiological study, we report the genetic characterization and geographic distribution of endemic, epidemic and imported HAV strains for the first time in Hungary with continuous co-circulation of sub-genotypes IA and IB HAV strains since 2003. These data provide basic information about the HAV situation in the country in an international context and can promote more effective national public health intervention strategies for the prevention of HAV transmissions and infections.

Molecular epidemiology of hepatitis A outbreaks and sporadic cases, Latvia, 2017 to 2019.

BackgroundHepatitis A is an acute infection of the liver caused by hepatitis A virus (HAV). Molecular detection and typing of the HAV VP1/P2A genomic region is used for genotyping and outbreak investigations. After a large hepatitis A outbreak in Latvia in 2007–08, only sporadic cases were registered until 2017 when a rise in cases occurred. During 2017–19, 179 laboratory-confirmed hepatitis A cases were notified in Latvia.AimTo investigate the observed increase in hepatitis A cases during 2017 and to determine whether these cases were linked to one another, to risk groups, or to other outbreaks. The majority of HAV samples (69.8%) were typed.MethodsThe VP1/P2A genomic region of HAV was amplified and sequenced for 125 case serum samples. Information about hepatitis-related symptoms, hospitalisation, vaccination, a possible source of infection and suspected countries of origin of the virus were analysed for sequenced cases.ResultsMost HAV strains were subgenotype IA (n = 77), of which 41 were strains circulating among men who have sex with men (MSM) populations in Europe (VRD_521_2016 (n = 32), RIVM-HAV16–090 (n = 7) or V16–25801 (n = 2)). Forty-four cases were subgenotype IB and four cases subgenotype IIIA. However, other clusters and sporadic cases were detected with or without identifying the epidemiological link.ConclusionThis work represents molecular epidemiological data of hepatitis A cases in Latvia from 2017 to 2019. Molecular typing methods allow identification of clusters for public health needs and establishing links with other outbreaks, and to compare Latvian strains with reported strains from other countries.

Potentially Infectious Novel Hepatitis A Virus Strains Detected in Selected Treated Wastewater Discharge Sources, South Africa.

Hepatitis A virus (HAV) is a waterborne pathogen of public health importance. In South Africa (SA), unique HAV subgenotype IB strains have been detected in surface and wastewater samples, as well as on fresh produce at the point of retail. However, due to the use of molecular-based assays, the infectivity of the detected strains was unknown. Considering the potential shift of HAV endemicity from high to intermediate, which could increase the risk of severe symptomatic disease, this study investigated the identity of HAV strains detected before and after viability treatment of selected wastewater discharge samples. For one year, 118 samples consisting of sewage, treated wastewater discharge and downstream dam water were collected from five wastewater treatment plants (WWTP 1, 2, 3, 4 and 5). Unique HAV IB strains were detected in samples from all five WWTPs, with 11 of these strains carrying amino acid mutations at the immunodominant and neutralisation epitopes. A quasispecies dynamic of HAV has also been detected in sewage samples. The subsequent application of viability PCR revealed that potentially infectious HAV strains were discharged from WWTP 1, 2, 4 and 5 into the dam. Therefore, there is a potential risk of HAV exposure to communities using water sources downstream the WWTPs.

Detection of Piscine orthoreoviruses (PRV-1b AND PRV-3a) in farmed Coho salmon with jaundice syndrome from Chile

Piscine orthoreoviruses (PRVs) represent a group of marine viruses belonging to Reoviridae family, which are associated to emerging diseases in farmed Atlantic salmon, Rainbow trout and Coho salmon around the world. In Chile, the prevalence and deleterious effects of PRV infection in farmed salmonids had been studied recently, showing the occurrence of PRV-1 (formerly PRV-Ss) in Atlantic salmon with Heart and Skeletal Muscle Inflammation (HSMI), PRV-3 (formerly PRV-Om) in Rainbow trout with HSMI-like disease, and both viruses (PRV-1 and PRV-3) in Coho salmon with HSMI-like disease. In 1997, Jaundice Syndrome in Coho salmon was first described in Chile associated to outbreaks with high mortality, which occur during autumn season. Then, it was reported in 2006 that the jaundice condition in Coho salmon is consistent with an infectious form of hemolytic anemia and probably a virus was the causative agent, however the identification of the putative virus was not possible. Currently, the etiology of this syndrome is unknown, but several of the clinical signs exhibited by diseased Coho salmon are comparable to the signs showed by salmonids affected with some disease which are caused by PRVs. Therefore, it was of interest to determine if some PRVs could be related to Jaundice Syndrome in Coho salmon. In this manner, heart and head kidney tissue samples obtained from Coho salmon mortalities were analyzed in search of viral pathogens, where the presence of both PRV-1 and PRV-3 was confirmed by RT-qPCR and Sanger sequencing. Moreover, full-length genomic segment S1 sequences were obtained to determine the phylogenetic relationship with others PRV isolates previously described in Europe, America and Japan. One group of sequences found in Coho salmon with Jaundice Syndrome clustered into Sub-genotype Ib together with PRV-1 isolate described in Coho salmon with HSMI-like condition from Chile, and other group of sequences clustered into Genotype II with the first PRV-3 detected in Rainbow trout from Norway into previously described PRV-3a clade. Our results also suggest that PRVs has developed an infection cycle, which involves to Atlantic salmon, Rainbow trout and Coho salmon.

The excretion rate and stability of HAAg in human fecal samples after live attenuated hepatitis A vaccination.

The live attenuated hepatitis A virus vaccine (HA-L) is in routine use in the Chinese national immunization program (NIP). The major disadvantages of HA-L include that theoretically, it may be possible for mutation shifts and secondary infections of the live vaccine viral strain. The aim of this study was to explore variation in the viral strain after vaccination with the HA-L. A total of 1297 fecal samples (including 470 for the 18 to 36-month-old age group, 527 for the 3 to 16-year-old group, and 300 for the 16 years and older group) were collected in the study, and the rate of hepatitis A virus (HAV) positivity in fecal samples was 11.36% (31/273), 11.44% (31/271), 9.70% (26/268), 8.47% (21/248), and 9.70% (23/237) on days 0, 7, 14, 21 and 28, respectively. A total of 77 HAV positive samples were randomly selected for VP1/2A (360 bp, 2218-2577) gene analysis. Phylogenetic trees were then constructed by the neighbor-joining method. Phylogenetic analyses showed that all the isolated HAV strains belonged to sub-genotype IB, which was the same as the vaccine strain. Compared with the vaccine strain, HM-175/7MK-5 (M16632.1), there were only two base mutations discovered, at 2291 and 2568. However, the amino acid mutation analysis showed that those base mutations were synonymous mutations. The isolated HAV strains were genetically stable. This study provides a reference for the safety concern regarding the routine and wide-range use in people older than 18 months.

Near-Complete Genome Sequence of a Hepatitis A Subgenotype IB Virus Isolated from Frozen Raspberries

Hepatitis A virus is one of the most common causes of foodborne viral illness. Here, we report the nearly complete genome sequence of a hepatitis A virus (subgenotype IB) isolated from frozen raspberries using RNA sequencing-based metagenomics.

2192. Hepatitis A in Greece 2009–2017: Time for Setting New Priorities

BackgroundThe aim of this study was to present the epidemiological data and describe the outbreaks of hepatitis A identified over the period 2009–2017 in Greece.MethodsCases recorded through the mandatory notification system were classified in accordance with the European case definition. Data were analyzed by population group (general population, Roma, refugees / migrants, travelers to endemic countries, and MSM).ResultsBetween 2009 and 2017, 1,072 cases were reported to the Department of Epidemiological Surveillance and Intervention of the Hellenic Center for Disease Control and Prevention. The median annual number of reported cases was 86 (41–277). Reported cases exceeded the expected number in 2013, 2016 and 2017. In 2013 165 cases were reported [three times higher than the mean annual n reported for 2005–2012; 101 (61.6%) among Roma]. Three clusters were identified accounting for 50% of reported cases. Genotyping showed that clusters were due to hepatitis A virus subgenotype IA. In 2016, after the closure of the northern borders of Greece in March, 177 cases were reported among refugees (subgenotype IB, the majority of affected of Syrian origin). In 2017, an outbreak among men having sex with men (MSM) was recorded as part of a European HAV outbreak. One hundred and seventy-six male cases were recorded (median age; 38 years old, range; 21–55); 81 identified themselves as MSM. Genotyping data showed linkage to identified European clusters (subgenotype IA).ConclusionHepatitis A remains a significant Public Health threat in Greece. Preventive interventions should target populations with low access to healthcare, as well as MSM.Disclosures All authors: No reported disclosures.

Two concurrent outbreaks of hepatitis A highlight the risk of infection for non-immune travellers to Morocco, January to June 2018.

From January to June 2018, two ongoing hepatitis A outbreaks affected travellers returning from Morocco and cases in Europe without travel history, resulting in 163 patients in eight European countries. Most interviewed travel-related cases were unaware of the hepatitis A risk in Morocco. Molecular analysis revealed two distinct hepatitis A virus (HAV) strains (subgenotype IA DK2018_231; subgenotype IB V18–16428). Vaccination recommendations should be emphasised to increase awareness among non-immune travellers to Morocco and HAV-endemic countries.

Monitoring of Foodborne Hepatitis A virus Outbreaks in the Fresh Foods

HEPATITIS A virus (HAV) is one of the most widespread foodborne pathogens and the cause of viral hepatitis. Fresh foods can be considered as a vector of transmission for HAV when contaminated by spoiled irrigation water or when prepared by infected food handlers. To improve microbiological detection and to increase insights into the contribution of fruit and vegetables to foodborne viral transmission, sensitive and standardized methods are needed. Two outlet drainage water samples from El-Mariotia and El-Gable Elasfar canals (Nile River) were collected in each month starting from March 2015 over a period of 12 months. As well as vegetables and fruit samples (lettuce, green onion and strawberry) were irrigated and washed with drainage water were also collected. Samples were extracted and concentrated for viral analysis. Strawberry and lettuce foods collected from El-Mariotia and lettuce from El-Gable Elasfar gave positive serological results by enzyme-linked immunosorbent assay (ELISA) test.Reverse transcription polymerase chain reaction (RT-PCR) was successfully used to detect the virus in strawberry and lettuce samples using HAV-specific primers, designed to amplify a 500 bp fragment covering VP1/2A gene in HAV. The VP1/2A gene was sequenced and the nucleotide sequence similarity was in the range of 95- 99.1% between HAV-Eg isolate and 30 HAV sequences retrieved from GenBank. Phylogenetic analysis revealed that HAV-Eg isolate was grouped into a clade comprising Egyptian HAV isolates sub-genotype IB. Phylogenetic tree of nucleotides sequence showed that HAV sub- genotype IB is the circulating strain.

Genotypes of hepatitis a virus in Turkey: first report and clinical profile of children infected with sub-genotypes IA and IIIA

BackgroundHepatitis A virus (HAV) is a food and water-borne virus causing clinical (mainly hepatitis) and subclinical disease in humans. It is important to characterize circulating strains of HAV in order to prevent HAV infections using efficacious vaccines. The aim of this study was the detection and characterization of the circulating strains of HAV in Turkey by performing serology, RT-PCR, sequencing and phylogenetic analysis.MethodsIn this study, 355 HAV suspected cases were analysed by ELISA for the presence of antibodies to HAV. RNA was extracted from 54 HAV IgM positive human sera. None of the suspect cases were vaccinated against HAV and they never received blood transfusions. Samples found positive by RT-PCR using primers targeting the VP1/VP2A junction and VP1/VP3 capsid region of HAV, were subjected to sequencing and phylogenetic analyses.ResultsIgM type antibodies to HAV were detected in 54 patients. Twenty one of them were students. The age of IgM positive cases was between 3 and 60 years. IgM positivity differed in age groups and was higher in the age group 3 to 10 years. Phylogenetic analysis showed that the majority of HAV strains detected in this study belong to the “HAV 1B” cluster. In addition, the HAV sub-genotypes IA (KT874461.1) and IIIA (KT222963.1) were found in 2 children. These sub-genotypes were not previously reported in Turkey. The child who carried sub-genotype IIIA travelled to Afghanistan and presented with abdominal pain, icterus and vomitus. He was positive for anti-HAV IgM and IgG but negative for hepatitis B and C. Liver enzymes like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and lactate dehydrogenase were severely elevated. Bilirubin levels were also increased. White blood cells, neutrophils and hemoglobin were decreased while lymphocytes and monocytes were increased. Similar clinical signs and laboratory findings were reported for the child infected with sub-genotype IA but aspartate aminotransferase and alanine aminotransferase were not severely elevated.ConclusionsThe results indicate that molecular studies determining the HAV genotype variation in Turkey are timely and warranted. The majority of IgM positive cases in 3–10 year old patients indicate that childhood vaccination is important. Sub-genotype IB is the most prevalant genotype in Turkey. Surprisingly, sub-genotype IA and IIIA are also present in Turkey; future diagnostic efforts need to include diagnostic methods which can identify this emerging HAV genotypes. Our results also show that one important risk factor for contracting hepatitis A virus is international travel since genotype IIIA was detected in a child who had travelled to Afghanistan.

Detection and characterization of hepatitis A virus circulating in Egypt.

Hepatitis A virus (HAV) still poses a considerable problem worldwide. In the current study, hepatitis A virus was recovered from wastewater samples collected from three wastewater treatment plants over one year. Using RT-PCR, HAV was detected in 43 out of 68 samples (63.2%) representing both inlet and outlet. Eleven positive samples were subjected to sequencing targeting the VP1-2A junction region. Phylogenetic analysis revealed that all samples belonged to subgenotype IB with few substitutions at the amino acid level. The complete sequence of one isolate (HAV/Egy/BI-11/2015) showed that the similarity at the amino acid level was not reflected at the nucleotide level. However, the deduced amino acid sequence derived from the complete nucleotide sequence showed distinct substitutions in the 2B, 2C, and 3A regions. Recombination analysis revealed a recombination event between X75215 (subgenotype IA) and AF268396 (subgenotype IB) involving a portion of the 2B nonstructural protein coding region (nucleotides 3757-3868) assuming the herein characterized sequence an actual recombinant. Despite the role of recombination in picornaviruses evolution, its involvement in HAV evolution has rarely been reported, and this may be due to the limited available complete HAV sequences. To our knowledge, this represents the first characterized complete sequence of an Egyptian isolate and the described recombination event provides an important update on the circulating HAV strains in Egypt.

Hepatitis A virus infections and outbreaks in asylum seekers arriving to Germany, September 2015 to March 2016

From September 2015 to March 2016, hepatitis A notifications in Germany increased by 45% to 699 cases compared to 482 cases in the same period of the previous year. Children aged five to nine years were predominantly affected (22% of all cases in this period). We hypothesized that this increase could be explained by the marked influx of asylum seekers in this time period. We analysed national surveillance data and estimated the number of imported and autochthonous hepatitis A cases in asylum seekers. We also investigated molecular signatures of hepatitis A viruses sampled from asylum seekers to identify chains of transmission. We found that 40% (278 cases) of all 699 hepatitis A cases notified between September 2015 and March 2016 in Germany concerned asylum seekers. Most infections were acquired abroad, but at least 24% accounted for autochthonous infections. Among asylum seekers, children aged five to nine years were overrepresented with 97 of 278 (35%) notified cases. The analysed hepatitis A virus sequences were primarily subgenotype IB strains and clustered with previously isolated samples from the Middle East, Turkey, Pakistan and East Africa. Except one transmission from an asymptomatic child to a nursery nurse working in a mass accommodation, we are not aware of infection chains involving asylum seekers and German residents. We conclude that asylum-seeking children and adolescents are susceptible to hepatitis A virus infections, particularly children aged five to nine years. Measures to prevent secondary infections in asylum seekers such as extended hygiene measures and post-exposure prophylaxis seem advisable.Emerging Microbes & Infections (2017) 6, e26; doi:10.1038/emi.2017.11; published online 26 April 2017

First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV.

BackgroundHeart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic salmon Salmo salar, first recognized in 1999 in Norway, and recently associated with piscine orthoreovirus (PRV) infection. To date, HSMI lesions with presence of PRV have only been described in marine-farmed Atlantic salmon in Norway. A new HSMI-like disease in rainbow trout Oncorhynchus mykiss associated with a PRV-related virus has also been reported in Norway.MethodsSampling of Atlantic salmon and coho salmon was done during potential disease outbreaks, targeting lethargic/moribund fish. Fish were necropsied and tissues were taken for histopathologic analysis and testing for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons.ResultsThe Atlantic salmon manifested the classical presentation of HSMI with high PRV virus loads (low Ct values) as described in Norway. The coho salmon with low Ct values had myocarditis but only in the spongy layer, the myositis of red muscle in general was mild, and the hepatic necrosis was severe. Upon phylogenetic analysis of PRV segment S1 sequences, all the Chilean PRV strains from Atlantic salmon grouped as sub-genotype Ib, whereas the Chilean PRV strains from coho salmon were more diversified, grouping in both sub-genotypes Ia and Ib and others forming a distinct new phylogenetic cluster, designated Genotype II that included the Norwegian PRV-related virus.ConclusionsTo our knowledge the present work constitutes the first published report of HSMI lesions with presence of PRV in farmed Atlantic salmon outside of Europe, and the first report of HSMI-like lesions with presence of PRV in coho salmon in Chile. The Chilean PRV strains from coho salmon are more genetically diversified than those from Atlantic salmon, and some form a distinct new phylogenetic cluster, designated Genotype II.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0554-y) contains supplementary material, which is available to authorized users.

Detection and Molecular Characterization of Hepatitis A Virus from Tunisian Wastewater Treatment Plants with Different Secondary Treatments.

Hepatitis A virus (HAV) is the main causative agent of hepatitis infection associated with waterborne outbreaks worldwide. In Tunisia, there is no specific surveillance system for HAV and current secondary wastewater treatment processes are unable to remove viral particles, which present a potential public health problem. Qualitative and quantitative analysis of HAV in 271 raw and treated wastewater samples from five sewage treatment plants (STPs) during 13 months was performed. Moreover, the efficiency of three secondary wastewater treatment processes (conventional activated sludge, extended aeration, and oxidation ditch activated sludge) was evaluated. Data obtained demonstrated that HAV is endemic in Tunisia and circulates with high prevalence in both raw (66.9%) and treated (40.7%) wastewater. HAV circulates throughout the year in the coastal areas, with the highest rates found during summer and autumn, whereas in central Tunisia, high levels were shown in autumn and winter. Total virus removal was not achieved, since no difference in mean HAV loads was observed in effluents (6.0 × 10(3) genome copies [GC]/ml) and influents (2.7 × 10(3) GC/ml). The comparison of the HAV removal values of the three different wastewater treatment methods indicates that extended aeration and oxidation ditch activated sludge had better efficiency in removing viruses than conventional activated sludge did. Molecular characterization revealed that the vast majority of HAV strains belonged to subgenotype IA, with the cocirculation of subgenotype IB in wastewater treatment plants that collect tourism wastewater. This report provides important data on the incidence, behavior, seasonality, and genotype distribution of HAV in the environment in Tunisia, as well as the risk of infection derived from its occurrence in effluents due to inadequate wastewater treatment. In addition, these findings seem to confirm that the prevalence of HAV depends on socioeconomic level, sanitary conditions in the communities, sewage facilities, the locality, and the climate. The wide dispersion of HAV in effluents proves the inefficacity of the current wastewater treatment processes used in Tunisia to remove virus; therefore, establishment of tertiary treatment processes or replacement of the medium-charge activated sludge (conventional activated sludge) by the low-charge version (oxidation ditch activated sludge) is absolutely needed. Rapid detection of the HAV genome in wastewater may provide a timely warning sign to health authorities to implement population protection measures.

High Genetic Diversity of Newcastle Disease Virus in Wild and Domestic Birds in Northeastern China from 2013 to 2015 Reveals Potential Epidemic Trends.

Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is one of the most important viral diseases of birds globally, but little is currently known regarding enzootic trends of NDV in northeastern China, especially for class I viruses. Thus, we performed a surveillance study for NDV in northeastern China from 2013 to 2015. A total 755 samples from wild and domestic birds in wetlands and live bird markets (LBMs) were collected, and 10 isolates of NDV were identified. Genetic and phylogenetic analyses showed that five isolates from LBMs belong to class I subgenotype 1b, two (one from wild birds and one from LBMs) belong to the vaccine-like class II genotype II, and three (all from wild birds) belong to class II subgenotype Ib. Interestingly, the five class I isolates had epidemiological connections with viruses from southern, eastern, and southeastern China. Our findings, together with recent prevalence trends of class I and virulent class II NDV in China, suggest possible virus transmission between wild and domestic birds and the potential for an NDV epidemic in the future.

Efficiency of hepatitis A virus removal in six sewage treatment plants from central Tunisia.

The efficiency of six Tunisian sewage treatment plants (STP) for the removal of hepatitis A virus (HAV) from wastewater was analysed in order to evaluate the potential risk for human health linked to reuse or discharge of treated wastewater into the environment. The STP utilize different biological wastewater treatments including primary treatment, which involves the physical removal of organic and inorganic solids, and secondary treatment that involves different processes, such as activated sludge or lagoon. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and conventional RT-PCR were used for the analysis of the 325 wastewater samples (163 raw and 162 treated) obtained. Results revealed highest contamination in west-central of Tunisia in raw wastewater with 62.96% of samples positive for HAV and predominance during winter and autumn, whereas east-central region showed 50.62% of positive samples with high prevalence from winter through summer. The quantitative analysis revealed a range between 4.29×10(1) and 1.24×10(5)RNAcopies/mL in treated wastewater, showing clearly the inefficiency for total removal of HAV regardless of the treatment method used. The vast majority of HAV sequences belonged to the sub-genotype IA, except one that was assigned to sub-genotype IB.