A highly contagious disease commonly found in winged animals infected a human being for the first time in mainland China.

A 4-year-old boy was recently diagnosed with the H3N8 virus after coming in close contact with chickens and crows his family raised at their Henan province home.

The country’s National Health Commission (NHC) confirmed to Reuters the child suffered from a fever and other bird flu-like symptoms on April 5. However, no close contacts were infected with the virus at the time of publication.

The case seemingly contradicts an earlier initial assessment that ruled out the possibility of H3N8 infecting many people. The commission only identified a low risk of a major epidemic developing like COVID-19, also known as the Chinese Communist Party (CCP) virus.

NHC previously detected the H3N8 variant in horses, dogs, birds, and seals outside China. However, no human cases of H3N8 were reported before April 5.

Middle-aged man infected

Other bird flu strains have infected those working directly with live poultry across the East Asian nation.

A 41-year-old man earlier became the first human to contract the rare H10N3 variant.

NHC revealed the Zhenjiang resident from eastern Jiangsu province was hospitalized on April 28, 2021. He was diagnosed with H10N3 one month later.

According to Reuters, the organization disclosed no details on exactly how he contracted the variant or whether H10N3 can easily spread to humans.

His condition has since stabilized, allowing for his discharge from the hospital. A thorough investigation of close contacts found no other H10N3 cases. In addition, no relevant human cases were reported elsewhere in the world.

The commission still maintains the variant is “low pathogenic” and suggests the virus causes relatively minor illnesses among domestic fowl. The disease is also unlikely to cause a large-scale outbreak.

Very little known

The World Health Organization (WHO) indicated it had very little information on bird-to-human cases in mainland China.

“The source of the patient’s exposure to the H10N3 virus is not known at this time, and no other cases were found in emergency surveillance among the local population–at this time there is no indication of human to human transmission,” WHO said in a statement obtained by the newswire agency.

The United Nations (UN) agency agreed with the CCP’s assessment that only a few human H10N3 cases occurred at irregular intervals in either scattered or isolated locations.

“As long as avian influenza viruses circulate in poultry, sporadic infection of avian influenza in humans is not surprising, which is a vivid reminder that the threat of an influenza pandemic is persistent,” it said.

However, the UN’s Food and Agriculture Organization identified at least 160 isolated microorganism cultures between 1978 and 2018. Viral cases mainly occurred in wild birds or waterfowl in Asia and North America. None were detected in chickens.

Emergency Center for Transboundary Animal Diseases regional laboratory coordinator Filip Claes agrees the variant is “not a very common” one. However, genetic data should still be analyzed to help determine whether H10N3 resembles older viruses or is a novel mix of different viruses.

No significant human bird flu infections have been recorded since the H7N9 strain killed about 300 people in 2016 and 2017.

Symptoms explained

An earlier study called “Pathology of Equine Influenza virus (H3N8) in Murine Model” revealed possible symptoms of the H3N8 virus in horses and dogs.

Researchers from the National Research Centre on Equines, Lala Lajpat Rai University, and the Indian Veterinary Research Institute exposed laboratory mice to the variant. As a result, rodents experienced less movement, weight loss, respiratory complaints, and other symptoms.

“Mice inoculated with equine influenza viruses (group one) began to show respiratory distress and crouching at corners from two days post infection (dpi) onwards,” the research report said.

“Clinical signs were characterized by lethargy, forced expiration, ruffled coat, decreased activity and crouching at the corners with reduced feed and water intake between three and five dpi,” it added.

Mouse health conditions further deteriorated after about one week of exposure to the virus. They lost up to 6% of their original body mass, reflecting previous test results for the H1N1, H3N2, H5N1, and H9N2 variants.

“Severity of the clinical signs increased up to seven dpi, followed by a progressive decline and no clinical signs were observed at 14 dpi,” the report said.

“Reduction in the body weight is considered an important pathologic feature of influenza infection … [and] mice in the present study displayed a maximum of 5% to 6% weight reduction between two to eight dpi.”

Rodents also developed “lung lesions” with bronchitis, bronchiolitis, and interstitial pneumonia.

“Gross and histopathological lesions were confined to respiratory tract including nasal mucosa, trachea and lungs between two and 10 dpi, and comprised of congestion and gray discoloration of whole lung parenchyma with small focal areas of consolidation and red hepatization,” the report said.

Lab mice only began to increase their weight during the second week of infection. They did not regain original body mass until two weeks later.

“Mice started regaining body weight eight dpi onwards but failed to reach original body weight until the end of the experiment, i.e. 14 dpi,” researchers said.

“No mortality was observed in the infected mice. None of the mice from mock inoculated group (group two) showed any clinical signs or significant changes in the body weight,” they added.

They conceded the amount of exposure to H3N8 could have influenced the amount of weight each mouse lost.

“Reduction in body weight of mice in current experiment might be due to difference in dose of virus used for inoculation,” they said.

Degenerative effects

Infected horses experienced adverse pathological changes to the respiratory system. Larger rodents also experienced influenza-like symptoms and immune responses.

“Equines experimentally infected with eq/IB/07 exhibited similar degenerative lesions in the nasal turbinate and trachea,” the report said.

“Pandemic influenza virus infection in humans, H1N1 and H3N2 in guinea pigs, classical swine H1N1 in ferrets and mice, HPAI H5N1 in mice resulted in comparable lesions and antigen distribution as observed in our studies.”

Many toxins and other foreign substances were also present in infected swine and human respiratory systems.

“Abundant EIV antigen could be detected in nasal turbinate, tracheal, bronchial and bronchiolar epithelial cells, alveoli and interstitial macrophages which was in agreement with infection with H1N1 and HPAI H5N1 in human and pandemic swine H1N1 in pigs,” the study said.

No laboratory mice died from this H3N8 experiment, according to the document.

“No mortality and systemic spread of virus could be observed in any of the mice in present investigation with EIV,” it said.

Researchers concluded that EIVs could infect mice and lead to a rapid onset of respiratory illness.

“Clinical signs of seroconversion, substantial virus shedding associated with respiratory illness [are] characterized by gross and histopathological lesions in upper respiratory tract and lungs, no mortality and slow recovery,” they said.

They also believe lab mice can effectively demonstrate H3N8’s effect on human hosts. Test results showed “sufficient evidence” to qualify mice as effective models for studying EIVs.

“[This] mouse model for EI infection offers an attractive valuable tool for studying the interaction of EIV and host immune system, dissecting pathophysiology and molecular pathogenesis of EIV,” they said.

Methodology

The Institute Animal Ethical Committee and Institute Bio-safety Committee approved animal experimentation for this study.

Mice were acclimatized, “ad libitum” fed, and hydrated in micro-ventilator cages. Permission was also granted to use embryonated chicken eggs for virus isolation. Infected tissues and other bio-waste materials were safely disposed of via private partner Synergy Waste Management in India.

The Institute of Microbial Technology supplied both male and female rodents between four and six weeks old. Mice were weighed, and routine health care management was practiced daily.

Test subjects were divided into EIV inoculated (48) and mock-inoculated (24). Before experiments, mice were anesthetized with a mixture of Xylazine and Ketamine. Then, EIV inoculated mice were exposed to 20 microliters of H3N8 through an intranasal route, while the rest were mock inoculated with 20 microliters of allantoic fluid from healthy embryonated chicken eggs.

Mice were closely monitored for clinical signs and weight loss. Six EIV inoculated mice, and three mock-inoculated mice were euthanized at intervals via cervical dislocation. Internal organs were examined for the presence of gross lesions. In addition, blood, serum, and tissue samples were collected for further analysis.

Tissue was gathered from the nasal turbinate, trachea, lung left lobe, heart, liver, kidneys, spleen, brain, stomach, intestine, pancreas, and lymph nodes. Samples were immersed in 10% neutral buffered formalin for at least 96 hours.

Further, specimens were processed via embedding in paraffin, sectioned at three to four-micrometer thickness, and stained with hematoxylin and eosin. Stained slides were examined under a Nikon microscope.

The hyperimmune serum was directed against whole EIV in rabbits to confirm active viral infection in mice, localizing viral antigens in tissue, and immunohistochemical staining of tissue sections. Re-hydrated tissue sections were placed in 3% hydrogen peroxide solution in methanol for antigen retrieval work. The mixture was treated with trypsin and calcium chloride solution for 30 minutes at 37C (98.6F).

Sections were also exposed to primary rabbit hyperimmune serum raised against EIV at a dilution rate of one per 60 parts for an hour at 37C (98.6F). Some sections were treated with horseradish peroxidase. Immuno-reactivity was detected using hydrogen peroxide and a diaminobenzidine tetrahydrochloride substrate. Sections were counterstained with 10% Harris Haematoxylin stain and then examined under a microscope.

Electron microscopic studies on mice tissues were performed at the National Institute of High-Security Animal Diseases. Trachea and lung tissues were collected from mice and immersion-fixed in 2.5% glutaraldehyde solution in phosphate buffer at 4C (39F) for 12 hours. After dehydration and clearing, fixed tissues were embedded in Araldite 502. Semi-thin sections of 400-nanometer thickness were cut using an ultra-microtome and stained.

Stained sections were examined under a light microscope to identify areas of interest for ultrathin sectioning and transmission electron microscopy—an ultra-microtome cut ultrathin sections measuring between 60 and 90 nanometers. Sections were stained with saturated uranium acetate and lead citrate solution. Grids were examined for ultrastructural changes with a transmission electron microscope.

EIV replication in mice respiratory tract was assessed by determining the virus titer in the nasal wash solution and lung tissue collected at different intervals. Nasal washings were collected after sacrifice by tying the trachea towards the trachea-bronchial junction and injecting 300 microliters of Hanks balanced saline solution containing an antibiotic and antifungal solution. Nasal wash was collected from external nares and stored at –70C (–94F). For virus isolation and titration, lung tissue (25 micrograms) was homogenized in cold phosphate-buffered saline, followed by serial 10-fold dilutions and inoculation in embryonated chicken egg via an intra-allantoic route.

A QIAamp Viral RNA mini kit helped extract ribonucleic acid (RNA) from lung homogenate (25 milligrams) and nasal washings (140 microliters). The quality and quantity of isolated RNA were monitored in BioPhotometer Plus and stored at –80C (–112F) for further analysis.

Airborne transmission

different study called “Respiratory transmission of an avian H3N8 influenza virus isolated from a harbor seal” confirmed the same variant could be transmitted via airborne droplets.

Researchers from St. Jude Children’s Research Hospital, the University of North Carolina, and the U.S. Geological Survey discovered that H3N8 could quickly spread with limited direct contact.

They found seals, ferrets, and mice effectively transmitted the disease through respiratory moisture, indicating the variant could quickly spread through large populations.

“The harbor seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells,” they said in the research report.

“Although only the seal virus transmitted via respiratory droplets, the other avian viruses within this clade efficiently transmitted via contact [and] this may be a unique feature of this clade worthy of further studies.”

Researchers believe the strain poses considerable risks to humans since test subjects showed weak disease resistance. In addition, lab mice even experienced a jump in morbidity.

“Transmissibility of the seal H3N8 virus, coupled with the apparent lack of immunity makes these strains a concern,” they said.

“Implication of the H3N8 virus as a past human pandemic virus, the recent isolation of this subtype in pigs–and the potential acquisition of mammalian adaptation phenotypes–make the need for surveillance and risk analysis of these viruses of public health importance.”

They found proof the disease previously infected large numbers of people and urged authorities to closely monitor infections in the future to prevent history from repeating.

“Prevalence of H3N8 viruses in birds and multiple mammalian species, including recent isolations from pigs and evidence that it was a past human pandemic virus, make the need for surveillance and risk analysis of these viruses of public health importance,” they said.

The study also found the H3N8 variant seemingly underwent genetic changes that could increase the chance of a future outbreak.

“In 2011 an avian H3N8 influenza virus, isolated from [more than 160] moribund New England harbor seals, was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses,” the report said.

“Further, the virus acquired a substitution in the PB2 gene known to increase pathogenicity in mice [and, when] combined, these studies suggest that the seal H3N8 virus—although wholly derived from birds—may have the ability to cause enhanced disease and potentially transmit among mammals,” it added.

Global implications

Researchers speculate other airborne variants might already exist around the world.

“Indeed, the efficient respiratory droplet transmission and replication of this clade of H3N8 virus indicate that mammalian-transmissible, avian influenza viruses may already exist in nature,” they said.

Influenza H3N8 viruses are some of the most commonly found subtypes in wild birds. They are associated with mild to no disease in infected creatures. Unlike many avian subtypes, the strains have a broad host range relating to ongoing outbreaks in dogs and horses. They have also been isolated from pigs, donkeys, and seals.

“This suggests that these predominantly avian viruses easily cross species-barriers and establish lineages in mammals,” the study said.

The report also identified a direct link between infected seals and close contact with symptomatic humans.

“During one of the H7N7 seal outbreaks four persons in close contact with diseased seals developed conjunctivitis, demonstrating that direct transmission to humans can occur,” it said.

“The full extent of H3N8 virus exposure in harbor seals is unknown,” it added.
Researchers revealed variants like H3N3, H4N5, H4N6, and H7N7 were earlier isolated from seals with respiratory symptoms. In addition, both H4N5 and H7N7 have already been connected to large-scale mortality events.

“In previous mortality events, up to 20% of the local seal population died, and it was estimated that between 3% and 5% of the total harbor seal population in the northeast perished,” they said.

“Depending on location, timing and species tested, serological surveys in the United States and Canada have reported between zero and 26% of the seals had prior exposure to influenza viruses,” they added.

Fully approved study

St. Jude Children’s Research Hospital’s animal care and use committee approved all animal experiments. Jackson Laboratory supplied 16 six to eight-week-old female mice per group. They were lightly anesthetized with isofluorane and intranasally inoculated with phosphate-buffered saline or 105 tissue culture infectious dose units of virus in 25 microliters of phosphate-buffered saline.

Mice were monitored daily for clinical signs of infection and weighed every 48 hours post-infection (hpi). On days three and six post-infection, three control and diseased mice were euthanized, and lungs were collected and homogenized in 1 milliliter of phosphate-buffered saline. Viral titers are determined by tissue culture infectious dose analysis.

Triple F Farms supplied three 9 to 15-week-old male ferrets for transmission studies. They were inoculated intranasally with 106 tissue culture infectious dose units in 1 milliliter of phosphate-buffered saline.

Twenty-four hours later, three naïve ferrets per group were either housed in separate cages or placed in direct contact with the infected group. Bodyweight and temperature were measured every 48 hours, and ferrets were monitored for anorexia, sneezing, nasal discharge, and lethargy.

Nasal washes were collected every two dpi for viral titration. Sera was gathered at 14 dpi for analysis. Experiments were repeated three times for harbor seal virus and twice for other diseases, with six to nine ferrets in each group.

Based on differences in receptor specificity among avian H3N8 viruses, ferrets were intranasally inoculated with 106, 50% tissue culture infectious dose units of the duck/Ukraine, mallard/50, mallard/551, ruddy duck, long-tailed duck, and seal viruses.

Naïve ferrets were placed with the infected group for direct contact transmission at one dpi. In the seal virus’s case, they were housed in cages adjacent to donor ferrets and separated by grills. This allowed for unobstructed airflow while preventing direct contact (respiratory droplet transmission).

Animals were monitored for disease signs until 14 dpi. Nasal washes were collected every two dpi to assess viral shed and seroconversion determined at 14 dpi. Although all inoculated animals shed the virus between two and six dpi and seroconverted, only strains genetically related to the seal virus were transmitted to direct-contact animals.

“Interestingly, the long-tailed duck virus only transmitted to two out of three direct contacts, while the ruddy duck virus transmitted to 100% of contacts,” the study said.

“Most importantly, the seal virus transmitted by respiratory droplet to two out of three contacts between days five and seven post exposure,” it added.

No overt clinical signs of disease were observed in animals following infection. Ferrets inoculated with the harbor seal virus sneezed between two and four dpi. One of the aerosol contact animals sneezed on day six post-infection.

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