Bats Worldwide Carry Hepatitis E Virus-Related Viruses That Form a Putative Novel Genus within the Family Hepeviridae

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis in tropical and temperate climates. Tropical genotypes 1 and 2 are associated with food-borne and waterborne transmission. Zoonotic reservoirs (mainly pigs, wild boar, and deer) are considered for genotypes 3 and 4, which exist in temperate climates. In view of the association of several zoonotic viruses with bats, we analyzed 3,869 bat specimens from 85 different species and from five continents for hepevirus RNA. HEVs were detected in African, Central American, and European bats, forming a novel phylogenetic clade in the family Hepeviridae. Bat hepeviruses were highly diversified and comparable to human HEV in sequence variation. No evidence for the transmission of bat hepeviruses to humans was found in over 90,000 human blood donations and individual patient sera. Full-genome analysis of one representative virus confirmed formal classification within the family Hepeviridae. Sequence- and distance-based taxonomic evaluations suggested that bat hepeviruses constitute a distinct genus within the family Hepeviridae and that at least three other genera comprising human, rodent, and avian hepeviruses can be designated. This may imply that hepeviruses invaded mammalian hosts nonrecently and underwent speciation according to their host restrictions. Human HEV-related viruses in farmed and peridomestic animals might represent secondary acquisitions of human viruses, rather than animal precursors causally involved in the evolution of human HEV.     

Genetic and Experimental Evidence for Cross-Species Infection by Swine Hepatitis E Virus

Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared ≥97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses.     

Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol

Influenza viruses resistant to the neuraminidase (NA) inhibitor oseltamivir arise under drug selection pressure both in vitro and in vivo. Several mutations in the active site of the viral NA are known to confer relative resistance to oseltamivir, and influenza viruses with certain oseltamivir resistance mutations have been shown to transmit efficiently among cocaged ferrets. However, it is not known whether NA mutations alter aerosol transmission of drug-resistant influenza virus. Here, we demonstrate that recombinant human influenza A/H3N2 viruses without and with oseltamivir resistance mutations (in which NA carries the mutation E119V or the double mutations E119V I222V) have similar in ovo growth kinetics and infectivity in guinea pigs. These viruses also transmit efficiently by the contact route among cocaged guinea pigs, as in the ferret model. However, in an aerosol transmission model, in which guinea pigs are caged separately, the oseltamivir-resistant viruses transmit poorly or not at all; in contrast, the oseltamivir-sensitive virus transmits efficiently even in the absence of direct contact. The present results suggest that oseltamivir resistance mutations reduce aerosol transmission of influenza virus, which could have implications for public health measures taken in the event of an influenza pandemic.     

No transmission of hepatitis E virus in pigs fed diets containing commercial spray-dried porcine plasma: a retrospective study of samples from several swine trials

Background

Hepatitis E virus (HEV) has been reported in the human population and pigs are a recognized reservoir for HEV and a possible source of HEV transmission to humans. Spray-dried porcine plasma (SDPP) is an ingredient commonly used in feed for pigs around the world. Even though processing conditions used to produce SDPP should be adequate to inactivate HEV, it was of interest to analyze commercial SDPP samples for presence of genome and antibodies (AB) against HEV and to retrospectively analyze serum samples collected from pigs used in past experiments that had been fed diets containing either 0% or 8% SDPP to detect potential transmission of HEV as determined by seroconversion.

Results

Eighty-five commercial SDPP samples were analyzed by ELISA and 100% of them contained AB against HEV, while 22.4% (11 of 49 samples analyzed) were positive for HEV RNA.Frozen sera samples (n?=?140) collected from 70 pigs used in past experiments that had been fed diets containing either 0% or 8% commercial SDPP was analyzed by ELISA for AB against HEV. Age of pigs at sera sampling ranged from 3 to 15 weeks and feeding duration of diets ranged from approximately 4 to 9 weeks. One lot of SDPP used in one experiment was analyzed and confirmed to contain HEV RNA. Regardless of the diet fed, some sera samples collected at the beginning of an experiment contained AB titer against HEV. These sera samples were collected from weaned pigs prior to feeding of the experimental diets and the HEV titer was probably from maternal origin. However, by the end of the experiments, HEV titer was not detected or had declined by more than 50% of the initial titer concentration.

Conclusions

To our knowledge, this is the first study reporting presence of HEV AB titer and RNA in SDPP. Retrospective analysis of serum collected from pigs fed diets with SDPP revealed no indication of seroconversion to HEV. The results indicate that feeding SDPP in diets for pigs does not represent a risk of transmitting HEV, even though HEV genome may be detected in SDPP.

     

Detection of Hepatitis E Virus Antibodies in Dogs in the United Kingdom

Hepatitis E virus (HEV) genotypes 3 and 4 are zoonotic pathogens, with pigs predominantly implicated in disease transmission. The rapid rise in human cases in developed countries over the past decade indicates a change in epidemiology of HEV, and it has been suggested that additional animal species may be involved in transmission of infection. Multiple studies have identified contact with dogs as a risk factor for HEV infection in industrialised nations, and a low seroprevalence to HEV has previously been reported in dogs in low-income countries. In this study we aimed to evaluate the possibility that dogs are susceptible to HEV, and determine the frequency with which this occurs. Serum samples from UK dogs with and without hepatitis were screened for HEV-specific antibodies, and canine liver and stool samples were analysed by qPCR for the presence of HEV RNA. We describe evidence to show HEV infection occurs at low levels in dogs in the UK, but the strain of origin is undetermined. The low seroprevalence level of HEV in dogs implies the risk of zoonotic disease transmission is likely to be limited, but further investigations will be required to determine if HEV-infected dogs can transmit HEV to man.     

Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil

Serological evidence of hepatitis E virus infection (HEV) has been observed in both humans and different animal species living in non-endemic areas, suggesting that animals could be important reservoir for virus transmission to man. Antibodies to HEV have been detected in some Brazilian population groups. Nevertheless, sporadic cases of acute HEV infection have never been reported. We collected 271 serum samples from several domestic animals and also from pig handlers from Southeast of Brazil in order to investigate the seroprevalence of HEV infection. Anti-HEV IgG was detected in cows (1.42%), dogs (6.97%), chickens (20%), swines (24.3%), and rodents (50%), as well as in pig handlers (6.3%). The recognition of swine HEV infections in pigs in many countries of the world led us to investigate a larger sample of pigs (n = 357) from the same Brazilian region with ages ranging from 1 to > 25 weeks. IgG anti-HEV was detected in 100% of 7-day old pigs. Following a gradual decline between weeks 2 and 8 (probably due to loss of maternal IgG), the prevalence then steady increased until it reached 97.3% of animals older than 25 weeks. Besides the detection of anti-HEV antibodies in different animal species, the results showed that swine HEV infection seems to be almost universal within this Brazilian pig population. This is the first report that shows evidences of HEV circulation in Brazilian animal species and pig handlers.     

Evolution of an Eurasian Avian-like Influenza Virus in Na?ve and Vaccinated Pigs

Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.     

Prevalence and transmission of hepatitis E virus in domestic swine populations in different European countries

ABSTRACT: BACKGROUND: Hepatitis E virus (HEV) genotype 3 and 4 can cause liver disease in human and has its main reservoir in pigs. HEV investigations in pigs worldwide have been performed but there is still a lack of information on the infection dynamics in pig populations. FINDINGS: The HEV transmission dynamics in commercial pig farms in six different European countries was studied. The data collected show prevalence in weaners ranging from 8% to 30%. The average HEV prevalence in growers was between 20% and 44%. The fatteners prevalence ranged between 8% and 73%. Sows prevalence was similar in all countries. Boar faeces were tested for HEV only in Spain and Czech Republic, and the prevalence was 4.3% and 3.5% respectively. The collected data sets were analyzed using a recently developed model to estimate the transmission dynamics of HEV in the different countries confirming that HEV is endemic in pig farms. CONCLUSIONS: This study has been performed using similar detection methods (real time RT-PCR) for all samples and the same model (SIR model) to analyse the data. Furthermore, it describes HEV prevalence and within-herd transmission dynamics in European Countries (EU): Czech Republic, Italy, Portugal, Spain, The Netherlands and United Kingdom, confirming that HEV is circulating in pig farms from weaners to fatteners and that the reproductive number mathematical defined as R0 is in the same range for all countries studied.     

Complete genome sequence of the genotype 4 hepatitis E virus strain prevalent in swine in Jiangsu Province, China, reveals a close relationship with that from the human population in this area

Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human viruses. In a previous study, we sequenced the complete genome of a human genotype 4 HEV strain (HM439284) that is prevalent in Jiangsu Province, China. Here we report the complete genome of one genotype 4 HEV strain which is prevalent in swine herds in Jiangsu Province. Phylogenetic analysis indicated that the swine HEV strain in the present study has high sequence homology (>92%) with the genotype 4 HEV strains prevalent in the human population of Jiangsu Province. These results suggested that the genotype 4 HEV strain in the present study is involved in cross-species transmission between swine and humans in this area.     

Cocirculation of Avian H9N2 and Contemporary “Human” H3N2 Influenza A Viruses in Pigs in Southeastern China: Potential for Genetic Reassortment?

Pigs are permissive to both human and avian influenza viruses and have been proposed to be an intermediate host for the genesis of pandemic influenza viruses through reassortment or adaptation of avian viruses. Prospective virological surveillance carried out between March 1998 and June 2000 in Hong Kong, Special Administrative Region, People's Republic of China, on pigs imported from southeastern China, provides the first evidence of interspecies transmission of avian H9N2 viruses to pigs and documents their cocirculation with contemporary human H3N2 (A/Sydney/5/97-like, Sydney97-like) viruses. All gene segments of the porcine H9N2 viruses were closely related to viruses similar to chicken/Beijing/1/94 (H9N2), duck/Hong Kong/Y280/97 (H9N2), and the descendants of the latter virus lineage. Phylogenetic analysis suggested that repeated interspecies transmission events had occurred from the avian host to pigs. The Sydney97-like (H3N2) viruses isolated from pigs were related closely to contemporary human H3N2 viruses in all gene segments and had not undergone genetic reassortment. Cocirculation of avian H9N2 and human H3N2 viruses in pigs provides an opportunity for genetic reassortment leading to the emergence of viruses with pandemic potential.     

High Prevalence of Hepatitis E Virus in Swedish Moose – A Phylogenetic Characterization and Comparison of the Virus from Different Regions

Background

Hepatitis E virus (HEV) infects a range of species, including humans, pigs, wild boars and deer. Zoonotic transmission may contribute to the high HEV seroprevalence in the human population of many countries. A novel divergent HEV from moose (Alces alces) in Sweden was recently identified by partial genome sequencing. Since only one strain was found, its classification within the HEV family, prevalence in moose and zoonotic potential was unclear. We therefore investigated samples from 231 moose in seven Swedish counties for HEV, and sequenced a near complete moose HEV genome. Phylogenetic analysis to classify this virus within the family Hepeviridae and to explore potential host specific determinants was performed.

Methods and Findings

The HEV prevalence of moose was determined by PCR (marker for active infection) and serological assays (marker of past infection) of sera and 51 fecal samples from 231 Swedish moose. Markers of active and past infection were found in 67 (29%) animals, while 34 (15%) were positive for HEV RNA, 43 (19%) were seropositive for anti-HEV antibodies, and 10 (4%) had both markers. The number of young individuals positive for HEV RNA was larger than for older individuals, and the number of anti-HEV antibody positive individuals increased with age. The high throughput sequenced moose HEV genome was 35-60% identical to existing HEVs. Partial ORF1 sequences from 13 moose strains showed high similarity among them, forming a distinct monophyletic clade with a common ancestor to HEV genotype 1-6 group, which includes members known for zoonotic transmission.

Conclusions

This study demonstrates a high frequency of HEV in moose in Sweden, with markers of current and past infection demonstrated in 30% of the animals. Moose is thus an important animal reservoir of HEV. The phylogenetic relationship demonstrated that the moose HEV belonged to the genotype 1-6 group, which includes strains that also infect humans, and therefore may signify a potential for zoonotic transmission of this HEV.     

Vectoring ability of aphid clones of Rhopalosiphum padi (L.) and Sitobion avenae (Fabr.) and their capacity to retain barley yellow dwarf virus

Vectoring ability of four aphid clones, Rp-M and Rp-R26 of Rhopalosiphum padi and Sa-R1 and Sa-V of Sitobion avenae, to transmit barley yellow dwarf (PAV, MAV and RPV) luteoviruses (BYDV) was compared in controlled conditions. Significant differences between highly efficient vectors (HEV), Rp-M and Sa-Rl, and poorly efficient vectors (PEV), Rp-R26 and Sa-V, were found in transmission of their specific viruses with acquisition and inoculation access periods (AAP, IAP) of 5 days. BYD-RPV was occasionally transmitted by both clones of S. avenae. None of 150 tested apterous adults of the Rp-R26 transmitted BYD-MAV, while 10% of transmission was observed from those of the Rp-M in a parallel test. An improved ELISA and immuno-PCR were adapted to test for viruses in aphids. The results obtained by the improved ELISA indicated there was a good correlation between virus detection in single aphids of HEV clones after a 5 day AAP and virus transmission by them. In contrast, the percentages of virus-carrying aphids of PEV clones were generally higher than those of their transmission rates. BYD-MAV and BYD-RPV were also detected by the improved ELISA in single aphids of their PEV clones, with the exception of BYD-RPV in those of Sa-V. However, after a 2-day IAP, the improved ELISA in most cases failed to detect these viruses in single aphids of PEV clones. Detection by immuno-PCR demonstrated that all three viruses could be acquired and retained by the aphids of both HEV and PEV clones. But, as visualised from electrophoretic bands, after the 2-day IAP the amplified products from aphid extracts of PEV clones were reduced. The detection in a batch of nine aphids by the improved ELISA revealed that virus content in PEV clones decreased more rapidly than that in HEV clones during transmission. Thus, the difference in transmission efficiency of the aphid clones within species was not caused by an inability to acquire virus, but was determined by variation in vectoring ability between them. This was due to differences in ability to prevent the passage of virions from haemocoel to salivary duct and/or different capacities for the retention of BYDV.     

Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo

To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.     

Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.     

Influenza—an emerging and re-emerging disease

Influenza A viruses continue to emerge from the aquatic avian reservoir and cause pandemics. There are periodic exchanges of influenza virus genes or whole viruses between avians and other species giving rise to pandemics of diseases in humans, lower animals and birds. It is hypothesized that pigs are an intermediate host and that China is an epicenter for the evolution of human pandemic strains. However, the transmission of avian influenza viruses to pigs in Europe in 1979 and detection of reassortants with human influenza genes in pigs raises the question of whether the next pandemic of influenza will emerge in Europe!